Synergistic Antileukemic Effects of Hydroxyurea and SRJ23 in T-cell Acute Lymphoblastic Leukemia

Synergistic Antileukemic Effects of Hydroxyurea and SRJ23 in T-cell Acute Lymphoblastic Leukemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synergistic Antileukemic Effects of Hydroxyurea and SRJ23 in T-cell Acute Lymphoblastic Leukemia LAITH ALMARASHDEH, Johnson Stanslas, Thilakavathy Karuppiah, Bahariah Khalid This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5419120/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive blood cancer associated with high relapse rates, affecting about 15% of pediatric and 25% of adult cases. Initial management typically involves hydroxyurea (HU) to reduce elevated white blood cell counts before intensive chemotherapy. However, despite this approach, recurrence remains a substantial challenge, even with stem cell transplants. A promising therapeutic strategy for T-ALL might involve combining HU with an additional compound that can enhance cancer cell inhibition. SRJ23, a semi-synthetic andrographolide derivative, is a potential candidate due to its unique mechanism of inhibiting the GDP-GTP exchange, targeting the oncogenic K-Ras mutation pathway implicated in T-ALL progression. This study investigates the potential of HU and SRJ23 as a combined treatment to enhance apoptotic induction in T-ALL cells. Methods JCLs is an immortalized lines of human T lymphocyte cells that are used to study T-ALL. They were cultured and treated with varying concentrations of HU (7.8–1000 µM) and SRJ23 (0.1–100 µM), individually and in combination, for 96 hours. The cell viability was then assessed using the MTT assay. The drug interactions were calculated using the CompuSyn software, which runs the Chou-Talalay method to calculate the combination index (CI) and generate isobolograms, allowing for the quantitative, determination of synergistic, additive, or antagonistic effects between the two compounds. We used the FITC Annexin V/PI Apoptosis Detection Kit to analyse the treated cells with BD FACS LSRFortressa™ Cell Analyser and FACSDiva™ Software to quantify early and late apoptotic populations. Results Our study shows a significant decline in the JCLs viability resulting from the exposure of HU and SRJ23, especially when in both drugs were in combination. A synergistic effect was observed at two occasions that is when 10µM SRJ23 combined with 125µM HU and 10µM SRJ23 combined with 250µM of HU on the dose-response curves. These combination treatment induced apoptosis confirmed by Annexin V-FITC staining. Conclusion To our knowledge, our study is the first to prove that the combination of HU and SRJ23 exerts a synergistic effect against JCLs and has a significant inhibitory effect in vitro with a remarkable ability to induce apoptosis. Further in-vitro and animal research is deemed required to thoroughly understand the detailed impact of this novel HU and SRJ23 synergism before it can be embarked as a potential novel targeted T-ALL chemotherapy. T-ALL jurkat cell lines hydroxyurea SRJ23 growth inhibition assay synergism combination index apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia characterized by the abnormal growth and proliferation of early T-cell progenitors due to presence of K-Ras driver mutation. Overall incidence of T-ALL was reported to be 0.13 cases per 100000 population. The incidence decreased significantly after the age of 40 (IRR ranging from 0.51 to 0.55, p < 0.01) however with recent developments in diagnostic techniques, intensive chemotherapy and transplant protocols have not derived to the best ways yet for better overall survival, relapse and cure rates for T-ALL. Despite having a markedly enhanced understanding of molecular biology, there were only achievements of 85% five-year survival rate among pediatric populations [ 9 ] and less than 50% for adult population [ 14 ]. This disease typically manifests as patients complaining of pyrexia of unknown origin with or without night sweats, significant loss of weight more than 10% from the baseline weight 6 months prior and loss of appetite unintentionally or B symptoms. Clinical examination on these patients may reveal variable degrees of anemia, hyper-viscosity with or without lymphadenopathy and hepatosplenomegaly. At times, the lymphocytes dysregulation of their function can be apparent with anemia and jaundice in those patients that succumbed into autoimmune hemolytic anemia, bleeding episodes as in spontaneous bruises over the skin, in the muco-cutaneous tissue or other parts of the body either obvious or occult like immune thrombocytopenia presentation. Generalised lymphadenopathy may be apparent on radiology imaging like CAT scan and Pet-scan. T-ALL patients present largely with hyperleukocytosis with predominantly lymphocytes in their full blood picture with access lymphoblast. The flowcytometry or immunophenotyping can further identify the clusters of lymphoblasts accurately but it can never supersede a thorough bone marrow aspirate and trephine examination that of which inclusive of flowcytometric, cytogenetic and molecular testing of the abnormal lymphoblasts. The mainstay of urgent cytoreductive therapy in cytosis in acute and chronic haematological malignancies is by giving affordable and easily available oral genotoxic compound called hydroxycarbamide or generally called hydroxyurea (HU). HU has been traditionally used for decades to suppress any of the cell lineage excessiveness from the marrow of monoclonal in origin for example in acute myeloid leukemia (AML), ALL and chronic myeloproliferative neoplasm (MPN). It is used to control the blood counts therefore reducing the upregulation of malignant gene in polycythemia vera (PV), essential thrombocythemia (ET) and chronic myeloid leukemia (CML). The sequelaes due to uncontrolled blood counts is sometimes fatal and by doing so, it helps to prevent the risk of thrombosis and bleeding that can cause detrimental outcomes. HU is known to inhibit DNA replication [ 8 ] (Koç et al., 2004), and exhibits a short-term toxicity profile, which is generally well-tolerated among the majority of patients [ 4 , 5 ]. The compound exerts an impact on the enzymatic activity of RNR by interfering with the proton-coupled electron transfer process responsible for catalyzing the synthesis of new dNTPs [ 13 , 21 ]. A recent remarkable of scientific evidence to-date, had shown the K-Ras oncogenic mutation as the responsible genetic abnormality in the development of T-ALL. Coincidentally, SRJ23 which is an andrographolide (AG) semisynthetic compound had been reported to have a significant inhibitory effect to K-Ras oncogene in solid cancers, hence inducing apoptosis [ 6 , 7 , 15 , 20 ] (Fig. 1 ). As T-ALL being one of the haematological cancers with the worst outcome in survival despite with the availability of toxic treatment and haematopoeitic stem cell transplants, T-ALL remains to have the worst outcome possible even with the standard treatment. We embarked a study to evaluate and assess the effect of HU and SRJ23 in JCLs for their growth inhibition assay, presence of synergistic drug interactions and apoptosis potency both individually and in combination. 2 Methods 2.1 Growth inhibition assay The JCLs were obtained from the immunology laboratory of the Faculty of Medicine and Health Sciences at Universiti Putra Malaysia. The cells were grown in RPMI 1640 Medium (Sigma-Alderich, Malaysia) with the addition of 10% warm fetal bovine serum, 100 U/L penicillin, and 100 mg/L streptomycin (Nacalai Tesque). The culture was maintained at a temperature of 37 °C in an environment with 5% CO2. The cells were then distributed into 96-well, flat-bottom microtiter plates (TPP, Techno Plastic Products, Switzerland) at a density of 5.0×10^3 cells per well (Fig 2). After a 24-hour incubation period, the cells were treated with various concentrations of SRJ23 (0.1, 1, 10, and 100 µM) and HU (7.8, 15.6, 31.25, 62.5, 125, 250, 500, and 1000 μM) individually and in combination. Subsequently, the cells were incubated for 96 hours at a temperature of 37°C in an environment containing 5% CO2. Following a 96-hour incubation period, a volume of 50 µL of MTT solution with a concentration of 2 mg/mL was introduced to each well. Subsequently, the plates were reintroduced to the incubator for an extra four hours to foster the metabolic processes associated with the MTT. Then the 96-well plate was centrifuged at 300x g for 10 minutes at 4°C. Following centrifugation, the medium and excess MTT were aspirated and the crystals of formazon was dissolved in a volume of 100 µL of dimethyl sulfoxide (DMSO). The determination of the fraction of JCLs living cells was conducted by quantifying the absorbance of purple formazon at a wavelength of 550 nm using a Versamax microplate reader. The statistical analysis was conducted with the assistance of SOFTmax ® Pro software. 2.2 Combination index (CI) for drug-drug interactions analyses The identification of drug interactions was conducted through the utilization of the isobologram and CI technique, which was developed based on the median-effect concept proposed by Chou and Talalay [1,2]. This analysis was performed utilizing the CompuSyn software. The mathematical model employed for the analysis of drug-drug interactions utilizes the formula based on the principle of median effect. The aforementioned equation is expressed as fa/fu = [D/Dm]m in which fa represents the proportion of cells that experience an effect, fu denotes the proportion of cells that do not experience an effect and is equivalent to 1-fa, D signifies the concentration of the drug, Dm represents the dosage of the drug necessary for inhibiting 50% of the cells, and m denotes the slope of the median effect curve. The isobologram is a visual depiction of the pharmacological interaction, wherein a line is drawn connecting the Fa points and the fixed ratio combinations of HU and SRJ23, which are displayed on the x and y axes. The CI, a quantitative measure of drug combination effects obtained from the CompuSyn software, incorporates different concentrations of both drugs to correspond with the percentages of growth inhibition throughout the analysis. Table 1 shows the Chou-Talalay classification of synergism and antagonism, with CI values, preferred symbols, and descriptions. The CI 1 imply synergism, additive, and antagonism, respectively [1]. Table 1 Combination index (CI) values, along with corresponding symbols and descriptions, classify synergism or antagonism based on the Chou-Talalay method [11]. Range of CI Symbol Description <0.1 +++++ Very strong synergism 0.1-0.3 ++++ Strong synergism 0.3-0.7 +++ synergism 0.7-0.85 ++ Moderate synergism 0.85-0.90 + Slight synergism 0.90-1.10 ± Nearly additive 1.10-1.20 - Slight antagonism 1.20-1.45 -- Moderate antagonism 1.45-3.3 --- Antagonism 3.3-10 ---- Strong antagonism >10 ----- Very strong antagonism 2.3 Apoptosis assay The JCLs were cultured by seeding them in a 6-well plate with a flat bottom. The cells were allowed to proliferate exponentially at a density of roughly 1x10^6 cells per 1800 µl/well. The culture was then incubated for 24 hours at a temperature of 37°C (5% CO2 and 95% air). The concentration of synergism was determined by the MTT experiment, and the compounds were diluted in a serial manner to achieve the necessary concentration. These diluted compounds were then administered to the relevant wells in triplicate. In contrast, a concentration of 0.01% of dimethyl sulfoxide (DMSO) was utilized to treat the control wells, which corresponds to the concentration of the vehicle employed in the wells treated with the combination compound. The FITC Annexin V technique was conducted after a duration of 96 hours, following the guidelines specified by the manufacturer in the instructions accompanying the Annexin V-FITC/PI Apoptosis Detection Kit from BD Bioscience. The cells were collected, enumerated, and rinsed twice with cold phosphate-buffered saline (PBS) prior to being reconstituted at a concentration of 1x10^6 cells/ml in 1X Binding Buffer. Subsequently, a total of 100 µl of the solution was used to suspend 1 x 10^5 cells, which were then transferred into a 5 ml culture tube. Following the addition of 5 µl of FITC Annexin V and 5 µl of PI to each tube, a mild vortex was applied, and the mixtures were subsequently incubated at room temperature (25°C) in the absence of light for a duration of 15 minutes. Following the introduction of 400 µl of the 1X Binding Buffer into each tube, the samples were subjected to analysis using flowcytometry BD FACS LSRFortressa TM Cell Analyser and FACSDiva TM within 1 hour. 3 Results 3.1 JCLs growth inhibition assay with HU and (with or without) SRJ23 To evaluate the therapeutic potential efficacy of SRJ23 and HU, the JCL were subjected to a 24-hour cultivation period. Subsequently, these cells were exposed to varying concentrations of SRJ23 (0.1, 1, 10, and 100 µM) and HU (7.8, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 µM) either individually or in combination for a period of 96 hours. Following this treatment, the viability of the cells was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The findings indicated that the dose-response curves exhibited a decrease in cell viability within JCL with a synergistic effect with a combination of concentrations of 10 µM SRJ23 with 125 µM HU or 250 µM (Fig. 3 ). Table 2 Showing the statistical analysis of three replicates reveals the interaction patterns—synergistic, antagonistic, or additive—between SRJ23 (0.1, 1, 10, and 100 µM) and HU (ranging from 7.8 to 1000 µM) following 96 hours of exposure. Statistical significance among treatment groups (SRJ23, HU, and their combined treatment) is indicated by capital letters, with different letters representing significant differences (p < 0.05). Means that share the same letter are not significantly different from each other. The letter 'A' represents the group with the highest cell viability, followed by 'B,' 'C,' and so forth, each letter indicating a sequential decrease in viability. This lettering scheme allows for clear identification of which treatments resulted in the highest and lowest levels of cell viability among the tested groups. SRJ23 dose SRJ23 Mean Viability % HU dose HU Mean Viability % Combination Mean Viability % Combination Fa CI Interaction 0.1 93.76 ± 5.98 BCD 7.8 96.72 ± 4.26 BC 122.56 ± 12.90 A -0.22 ± 0.129 C ------------- antagonism 0.1 93.76 ± 5.98 BCD 15.625 85.53 ± 7.73 CDE 112.90 ± 7.30 AB -0.12 ± 0.070 C ------------- antagonism 0.1 93.76 ± 5.98 BCD 31.25 83.21 ± 9.70 CDE 88.42 ± 3.49 CDE 0.11 ± 0.035 B 3.13 ± 0.925 A antagonism 0.1 93.76 ± 5.98 BCD 62.5 72.24 ± 6.63 EF 73.48 ± 0.50 DEF 0.27 ± 0.004 B 1.87 ± 0.051 AB antagonism 0.1 93.76 ± 5.98 BCD 125 54.77 ± 2.03 F 23.12 ± 0.32 G 0.77 ± 0.003 A 2.16 ± 0.227 AB antagonism 0.1 93.76 ± 5.98 BCD 250 32.32 ± 16.30 G 15.94 ± 3.48 G 0.84 ± 0.030 A 1. 20 ± 0.167 AB antagonism 0.1 93.76 ± 5.98 BCD 500 18.89 ± 0.68 G 12.87 ± 1.09 G 0.87 ± 0.011 A 0.89 ± 0.197 B synergism 0.1 93.76 ± 5.98 BCD 1000 15.90 ± 1.86 G 10.12 ± 0.62 G 0.90 ± 0.006 A 0.86 ± 0.165 B synergism 1 88.72 ± 3.44 ABC 7.8 96.72 ± 4.26 BC 111.28 ± 0.81 A -0.11 ± 0.008 D ------------- antagonism 1 88.72 ± 3.44 ABC 15.625 85.53 ± 7.73 CDE 95.54 ± 7.92 AB 0.04 ± 0.080 CD ------------- antagonism 1 88.72 ± 3.44 ABC 31.25 83.21 ± 9.70 CDE 71.95 ± 14.08 BCD 0.28 ± 0.141 C 1.88 ± 0.512 A antagonism 1 88.72 ± 3.44 ABC 62.5 72.24 ± 6.63 EF 44.34 ± 17.29 EFG 0.56 ± 0.173 B 1.62 ± 0.009 A antagonism 1 88.72 ± 3.44 ABC 125 54.77 ± 2.03 F 23.62 ± 4.31 GH 0.76 ± 0.043 AB 1.25 ± 0.012 A antagonism 1 88.72 ± 3.44 ABC 250 32.32 ± 16.30 G 12.87 ± 1.30 H 0.87 ± 0.012 A 1.31 ± 0.062 A antagonism 1 88.72 ± 3.44 ABC 500 18.89 ± 0.68 G 13.37 ± 4.08 H 0.87 ± 0.041 A 0.77 ± 0.026 A synergism 1 88.72 ± 3.44 ABC 1000 15.90 ± 1.86 G 8.90 ± 0.43 H 0.94 ± 0.020 A 0.70 ± 0.007 A synergism 10 66.07 ± 15.15 C 7.8 96.72 ± 4.26 BC 5.49 ± 0.83 E 0.95 ± 0.008 ABC 0.52 ± 0.202 A synergism 10 66.07 ± 15.15 C 15.625 85.53 ± 7.73 CDE 6.30 ± 0.21 E 0.94 ± 0.002 ABCD 0.61 ± 0.245 A synergism 10 66.07 ± 15.15 C 31.25 83.21 ± 9.70 CDE 4.31 ± 0.53 E 0.96 ± 0.005 A 0.69 ± 0.257 A synergism 10 66.07 ± 15.15 C 62.5 72.24 ± 6.63 EF 5.23 ± 0.53 E 0.95 ± 0.005 AB 0.68 ± 0.226 A synergism 10 66.07 ± 15.15 C 125 54.77 ± 2.03 F 7.25 ± 0.67 E 0.93 ± 0.007 BCD 0.67 ± 0.108 A synergism 10 66.07 ± 15.15 C 250 32.32 ± 16.30 G 7.06 ± 1.25 E 0.93 ± 0.012 BCD 0.57 ± 0.226 A synergism 10 66.07 ± 15.15 C 500 18.89 ± 0.68 G 7.86 ± 1.45 E 0.92 ± 0.014 CD 0.75 ± 0.425 A synergism 10 66.07 ± 15.15 C 1000 15.90 ± 1.86 G 8.47 ± 0.57 E 0.92 ± 0.006 D 0.85 ± 0.399 A synergism 100 27.059 ± 0.24 D 7.8 96.72 ± 4.26 BC 3.49 ± 0.63 E 0.97 ± 0.001 A 0.79 ± 0.041 A synergism 100 27.059 ± 0.24 D 15.625 85.53 ± 7.73 CDE 3.99 ± 0.87 E 0.96 ± 0.009 AB 0.90 ± 0.075 A synergism 100 27.059 ± 0.24 D 31.25 83.21 ± 9.70 CDE 4.17 ± 0.34 E 0.99 ± 0.003 AB 0.88 ± 0.014 A synergism 100 27.059 ± 0.24 D 62.5 72.24 ± 6.63 EF 4.24 ± 0.07 E 0.96 ± 0.001 AB 0.80 ± 0.347 A synergism 100 27.059 ± 0.24 D 125 54.77 ± 2.03 F 5.27 ± 1.05 E 0.95 ± 0.010 AB 0.78 ± 0.405 A synergism 100 27.059 ± 0.24 D 250 32.32 ± 16.30 G 5.80 ± 1.01 E 0.94 ± 0.010 AB 0.81 ± 0.387 A synergism 100 27.059 ± 0.24 D 500 18.89 ± 0.68 G 5.92 ± 0.93 E 0.94 ± 0.009 AB 0.96 ± 0.552 A synergism 100 27.059 ± 0.24 D 1000 15.90 ± 1.86 G 6.28 ± 1.40 E 0.94 ± 0.014 B 0.99 ± 0.530 A synergism 3.2 CompuSyn drug-drug interaction assays After the acquisition of the MTT test results, they were entered into the CompSyn program to determine the CI. The CI was calculated for the synergistic impact of combining 10 µM SRJ23 with either 125 µM HU (resulting in a combination index of 0.67) or 250 µM HU (resulting in a combination index of 0.57). 3.3 Apoptosis Assay According to Wong et al. [ 19 ], SRJ23 exhibits the capacity to induce apoptosis in several cancer cell types. The aim of our study was to investigate the potential induction of apoptosis in JCs with exposure to SRJ23 in combination with HU, utilizing the Annexin V-FITC/PI double-staining method. Following a treatment duration of 96 hours, it is noteworthy to observe that the administration of 10 µM SRJ23 in combination with 125 or 250 µM HU resulted in a considerable induction of apoptosis in JCs, as depicted in (Figs. 4 and 5). Figure 5A histogram showing percentage of untreated JCs, DMSO, SRJ23, HU and SRJ23 10 µM in combination with HU 125 µM, and SRJ23 10 µM in combination with HU 250 µM combination. Notes: JCs were treated with different concentrations of SRJ23 and/or HU for 96 hours. Cells were stained according to the manufacturer’s instruction provided with the Annexin V-FITC/PI Apoptosis Detection kit using flow cytometry. Table 3 Showing value of JCs obtained from the Annexin V-PI staining assay and their significance. Different superscript of capital letters indicates a statistically significant difference (p < 0.05), with an uppercase letter between different treatments (in columns) and a lowercase letter between different viable stages (in rows). Treatment Viable cells Early apoptosis Late apoptosis Necrosis Control 99.90 ± 0.00 A(a) 0.00 ± 0.00 C(b) 0.00 ± 0.00 D(b) 0.00 ± 00.00 C(b) Co-DMSO 85.60 ± 1.91A B(a) 8.13 ± 1.27 BC(b) 5.43 ± 0.40 CD(b) 0.87 ± 0.29 BC(c) HU 125 µM 69.87 ± 15.85 B(a) 15.70 ± 14.72 BC(b) 10.73 ± 3.45 ABC(b) 3.73 ± 2.42 A(b) HU 250 µM 63.0 ± 26.40 B(a) 20.60 ± 21.00 BC(ab) 13.47 ± 6.98 AB(b) 2.90 ± 1.57 AB(b) SRJ23 10 µM 30.13 ± 2.19 CD(b) 57.00 ± 2.94 A(a) 9.80 ± 0.17 BC(c) 3.10 ± 0.52 AB(d) Co-drug 125 µM 21.67 ± 1.67 D(b) 70.93 ± 0.98 A(a) 6.17 ± 0.75 BCD(c) 1.23 ± 0.06 ABC(d) Co-drug 250 µM 20.53 ± 1.16 D(b) 72.77 ± 2.19 A(a) 5.43 ± 0.98 CD(c) 1.30 ± 0.00 ABC(d) 4 Discussion Several investigations have demonstrated the efficacy of SRJ23 in inhibiting the growth of breast, colon, pancreatic, and prostate cancer models [ 7 , 15 , 20 ]. A study evaluated the combined impact of SRJ23 and gemcitabine as well as the individual drug's effect on pancreatic cancer cells. The MTT and immunoblotting assays were performed, and the study findings verified that the combination of 10 µM SRJ23 and 10 µM gemcitabine had a synergistic impact in inhibiting the proliferation of the pancreatic cell line. While the medicines, when taken alone, were able to stimulate autophagy by increasing the protein expression of the autophagy marker, there was a significant increase in the protein expression of the apoptotic marker compared to individual therapy [ 16 ]. Multiple studies indicate the cytotoxic effects of the HU drug on MPN and sickle cell anemia. Additionally, HU has been investigated in combination therapies for brain malignancies and melanoma [ 10 , 18 , 19 ]. Therefore, an investigation was conducted to examine the cytotoxic effects of the combination of SRJ3 and HU on JCLs. In the in vitro setting, it was observed that SRJ23 had cytotoxic effects at concentrations of 0.1, 1, 10, and 100 µM. HU exhibited cytotoxicity at concentrations of 250, 500, and 1000 µM. Following the creation and experimentation of the combination, the JCL was subjected to dose-response testing in order to determine the parameters of growth inhibition. The inhibitory effect of SRJ23 on JCLs growth was shown to be superior to that of HU at concentrations of 0.1, 1, 10, and 100 µM. In this investigation, HU was employed to impede DNA synthesis by disrupting the proton-coupled electron transfer process necessary for the enzymatic activity of RNR in the production of dNTPs. During the S phase, the activity of HU serves to impede DNA synthesis, halt the advancement of the cell cycle, and trigger checkpoints by inhibiting the replication of chromosomes [ 8 , 17 , 21 ]. The study conducted by wong et al [ 20 ] demonstrates that SRJ23, a derivative of AGP, induces mitochondrial apoptosis through the activation of caspase-8. Additionally, it was shown that SRJ23 causes cell cycle arrest in the G2/M and G1 phases by downregulating the expression of CDK1, CDK4, and cyclin [ 20 ]. In this study, the dose-response relationship of SRJ23 and HU was assessed in terms of their anticancer properties. The doses used for evaluation were 0.1, 1, 10, and 100 µM for SRJ23, and 7.8, 15.6, 31.25, 62.5, 125, 250, 500, and 1000 µM for HU. In order to assess the combined anticancer effects of the compounds, the selected dosages were intended to potentially yield a decrease in the conventional therapeutic doses administered to patients. This reduction in dosage has the potential to enhance cancer treatment outcomes and mitigate associated toxicities. Since there is no existing research on the combined effects of SRJ23 and HU, we conducted a study using the MTT test to generate dose-response curves for SRJ23, HU, and the combined therapy. The calculation of the proportion of cells fraction affected (FA) by the two drugs SRJ23 and HU was performed utilizing Compusyn software, accessible at http://www.combosyn.com . An examination of the synergy of several pharmacokinetic equations derived from the enzyme kinetic model was conducted using the Chou and Talalay (1984) [ 3 ] technique as implemented in the Compusyn computer. The investigation led to the creation of the CI. According to the Chou approach and the use of Compusyn software, synergy is present when the CI is less than 1. The CI was determined by evaluating the synergistic effect of co-administering 10 µM SRJ23 with either 125 µM HU (yielding a CI of 0.67) or 250 µM HU (yielding a CI of 0.57). The confidence interval values can be utilized to evaluate the magnitude of a synergistic or antagonistic effect. If the confidence interval falls within the range of 0.90 to 0.85, it indicates the presence of slight synergies between the two groups. A CI ranging from 0.7 to 0.85 suggests moderate synergies, while a CI between 0.3 and 0.7 indicates the presence of synergies. Strong synergies are observed when the CI falls within the range of 0.1 to 0.3, and very strong synergies are indicated when the CI is less than 0.1. On the other hand, when comparing the confidence interval values, those falling within the range of 0.9 to 1.1 indicate a substantially additive effect, while values over 1.1 indicate antagonism. [ 2 , 12 ]. The FITC-Annexin V test was conducted to detect apoptotic cells in the JCLs following treatment with the drugs SRJ23 and HU, both separately and in combination. The cells were exposed to a combination of synergistic substances at certain doses (10 µM SRJ23 with 125, 250 µM HU) for a duration of 96 hours to assess the presence of apoptotic cells. Upon treatment of JCLs with SRJ23 at a concentration of 10 µM, the results revealed that 30.13% of the cells remained alive, while 57% exhibited early apoptosis, 9.8% displayed late apoptosis, and 3.1% underwent necrosis. HU at a concentration of 125 µM resulted in 69.87% viable cells, 15.7% early apoptosis, 10.7% late apoptosis, and 3.7% necrosis. On the other hand, HU at a concentration of 250 µM resulted in 63% viable cells, 20.6% early apoptosis, 13.47% late apoptosis, and 2.9% necrosis. The initial synergistic combination drug (125 µM HU + 10 µM SRJ23) exhibited 21.67% viable cells, 70.93% early apoptosis, 6.17% late apoptosis, and 1.23% necrosis. Similarly, the second synergistic combination drug (250 µM HU + 10 µM SRJ23) demonstrated 20.53% viable cells, 712.77% early apoptosis, 5.43% late apoptosis, and 1.3% necrosis. When JCLs were exposed to SRJ23 and HU alone or in a synergistic combination (10 µM SRJ23 with 125 or 250 µM HU), the synergistic combination exhibited a significant augmentation in apoptotic cells compared to the individual drugs. 5 Limitations There are several limitations that should be taken seriously before any further recommendation advocating the combination therapy of HU and SRJ23 to be incorporated for in vivo or clinical studies. There were other concentrations of HU and SRJ23 combinations that have yet to be studied that may exert a better growth inhibition or perhaps will be more ideal to be explored. Allocation of fundings adequately to further investigate the synergism of the two combinations is crucial to improvise treatment of T-ALL in the future. To thoroughly understand the detailed impact of this novel synergistic combination and to assess its potential as a targeted cancer therapy, further in vitro testing is recommended. Cell Cycle Analysis: Conduct cell cycle analysis tounderstand how the treatment affects cell growth and development. By examining thedistribution of cells in different stages of the cell cycle (G1, S, and G2/M), researcherscan determine if the drug causes cell cycle arrest or stimulates cell death. Autophagy detection assays need to be done to evaluate how T-ALL responds to the treatment and whether modulating autophagy could be a viabletherapeutic strategy. This can also provide insights into the mechanisms of drug resistance and help identify potential targets for anti-T-ALL therapy. We can utilise Western blotting to analyse protein expression levels, identify therapeutic targets, assess signalling pathway activity, and evaluate treatment responses. Probing for specific proteins involved in T-ALL progression can help determine treatment efficacy and identify potential biomarkers for prognosis or treatment response prediction. This technique also aids in identifying dysregulated signalling pathways, guiding the development of targeted therapies. By implementing transgene techniques to introduce exogenous genes into T-ALL, we can study their impact on cellular behaviour and function. This method allows manipulation and control of gene expression levels, providing insights into changes in cell proliferation, apoptosis, or drug sensitivity, and aiding in the discovery of promising therapeutic targets. A gene knockdown experiments using RNA interference (RNAi) or CRISPR-Cas9 technology to reduce the expression of specific genes aids in understanding gene function in T-ALL. RNAi involves introducing small RNA molecules to degrade the mRNA of the gene of interest, while CRISPR-Cas9 enables precise genomic editing to disrupt specific genes. These recommended assays and techniques will enhance the understanding of the mechanistic effects of SRJ23 and HU on T-ALL and support the development of effective targeted therapies against T-ALL. 6 Conclusion The combination of SRJ23 at a concentration of 10 µM with HU at concentrations of 125 µM and 250 µM had a significant synergistic effect, as shown by the calculated CI values of 0.67 and 0.57, respectively. The combined doses of these compounds resulted in a significant decrease in JC viability compared to the HU and SRJ23 alone. Our study also demonstrates evidence of synergistic inhibitory effect and apoptosis initiation with a certain specified combination of HU and SRJ23 concentrations against T-ALL/ JC, leading to a noteworthy inhibitory impact in vitro., suggesting a promising new approach to targeted chemotherapy. Further studies need to be taken place to investigate other concentrations of the combination of HU and SRJ23 with the best synergistic and growth inhibitory effect before any in vivo and animal studies can be done. Abbreviations AGP Andrographolide CDK Cyclin-dependent kinase CI Combination index DMSO Dimethyl sulfoxide dNTPs Deoxyribonucleotides FA Fraction affected FITC Fluorescein isothiocyanate GDP Guanosine diphosphate GTP Guanosine triphosphate HU Hydroxyurea JCLs jurkat cell lines K-Ras Kirsten rat sarcoma virus MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide MPN Myeloproliferative neoplasm PI Propidium Iodide RNR Ribonucleotide reductase SD Standard deviation SRJ23 3,19-(3-Chloro-4-fluorobenzylidene) andrographolide T-ALL T cell acute lymphoblastic leukemia Declarations Author contribution Laith Marashdeh wrote the manuscript; Johnson Stanslas and Thilakavathy Karuppiah reviewed and validated the methodology and investigations; Bahariah Khalid critically revised the manuscript, contributed to the figures and tables, and approved its structure and content. All authors have reviewed and approved the final version for publication. Funding This study was supported by the Pharmacotherapeutics Unit of the Faculty of Medicine and Health Sciences at Universiti Putra Malaysia. Data Availability All data associated with this study are presented within the manuscript. Additional details can be obtained by contacting the corresponding author. Competing interests The authors declare no competing interests. References Chou, T.-C. (2006). Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacological Reviews, 58(3), 621–681. DOI: 10.1124/pr.58.3.10 Chou, T.-C. (2010). Drug Combination Studies and Their Synergy Quantification Using the Chou-Talalay MethodSynergy Quantification Method. Cancer Research, 70(2), 440–446. DOI: 10.1158/0008-5472.CAN-09-1947 Chou, T.-C., & Talalay, P. (1984). Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Advances in Enzyme Regulation, 22, 27–55. DOI: 10.1016/0065-2571(84)90007-4 Coache, D., Friciu, M., Bernine Marcellin, R., Bonnemain, L., Viau, A., Roullin, V. G., Forest, J.-M., & Leclair, G. (2022). Stability evaluation of compounded hydroxyurea 100 mg/mL oral liquids using a novel analytical method involving chemical derivatization. Plos One, 17(6), e0270206. DOI: 10.1371/journal.pone.0270206 Heeney, M. M., Whorton, M. R., Howard, T. A., Johnson, C. A., & Ware, R. E. (2004). Chemical and functional analysis of hydroxyurea oral solutions. Journal of Pediatric Hematology/Oncology, 26(3), 179–184. DOI: 10.1097/00043426-200403000-00007 Jada, S. R., Matthews, C., Saad, M. S., Hamzah, A. S., Lajis, N. H., Stevens, M. F. G., & Stanslas, J. (2008). Benzylidene derivatives of andrographolide inhibit growth of breast and colon cancer cells in vitro by inducing G1 arrest and apoptosis. British Journal of Pharmacology, 155(5), 641–654. DOI: 10.1038/bjp.2008.368 Jada, S. R., Subur, G. S., Matthews, C., Hamzah, A. S., Lajis, N. H., Saad, M. S., Stevens, M. F. G., & Stanslas, J. (2007). Semisynthesis and in vitro anticancer activities of andrographolide analogues. Phytochemistry, 68(6), 904–912. DOI: 10.1016/j.phytochem.2006.11.031 Koç, A., Wheeler, L. J., Mathews, C. K., & Merrill, G. F. (2004). Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools. Journal of Biological Chemistry, 279(1), 223–230. DOI: 10.1074/jbc.M303952200 Lato, M. W., Przysucha, A., Grosman, S., Zawitkowska, J., & Lejman, M. (2021). The new therapeutic strategies in pediatric t-cell acute lymphoblastic leukemia. International Journal of Molecular Sciences, 22(9), 4502. DOI: 10.3390/ijms22094502 Madaan, K., Kaushik, D., & Verma, T. (2012). Hydroxyurea: a key player in cancer chemotherapy. Expert Review of Anticancer Therapy, 12(1), 19–29. DOI: 10.1586/era.11.175 Matthews, H., Deakin, J., Rajab, M., Idris-Usman, M., & Nirmalan, N. J. (2017). Investigating antimalarial drug interactions of emetine dihydrochloride hydrate using CalcuSyn-based interactivity calculations. PloS one, 12(3), e0173303. Nautiyal, J., Kanwar, S. S., Yu, Y., & Majumdar, A. P. N. (2011). Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells. Journal of Molecular Signaling, 6(1), 1–11. DOI: 10.1186/1750-2187-6-7 Offenbacher, A. R., & Barry, B. A. (2020). A proton wire mediates proton coupled electron transfer from hydroxyurea and other hydroxamic acids to tyrosyl radical in class Ia ribonucleotide reductase. The Journal of Physical Chemistry B, 124(2), 345–354. DOI: 10.1021/acs.jpcb.9b08587 Patel, A. A., Thomas, J., Rojek, A. E., & Stock, W. (2020). Biology and treatment paradigms in T cell acute lymphoblastic leukemia in older adolescents and adults. Current Treatment Options in Oncology, 21(7), 1–21. DOI: 10.1007/s11864-020-00757-5 Quah, S. Y., Wong, C. C., Wong, H. C., Ho, K. L., Manan, N. A., Deb, P. K., Sagineedu, S. R., & Stanslas, J. (2021). Microarray-based identification of differentially expressed genes associated with andrographolide derivatives-induced resistance in colon and prostate cancer cell lines. Toxicology and Applied Pharmacology, 425, 115605. DOI: 10.1016/j.taap.2021.115605 Selvarajoo, N., Sagineedu, S. R., Lim, W., Chee, J., Ho, K. L., & Stanslas, J. (2022). Interplay Between Autophagy And Apoptosis In A Pancreatic Cancer Cell Line Treated With SRJ23, Gemcitabine Or Combination Of Both Agents. Malaysian Journal of Medicine & Health Sciences, 18. DOI: 10.1007/s11523-013-0278-5 Singh, A., & Xu, Y.-J. (2016). The cell killing mechanisms of hydroxyurea. Genes, 7(11), 99. DOI: 10.3390/genes7110099 Spivak, J. L., & Hasselbalch, H. (2011). Hydroxycarbamide: a user’s guide for chronic myeloproliferative disorders. Expert Review of Anticancer Therapy, 11(3), 403–414. DOI: 10.1586/era.11.10 Stearns, B., Losee, K. A., & Bernstein, J. (1963). Hydroxyurea. A new type of potential antitumor Agent1. Journal of Medicinal Chemistry, 6(2), 201. DOI: 10.1021/jm00338a026 Wong, H. C., Wong, C. C., Sagineedu, S. R., Loke, S. C., Lajis, N. H., & Stanslas, J. (2014). SRJ23, a new semisynthetic andrographolide derivative: in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis in prostate cancer cells. Cell Biology and Toxicology, 30(5), 269–288. DOI: 10.1007/s10565-014-9282-5 Xu, Y., Singh, A., & Alter, G. M. (2016). Hydroxyurea induces cytokinesis arrest in cells expressing a mutated sterol-14α-demethylase in the ergosterol biosynthesis pathway. Genetics, 204(3), 959–973. DOI: 10.1534/genetics.116.191536 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 26 Dec, 2024 Reviews received at journal 03 Dec, 2024 Reviews received at journal 02 Dec, 2024 Reviewers agreed at journal 28 Nov, 2024 Reviewers agreed at journal 28 Nov, 2024 Reviewers invited by journal 26 Nov, 2024 Editor assigned by journal 22 Nov, 2024 Submission checks completed at journal 21 Nov, 2024 First submitted to journal 08 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5419120","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":385119667,"identity":"a1f83f95-8a12-43a0-a364-0b6bf0ff168a","order_by":0,"name":"LAITH ALMARASHDEH","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"LAITH","middleName":"","lastName":"ALMARASHDEH","suffix":""},{"id":385119668,"identity":"bdcc6914-58e1-4361-9552-f5e9501ae8b6","order_by":1,"name":"Johnson Stanslas","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Johnson","middleName":"","lastName":"Stanslas","suffix":""},{"id":385119669,"identity":"9d5ca0e7-e7cf-4d72-928f-21569f522720","order_by":2,"name":"Thilakavathy Karuppiah","email":"","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Thilakavathy","middleName":"","lastName":"Karuppiah","suffix":""},{"id":385119670,"identity":"dd230a64-0be1-409f-9c35-6c24b6bb90a8","order_by":3,"name":"Bahariah Khalid","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYBACAwYeMJnAD+Ezk6BFsoE0LQwMCQYHiNVizn724OeKArs84xu5RzcwVFgnNoidMcCrxbInL1nyjEFysdmNvLQbDGfSExukc/BrMTiQYyDZYMCcuO1GjtkNxrbDRGg5/8b4Z4NBfeLmGSAt/4jRAjQcaMvhxA0SIC0NRGixnPHGzLLB4HjijDPv0m4kHEs3bpNOK8CrxZw/x/hmw5/qxP723GM3PtRYy/ZLJ2/AqwUJACMoAUixMXDgdxiqFghgf0CsllEwCkbBKBgZAACRVUoAb6F7ygAAAABJRU5ErkJggg==","orcid":"","institution":"Universiti Putra Malaysia","correspondingAuthor":true,"prefix":"","firstName":"Bahariah","middleName":"","lastName":"Khalid","suffix":""}],"badges":[],"createdAt":"2024-11-09 00:23:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5419120/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5419120/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71890462,"identity":"660f918a-aac5-4c61-94c4-622804bace54","added_by":"auto","created_at":"2024-12-19 12:59:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9895,"visible":true,"origin":"","legend":"\u003cp\u003eThe crystals of AG.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5419120/v1/9a7d1e50eb28b728a043a58c.jpg"},{"id":71890464,"identity":"9d6f70dd-32f4-4dac-be9e-54c54a36422b","added_by":"auto","created_at":"2024-12-19 12:59:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":207651,"visible":true,"origin":"","legend":"\u003cp\u003eA cartoon illustration of how the workflow of culturing the JCs and running of the MTT assay protocol.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5419120/v1/c15e0464fef279983c7af46c.png"},{"id":71890904,"identity":"90e0775b-baa1-44f7-a021-ef19aacaca70","added_by":"auto","created_at":"2024-12-19 13:07:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":323673,"visible":true,"origin":"","legend":"\u003cp\u003eThe growth inhibitory effects of SRJ23 and HU on JC were evaluated using the MTT cell viability assay over a 96-hour treatment period. JCs, cultured in 96-well plates at a density of 1.0×10⁴ cells per well, were exposed to varying concentrations of the compounds. The dose-response curves illustrate the cytotoxic impact of SRJ23 and HU, highlighting a reduction in cell viability across treatments, as measured through MTT assays. Mean ± SD, n=3 independent experiments.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5419120/v1/71f957b0785a7753fc6040e7.jpg"},{"id":71890466,"identity":"f2a24cd8-cbb1-4611-adc4-ebe0c352b8f7","added_by":"auto","created_at":"2024-12-19 12:59:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":345783,"visible":true,"origin":"","legend":"\u003cp\u003eA-G Contour plots showing the percentage distribution of viable, early, late apoptotic and necrosis of JCs in the untreated cells, DMSO, HU, SRJ23, SRJ23 10 µM in combination with HU 125 µM, and SRJ23 10 µM in combination with HU 250 µM after 96 hours of exposure.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5419120/v1/c7153fb0c91db11d1dc46910.png"},{"id":71892033,"identity":"61d4de62-ddf3-4001-b083-a699854ed28f","added_by":"auto","created_at":"2024-12-19 13:15:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":194456,"visible":true,"origin":"","legend":"\u003cp\u003eA histogram showing percentage of untreated JCs, DMSO, SRJ23, HU and SRJ23 10 µM in combination with HU 125 µM, and SRJ23 10 µM in combination with HU 250 µM combination. Notes: JCs were treated with different concentrations of SRJ23 and/or HU for 96 hours. Cells were stained according to the manufacturer’s instruction provided with the Annexin V-FITC/PI Apoptosis Detection kit using flow cytometry.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5419120/v1/05d3a539283ba10474999392.jpg"},{"id":71892578,"identity":"eda614f5-1f3e-4e76-9dfc-7da4656a4bd7","added_by":"auto","created_at":"2024-12-19 13:23:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1862496,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5419120/v1/8352590d-d2f9-4bac-9a59-13d784a1b3f8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic Antileukemic Effects of Hydroxyurea and SRJ23 in T-cell Acute Lymphoblastic Leukemia","fulltext":[{"header":"1 Background","content":"\u003cp\u003eT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia characterized by the abnormal growth and proliferation of early T-cell progenitors due to presence of K-Ras driver mutation. Overall incidence of T-ALL was reported to be 0.13 cases per 100000 population. The incidence decreased significantly after the age of 40 (IRR ranging from 0.51 to 0.55, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) however with recent developments in diagnostic techniques, intensive chemotherapy and transplant protocols have not derived to the best ways yet for better overall survival, relapse and cure rates for T-ALL. Despite having a markedly enhanced understanding of molecular biology, there were only achievements of 85% five-year survival rate among pediatric populations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and less than 50% for adult population [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis disease typically manifests as patients complaining of pyrexia of unknown origin with or without night sweats, significant loss of weight more than 10% from the baseline weight 6 months prior and loss of appetite unintentionally or B symptoms. Clinical examination on these patients may reveal variable degrees of anemia, hyper-viscosity with or without lymphadenopathy and hepatosplenomegaly. At times, the lymphocytes dysregulation of their function can be apparent with anemia and jaundice in those patients that succumbed into autoimmune hemolytic anemia, bleeding episodes as in spontaneous bruises over the skin, in the muco-cutaneous tissue or other parts of the body either obvious or occult like immune thrombocytopenia presentation. Generalised lymphadenopathy may be apparent on radiology imaging like CAT scan and Pet-scan. T-ALL patients present largely with hyperleukocytosis with predominantly lymphocytes in their full blood picture with access lymphoblast. The flowcytometry or immunophenotyping can further identify the clusters of lymphoblasts accurately but it can never supersede a thorough bone marrow aspirate and trephine examination that of which inclusive of flowcytometric, cytogenetic and molecular testing of the abnormal lymphoblasts. The mainstay of urgent cytoreductive therapy in cytosis in acute and chronic haematological malignancies is by giving affordable and easily available oral genotoxic compound called hydroxycarbamide or generally called hydroxyurea (HU). HU has been traditionally used for decades to suppress any of the cell lineage excessiveness from the marrow of monoclonal in origin for example in acute myeloid leukemia (AML), ALL and chronic myeloproliferative neoplasm (MPN). It is used to control the blood counts therefore reducing the upregulation of malignant gene in polycythemia vera (PV), essential thrombocythemia (ET) and chronic myeloid leukemia (CML). The sequelaes due to uncontrolled blood counts is sometimes fatal and by doing so, it helps to prevent the risk of thrombosis and bleeding that can cause detrimental outcomes. HU is known to inhibit DNA replication [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] (Ko\u0026ccedil; et al., 2004), and exhibits a short-term toxicity profile, which is generally well-tolerated among the majority of patients [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The compound exerts an impact on the enzymatic activity of RNR by interfering with the proton-coupled electron transfer process responsible for catalyzing the synthesis of new dNTPs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA recent remarkable of scientific evidence to-date, had shown the K-Ras oncogenic mutation as the responsible genetic abnormality in the development of T-ALL. Coincidentally, SRJ23 which is an andrographolide (AG) semisynthetic compound had been reported to have a significant inhibitory effect to K-Ras oncogene in solid cancers, hence inducing apoptosis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As T-ALL being one of the haematological cancers with the worst outcome in survival despite with the availability of toxic treatment and haematopoeitic stem cell transplants, T-ALL remains to have the worst outcome possible even with the standard treatment. We embarked a study to evaluate and assess the effect of HU and SRJ23 in JCLs for their growth inhibition assay, presence of synergistic drug interactions and apoptosis potency both individually and in combination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2 Methods","content":"\u003ch2\u003e\u003cstrong\u003e2.1 Growth inhibition assay\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe JCLs were obtained from the immunology laboratory of the Faculty of Medicine and Health Sciences at Universiti Putra Malaysia. The cells were grown in RPMI 1640 Medium (Sigma-Alderich, Malaysia) with the addition of 10% warm fetal bovine serum, 100 U/L penicillin, and 100 mg/L streptomycin (Nacalai Tesque). The culture was maintained at a temperature of 37 \u0026deg;C in an environment with 5% CO2. The cells were then distributed into 96-well, flat-bottom microtiter plates (TPP, Techno Plastic Products, Switzerland) at a density of 5.0\u0026times;10^3 cells per well (Fig 2). \u0026nbsp; After a 24-hour incubation period, the cells were treated with various concentrations of SRJ23 (0.1, 1, 10, and 100 \u0026micro;M) and HU (7.8, 15.6, 31.25, 62.5, 125, 250, 500, and 1000 \u0026mu;M) individually and in combination. Subsequently, the cells were incubated for 96 hours at a temperature of 37\u0026deg;C in an environment containing 5% CO2. Following a 96-hour incubation period, a volume of 50 \u0026micro;L of MTT solution with a concentration of 2 mg/mL was introduced to each well. Subsequently, the plates were reintroduced to the incubator for an extra four hours to foster the metabolic processes associated with the MTT. Then the 96-well plate was centrifuged at 300x g for 10 minutes at 4\u0026deg;C. Following centrifugation, the medium and excess MTT were aspirated and the crystals of formazon was dissolved in a volume of 100 \u0026micro;L of dimethyl sulfoxide (DMSO). The determination of the fraction of JCLs living cells was conducted by quantifying the absorbance of purple formazon at a wavelength of 550 nm using a Versamax microplate reader. The statistical analysis was conducted with the assistance of SOFTmax\u003csup\u003e\u0026reg;\u003c/sup\u003e Pro software.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e2.2 Combination index (CI) for drug-drug interactions analyses\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe identification of drug interactions was conducted through the utilization of the isobologram and CI technique, which was developed based on the median-effect concept proposed by Chou and Talalay [1,2]. This analysis was performed utilizing the CompuSyn software. The mathematical model employed for the analysis of drug-drug interactions utilizes the formula based on the principle of median effect. The aforementioned equation is expressed as fa/fu = [D/Dm]m in which fa represents the proportion of cells that experience an effect, fu denotes the proportion of cells that do not experience an effect and is equivalent to 1-fa, D signifies the concentration of the drug, Dm represents the dosage of the drug necessary for inhibiting 50% of the cells, and m denotes the slope of the median effect curve. The isobologram is a visual depiction of the pharmacological interaction, wherein a line is drawn connecting the Fa points and the fixed ratio combinations of HU and SRJ23, which are displayed on the x and y axes.\u003c/p\u003e\n\u003cp\u003eThe CI, a quantitative measure of drug combination effects obtained from the CompuSyn software, incorporates different concentrations of both drugs to correspond with the percentages of growth inhibition throughout the analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1\u0026nbsp;shows the Chou-Talalay classification of synergism and antagonism, with CI values, preferred symbols, and descriptions. The CI \u0026lt; 1, = 1, and \u0026gt; 1 imply synergism, additive, and antagonism, respectively [1].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e Combination index (CI) values, along with corresponding symbols and descriptions, classify synergism or antagonism based on the Chou-Talalay method [11].\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRange of CI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSymbol\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e\u0026lt;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e+++++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003eVery strong synergism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e0.1-0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e++++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003eStrong synergism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e0.3-0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003esynergism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e0.7-0.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003eModerate synergism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e0.85-0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003eSlight synergism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e0.90-1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e\u0026plusmn;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003eNearly additive\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e1.10-1.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003eSlight antagonism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e1.20-1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003eModerate antagonism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e1.45-3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e---\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003eAntagonism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e3.3-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003eStrong antagonism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 191px;\"\u003e\n \u003cp\u003e\u0026gt;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e-----\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 193px;\"\u003e\n \u003cp\u003eVery strong antagonism\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003e\u003cstrong\u003e2.3 Apoptosis assay\u0026nbsp;\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe JCLs were cultured by seeding them in a 6-well plate with a flat bottom. The cells were allowed to proliferate exponentially at a density of roughly 1x10^6 cells per 1800 \u0026micro;l/well. The culture was then incubated for 24 hours at a temperature of 37\u0026deg;C (5% CO2 and 95% air). The concentration of synergism was determined by the MTT experiment, and the compounds were diluted in a serial manner to achieve the necessary concentration. These diluted compounds were then administered to the relevant wells in triplicate. In contrast, a concentration of 0.01% of dimethyl sulfoxide (DMSO) was utilized to treat the control wells, which corresponds to the concentration of the vehicle employed in the wells treated with the combination compound. The FITC Annexin V technique was conducted after a duration of 96 hours, following the guidelines specified by the manufacturer in the instructions accompanying the Annexin V-FITC/PI Apoptosis Detection Kit from BD Bioscience. The cells were collected, enumerated, and rinsed twice with cold phosphate-buffered saline (PBS) prior to being reconstituted at a concentration of 1x10^6 cells/ml in 1X Binding Buffer. Subsequently, a total of 100 \u0026micro;l of the solution was used to suspend 1 x 10^5 cells, which were then transferred into a 5 ml culture tube. Following the addition of 5 \u0026micro;l of FITC Annexin V and 5 \u0026micro;l of PI to each tube, a mild vortex was applied, and the mixtures were subsequently incubated at room temperature (25\u0026deg;C) in the absence of light for a duration of 15 minutes. Following the introduction of 400 \u0026micro;l of the 1X Binding Buffer into each tube, the samples were subjected to analysis using flowcytometry BD FACS LSRFortressa\u003csup\u003eTM\u003c/sup\u003e Cell Analyser and FACSDiva\u003csup\u003eTM\u003c/sup\u003e within 1 hour.\u003c/p\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 JCLs growth inhibition assay with HU and (with or without) SRJ23\u003c/h2\u003e \u003cp\u003eTo evaluate the therapeutic potential efficacy of SRJ23 and HU, the JCL were subjected to a 24-hour cultivation period. Subsequently, these cells were exposed to varying concentrations of SRJ23 (0.1, 1, 10, and 100 \u0026micro;M) and HU (7.8, 15.625, 31.25, 62.5, 125, 250, 500, and 1000 \u0026micro;M) either individually or in combination for a period of 96 hours. Following this treatment, the viability of the cells was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The findings indicated that the dose-response curves exhibited a decrease in cell viability within JCL with a synergistic effect with a combination of concentrations of 10 \u0026micro;M SRJ23 with 125 \u0026micro;M HU or 250 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eShowing the statistical analysis of three replicates reveals the interaction patterns\u0026mdash;synergistic, antagonistic, or additive\u0026mdash;between SRJ23 (0.1, 1, 10, and 100 \u0026micro;M) and HU (ranging from 7.8 to 1000 \u0026micro;M) following 96 hours of exposure. Statistical significance among treatment groups (SRJ23, HU, and their combined treatment) is indicated by capital letters, with different letters representing significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Means that share the same letter are not significantly different from each other. The letter 'A' represents the group with the highest cell viability, followed by 'B,' 'C,' and so forth, each letter indicating a sequential decrease in viability. This lettering scheme allows for clear identification of which treatments resulted in the highest and lowest levels of cell viability among the tested groups.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSRJ23 dose\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSRJ23 Mean Viability %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHU dose\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHU Mean Viability %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCombination Mean Viability %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCombination Fa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eInteraction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98\u003csup\u003e\u003cb\u003eBCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.72\u0026thinsp;\u0026plusmn;\u0026thinsp;4.26\u003csup\u003e\u003cb\u003eBC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e122.56\u0026thinsp;\u0026plusmn;\u0026thinsp;12.90\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.129\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-------------\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eantagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98\u003csup\u003e\u003cb\u003eBCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.53\u0026thinsp;\u0026plusmn;\u0026thinsp;7.73 \u003csup\u003e\u003cb\u003eCDE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e112.90\u0026thinsp;\u0026plusmn;\u0026thinsp;7.30\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.070\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-------------\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eantagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98\u003csup\u003e\u003cb\u003eBCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83.21\u0026thinsp;\u0026plusmn;\u0026thinsp;9.70 \u003csup\u003e\u003cb\u003eCDE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e88.42\u0026thinsp;\u0026plusmn;\u0026thinsp;3.49\u003csup\u003e\u003cb\u003eCDE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.035\u003csup\u003e\u003cb\u003eB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.925\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eantagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98\u003csup\u003e\u003cb\u003eBCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72.24\u0026thinsp;\u0026plusmn;\u0026thinsp;6.63\u003csup\u003e\u003cb\u003eEF\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e73.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003e\u003cb\u003eDEF\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003csup\u003e\u003cb\u003eB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eantagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98\u003csup\u003e\u003cb\u003eBCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003csup\u003e\u003cb\u003eF\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.227\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eantagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98\u003csup\u003e\u003cb\u003eBCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.32\u0026thinsp;\u0026plusmn;\u0026thinsp;16.30\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.94\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.030\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1. 20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.167\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eantagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98\u003csup\u003e\u003cb\u003eBCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.197\u003csup\u003e\u003cb\u003eB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.76\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98\u003csup\u003e\u003cb\u003eBCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.165\u003csup\u003e\u003cb\u003eB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44\u003csup\u003e\u003cb\u003eABC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.72\u0026thinsp;\u0026plusmn;\u0026thinsp;4.26\u003csup\u003e\u003cb\u003eBC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e111.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003e\u003cb\u003eD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-------------\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eantagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44\u003csup\u003e\u003cb\u003eABC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.53\u0026thinsp;\u0026plusmn;\u0026thinsp;7.73 \u003csup\u003e\u003cb\u003eCDE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95.54\u0026thinsp;\u0026plusmn;\u0026thinsp;7.92\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.080\u003csup\u003e\u003cb\u003eCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-------------\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eantagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44\u003csup\u003e\u003cb\u003eABC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83.21\u0026thinsp;\u0026plusmn;\u0026thinsp;9.70 \u003csup\u003e\u003cb\u003eCDE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e71.95\u0026thinsp;\u0026plusmn;\u0026thinsp;14.08\u003csup\u003e\u003cb\u003eBCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.141\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.512\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eantagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44\u003csup\u003e\u003cb\u003eABC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72.24\u0026thinsp;\u0026plusmn;\u0026thinsp;6.63\u003csup\u003e\u003cb\u003eEF\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44.34\u0026thinsp;\u0026plusmn;\u0026thinsp;17.29\u003csup\u003e\u003cb\u003eEFG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.173\u003csup\u003e\u003cb\u003eB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eantagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44\u003csup\u003e\u003cb\u003eABC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003csup\u003e\u003cb\u003eF\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.62\u0026thinsp;\u0026plusmn;\u0026thinsp;4.31\u003csup\u003e\u003cb\u003eGH\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.043\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eantagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44\u003csup\u003e\u003cb\u003eABC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.32\u0026thinsp;\u0026plusmn;\u0026thinsp;16.30\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003csup\u003e\u003cb\u003eH\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.062\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eantagonism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44\u003csup\u003e\u003cb\u003eABC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.37\u0026thinsp;\u0026plusmn;\u0026thinsp;4.08\u003csup\u003e\u003cb\u003eH\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.041\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e88.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.44\u003csup\u003e\u003cb\u003eABC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003e\u003cb\u003eH\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.07\u0026thinsp;\u0026plusmn;\u0026thinsp;15.15\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.72\u0026thinsp;\u0026plusmn;\u0026thinsp;4.26\u003csup\u003e\u003cb\u003eBC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003csup\u003e\u003cb\u003eABC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.202\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.07\u0026thinsp;\u0026plusmn;\u0026thinsp;15.15\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.53\u0026thinsp;\u0026plusmn;\u0026thinsp;7.73 \u003csup\u003e\u003cb\u003eCDE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003csup\u003e\u003cb\u003eABCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.245\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.07\u0026thinsp;\u0026plusmn;\u0026thinsp;15.15\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83.21\u0026thinsp;\u0026plusmn;\u0026thinsp;9.70 \u003csup\u003e\u003cb\u003eCDE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.257\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.07\u0026thinsp;\u0026plusmn;\u0026thinsp;15.15\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72.24\u0026thinsp;\u0026plusmn;\u0026thinsp;6.63\u003csup\u003e\u003cb\u003eEF\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.226\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.07\u0026thinsp;\u0026plusmn;\u0026thinsp;15.15\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003csup\u003e\u003cb\u003eF\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003csup\u003e\u003cb\u003eBCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.108\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.07\u0026thinsp;\u0026plusmn;\u0026thinsp;15.15\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.32\u0026thinsp;\u0026plusmn;\u0026thinsp;16.30\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003csup\u003e\u003cb\u003eBCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.226\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.07\u0026thinsp;\u0026plusmn;\u0026thinsp;15.15\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003csup\u003e\u003cb\u003eCD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.425\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.07\u0026thinsp;\u0026plusmn;\u0026thinsp;15.15\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003csup\u003e\u003cb\u003eD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.399\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.059\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003e\u003cb\u003eD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.72\u0026thinsp;\u0026plusmn;\u0026thinsp;4.26\u003csup\u003e\u003cb\u003eBC\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.041\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.059\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003e\u003cb\u003eD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.53\u0026thinsp;\u0026plusmn;\u0026thinsp;7.73 \u003csup\u003e\u003cb\u003eCDE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.075\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.059\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003e\u003cb\u003eD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83.21\u0026thinsp;\u0026plusmn;\u0026thinsp;9.70 \u003csup\u003e\u003cb\u003eCDE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.059\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003e\u003cb\u003eD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72.24\u0026thinsp;\u0026plusmn;\u0026thinsp;6.63\u003csup\u003e\u003cb\u003eEF\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.347\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.059\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003e\u003cb\u003eD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003csup\u003e\u003cb\u003eF\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.405\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.059\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003e\u003cb\u003eD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.32\u0026thinsp;\u0026plusmn;\u0026thinsp;16.30\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.387\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.059\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003e\u003cb\u003eD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003csup\u003e\u003cb\u003eAB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.552\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.059\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003e\u003cb\u003eD\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86\u003csup\u003e\u003cb\u003eG\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003csup\u003e\u003cb\u003eE\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014\u003csup\u003e\u003cb\u003eB\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.530\u003csup\u003e\u003cb\u003eA\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003esynergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 CompuSyn drug-drug interaction assays\u003c/h2\u003e \u003cp\u003eAfter the acquisition of the MTT test results, they were entered into the CompSyn program to determine the CI. The CI was calculated for the synergistic impact of combining 10 \u0026micro;M SRJ23 with either 125 \u0026micro;M HU (resulting in a combination index of 0.67) or 250 \u0026micro;M HU (resulting in a combination index of 0.57).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Apoptosis Assay\u003c/h2\u003e \u003cp\u003eAccording to Wong et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], SRJ23 exhibits the capacity to induce apoptosis in several cancer cell types. The aim of our study was to investigate the potential induction of apoptosis in JCs with exposure to SRJ23 in combination with HU, utilizing the Annexin V-FITC/PI double-staining method. Following a treatment duration of 96 hours, it is noteworthy to observe that the administration of 10 \u0026micro;M SRJ23 in combination with 125 or 250 \u0026micro;M HU resulted in a considerable induction of apoptosis in JCs, as depicted in (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and 5).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5A\u003c/b\u003e histogram showing percentage of untreated JCs, DMSO, SRJ23, HU and SRJ23 10 \u0026micro;M in combination with HU 125 \u0026micro;M, and SRJ23 10 \u0026micro;M in combination with HU 250 \u0026micro;M combination. Notes: JCs were treated with different concentrations of SRJ23 and/or HU for 96 hours. Cells were stained according to the manufacturer\u0026rsquo;s instruction provided with the Annexin V-FITC/PI Apoptosis Detection kit using flow cytometry.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eShowing value of JCs obtained from the Annexin V-PI staining assay and their significance. Different superscript of capital letters indicates a statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with an uppercase letter between different treatments (in columns) and a lowercase letter between different viable stages (in rows).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eViable cells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly apoptosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLate apoptosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNecrosis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e99.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eA(a)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eC(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eD(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;00.00\u003csup\u003eC(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCo-DMSO\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91A\u003csup\u003eB(a)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003csup\u003eBC(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003eCD(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003eBC(c)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHU 125 \u0026micro;M\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e69.87\u0026thinsp;\u0026plusmn;\u0026thinsp;15.85\u003csup\u003eB(a)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.70\u0026thinsp;\u0026plusmn;\u0026thinsp;14.72\u003csup\u003eBC(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.73\u0026thinsp;\u0026plusmn;\u0026thinsp;3.45\u003csup\u003eABC(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.73\u0026thinsp;\u0026plusmn;\u0026thinsp;2.42\u003csup\u003eA(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHU 250 \u0026micro;M\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.0\u0026thinsp;\u0026plusmn;\u0026thinsp;26.40\u003csup\u003eB(a)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.60\u0026thinsp;\u0026plusmn;\u0026thinsp;21.00\u003csup\u003eBC(ab)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.47\u0026thinsp;\u0026plusmn;\u0026thinsp;6.98\u003csup\u003eAB(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57\u003csup\u003eAB(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSRJ23 10 \u0026micro;M\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.13\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19\u003csup\u003eCD(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.94\u003csup\u003eA(a)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003eBC(c)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003csup\u003eAB(d)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCo-drug 125 \u0026micro;M\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003csup\u003eD(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003csup\u003eA(a)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003csup\u003eBCD(c)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eABC(d)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCo-drug 250 \u0026micro;M\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003csup\u003eD(b)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72.77\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19\u003csup\u003eA(a)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003csup\u003eCD(c)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eABC(d)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eSeveral investigations have demonstrated the efficacy of SRJ23 in inhibiting the growth of breast, colon, pancreatic, and prostate cancer models [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. A study evaluated the combined impact of SRJ23 and gemcitabine as well as the individual drug's effect on pancreatic cancer cells. The MTT and immunoblotting assays were performed, and the study findings verified that the combination of 10 \u0026micro;M SRJ23 and 10 \u0026micro;M gemcitabine had a synergistic impact in inhibiting the proliferation of the pancreatic cell line. While the medicines, when taken alone, were able to stimulate autophagy by increasing the protein expression of the autophagy marker, there was a significant increase in the protein expression of the apoptotic marker compared to individual therapy [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Multiple studies indicate the cytotoxic effects of the HU drug on MPN and sickle cell anemia. Additionally, HU has been investigated in combination therapies for brain malignancies and melanoma [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, an investigation was conducted to examine the cytotoxic effects of the combination of SRJ3 and HU on JCLs. In the in vitro setting, it was observed that SRJ23 had cytotoxic effects at concentrations of 0.1, 1, 10, and 100 \u0026micro;M. HU exhibited cytotoxicity at concentrations of 250, 500, and 1000 \u0026micro;M. Following the creation and experimentation of the combination, the JCL was subjected to dose-response testing in order to determine the parameters of growth inhibition. The inhibitory effect of SRJ23 on JCLs growth was shown to be superior to that of HU at concentrations of 0.1, 1, 10, and 100 \u0026micro;M. In this investigation, HU was employed to impede DNA synthesis by disrupting the proton-coupled electron transfer process necessary for the enzymatic activity of RNR in the production of dNTPs. During the S phase, the activity of HU serves to impede DNA synthesis, halt the advancement of the cell cycle, and trigger checkpoints by inhibiting the replication of chromosomes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The study conducted by wong et al [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] demonstrates that SRJ23, a derivative of AGP, induces mitochondrial apoptosis through the activation of caspase-8. Additionally, it was shown that SRJ23 causes cell cycle arrest in the G2/M and G1 phases by downregulating the expression of CDK1, CDK4, and cyclin [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, the dose-response relationship of SRJ23 and HU was assessed in terms of their anticancer properties. The doses used for evaluation were 0.1, 1, 10, and 100 \u0026micro;M for SRJ23, and 7.8, 15.6, 31.25, 62.5, 125, 250, 500, and 1000 \u0026micro;M for HU. In order to assess the combined anticancer effects of the compounds, the selected dosages were intended to potentially yield a decrease in the conventional therapeutic doses administered to patients. This reduction in dosage has the potential to enhance cancer treatment outcomes and mitigate associated toxicities. Since there is no existing research on the combined effects of SRJ23 and HU, we conducted a study using the MTT test to generate dose-response curves for SRJ23, HU, and the combined therapy. The calculation of the proportion of cells fraction affected (FA) by the two drugs SRJ23 and HU was performed utilizing Compusyn software, accessible at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.combosyn.com\u003c/span\u003e\u003cspan address=\"http://www.combosyn.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. An examination of the synergy of several pharmacokinetic equations derived from the enzyme kinetic model was conducted using the Chou and Talalay (1984) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] technique as implemented in the Compusyn computer. The investigation led to the creation of the CI. According to the Chou approach and the use of Compusyn software, synergy is present when the CI is less than 1. The CI was determined by evaluating the synergistic effect of co-administering 10 \u0026micro;M SRJ23 with either 125 \u0026micro;M HU (yielding a CI of 0.67) or 250 \u0026micro;M HU (yielding a CI of 0.57). The confidence interval values can be utilized to evaluate the magnitude of a synergistic or antagonistic effect. If the confidence interval falls within the range of 0.90 to 0.85, it indicates the presence of slight synergies between the two groups. A CI ranging from 0.7 to 0.85 suggests moderate synergies, while a CI between 0.3 and 0.7 indicates the presence of synergies. Strong synergies are observed when the CI falls within the range of 0.1 to 0.3, and very strong synergies are indicated when the CI is less than 0.1. On the other hand, when comparing the confidence interval values, those falling within the range of 0.9 to 1.1 indicate a substantially additive effect, while values over 1.1 indicate antagonism. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The FITC-Annexin V test was conducted to detect apoptotic cells in the JCLs following treatment with the drugs SRJ23 and HU, both separately and in combination. The cells were exposed to a combination of synergistic substances at certain doses (10 \u0026micro;M SRJ23 with 125, 250 \u0026micro;M HU) for a duration of 96 hours to assess the presence of apoptotic cells. Upon treatment of JCLs with SRJ23 at a concentration of 10 \u0026micro;M, the results revealed that 30.13% of the cells remained alive, while 57% exhibited early apoptosis, 9.8% displayed late apoptosis, and 3.1% underwent necrosis. HU at a concentration of 125 \u0026micro;M resulted in 69.87% viable cells, 15.7% early apoptosis, 10.7% late apoptosis, and 3.7% necrosis. On the other hand, HU at a concentration of 250 \u0026micro;M resulted in 63% viable cells, 20.6% early apoptosis, 13.47% late apoptosis, and 2.9% necrosis. The initial synergistic combination drug (125 \u0026micro;M HU\u0026thinsp;+\u0026thinsp;10 \u0026micro;M SRJ23) exhibited 21.67% viable cells, 70.93% early apoptosis, 6.17% late apoptosis, and 1.23% necrosis. Similarly, the second synergistic combination drug (250 \u0026micro;M HU\u0026thinsp;+\u0026thinsp;10 \u0026micro;M SRJ23) demonstrated 20.53% viable cells, 712.77% early apoptosis, 5.43% late apoptosis, and 1.3% necrosis. When JCLs were exposed to SRJ23 and HU alone or in a synergistic combination (10 \u0026micro;M SRJ23 with 125 or 250 \u0026micro;M HU), the synergistic combination exhibited a significant augmentation in apoptotic cells compared to the individual drugs.\u003c/p\u003e"},{"header":"5 Limitations","content":"\u003cp\u003eThere are several limitations that should be taken seriously before any further recommendation advocating the combination therapy of HU and SRJ23 to be incorporated for in vivo or clinical studies. There were other concentrations of HU and SRJ23 combinations that have yet to be studied that may exert a better growth inhibition or perhaps will be more ideal to be explored. Allocation of fundings adequately to further investigate the synergism of the two combinations is crucial to improvise treatment of T-ALL in the future. To thoroughly understand the detailed impact of this novel synergistic\u003c/p\u003e \u003cp\u003ecombination and to assess its potential as a targeted cancer therapy, further in vitro testing is recommended. Cell Cycle Analysis: Conduct cell cycle analysis tounderstand how the treatment affects cell growth and development. By examining thedistribution of cells in different stages of the cell cycle (G1, S, and G2/M), researcherscan determine if the drug causes cell cycle arrest or stimulates cell death. Autophagy detection assays need to be done to evaluate how T-ALL responds to the treatment and whether modulating autophagy could be a viabletherapeutic strategy. This can also provide insights into the mechanisms of drug resistance and help identify potential targets for anti-T-ALL therapy. We can utilise Western blotting to analyse protein expression levels, identify therapeutic targets, assess signalling pathway activity, and evaluate treatment responses. Probing for specific proteins involved in T-ALL progression can help determine treatment efficacy and identify potential biomarkers for prognosis or treatment response prediction. This technique also aids in identifying dysregulated signalling pathways, guiding the development of targeted therapies. By implementing transgene techniques to introduce exogenous genes into T-ALL, we can study their impact on cellular behaviour and function. This method allows manipulation and control of gene expression levels, providing insights into changes in cell proliferation, apoptosis, or drug sensitivity, and aiding in the discovery of promising therapeutic targets. A gene knockdown experiments using RNA interference (RNAi) or CRISPR-Cas9 technology to reduce the expression of specific genes aids in understanding gene function in T-ALL.\u003c/p\u003e \u003cp\u003eRNAi involves introducing small RNA molecules to degrade the mRNA of the gene of interest, while CRISPR-Cas9 enables precise genomic editing to disrupt specific\u003c/p\u003e \u003cp\u003egenes. These recommended assays and techniques will enhance the understanding of the mechanistic effects of SRJ23 and HU on T-ALL and support the development of effective targeted therapies against T-ALL.\u003c/p\u003e"},{"header":"6 Conclusion","content":"\u003cp\u003eThe combination of SRJ23 at a concentration of 10 \u0026micro;M with HU at concentrations of 125 \u0026micro;M and 250 \u0026micro;M had a significant synergistic effect, as shown by the calculated CI values of 0.67 and 0.57, respectively. The combined doses of these compounds resulted in a significant decrease in JC viability compared to the HU and SRJ23 alone. Our study also demonstrates evidence of synergistic inhibitory effect and apoptosis initiation with a certain specified combination of HU and SRJ23 concentrations against T-ALL/ JC, leading to a noteworthy inhibitory impact in vitro., suggesting a promising new approach to targeted chemotherapy. Further studies need to be taken place to investigate other concentrations of the combination of HU and SRJ23 with the best synergistic and growth inhibitory effect before any in vivo and animal studies can be done.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAGP \u0026nbsp; \u0026nbsp; \u0026nbsp;Andrographolide\u003c/p\u003e\n\u003cp\u003eCDK \u0026nbsp; \u0026nbsp; \u0026nbsp;Cyclin-dependent kinase\u003c/p\u003e\n\u003cp\u003eCI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Combination index\u003c/p\u003e\n\u003cp\u003eDMSO \u0026nbsp; Dimethyl sulfoxide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003edNTPs \u0026nbsp; Deoxyribonucleotides\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Fraction affected\u003c/p\u003e\n\u003cp\u003eFITC \u0026nbsp; \u0026nbsp; \u0026nbsp;Fluorescein isothiocyanate\u003c/p\u003e\n\u003cp\u003eGDP \u0026nbsp; \u0026nbsp; \u0026nbsp;Guanosine diphosphate\u003c/p\u003e\n\u003cp\u003eGTP \u0026nbsp; \u0026nbsp; \u0026nbsp;Guanosine triphosphate\u003c/p\u003e\n\u003cp\u003eHU \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Hydroxyurea\u003c/p\u003e\n\u003cp\u003eJCLs \u0026nbsp; jurkat cell lines\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eK-Ras \u0026nbsp; \u0026nbsp;Kirsten rat sarcoma virus\u003c/p\u003e\n\u003cp\u003eMTT \u0026nbsp; \u0026nbsp; \u0026nbsp; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide\u003c/p\u003e\n\u003cp\u003eMPN \u0026nbsp; \u0026nbsp; Myeloproliferative neoplasm\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Propidium Iodide\u003c/p\u003e\n\u003cp\u003eRNR \u0026nbsp; \u0026nbsp; \u0026nbsp;Ribonucleotide reductase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSD \u0026nbsp; \u0026nbsp; \u0026nbsp; Standard deviation\u003c/p\u003e\n\u003cp\u003eSRJ23 \u0026nbsp; 3,19-(3-Chloro-4-fluorobenzylidene) andrographolide\u003c/p\u003e\n\u003cp\u003eT-ALL \u0026nbsp; \u0026nbsp; T cell acute lymphoblastic leukemia\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e Laith Marashdeh wrote the manuscript; Johnson Stanslas and Thilakavathy Karuppiah reviewed and validated the methodology and investigations; Bahariah Khalid critically revised the manuscript, contributed to the figures and tables, and approved its structure and content. All authors have reviewed and approved the final version for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was supported by the Pharmacotherapeutics Unit of the Faculty of Medicine and Health Sciences at Universiti Putra Malaysia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003eAll data associated with this study are presented within the manuscript. Additional details can be obtained by contacting the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChou, T.-C. (2006). Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacological Reviews, 58(3), 621\u0026ndash;681. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1124/pr.58.3.10\u003c/span\u003e\u003cspan address=\"10.1124/pr.58.3.10\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChou, T.-C. (2010). Drug Combination Studies and Their Synergy Quantification Using the Chou-Talalay MethodSynergy Quantification Method. Cancer Research, 70(2), 440\u0026ndash;446. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1158/0008-5472.CAN-09-1947\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.CAN-09-1947\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChou, T.-C., \u0026amp; Talalay, P. (1984). Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Advances in Enzyme Regulation, 22, 27\u0026ndash;55. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/0065-2571(84)90007-4\u003c/span\u003e\u003cspan address=\"10.1016/0065-2571(84)90007-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoache, D., Friciu, M., Bernine Marcellin, R., Bonnemain, L., Viau, A., Roullin, V. G., Forest, J.-M., \u0026amp; Leclair, G. (2022). Stability evaluation of compounded hydroxyurea 100 mg/mL oral liquids using a novel analytical method involving chemical derivatization. Plos One, 17(6), e0270206. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pone.0270206\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0270206\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeeney, M. M., Whorton, M. R., Howard, T. A., Johnson, C. A., \u0026amp; Ware, R. E. (2004). Chemical and functional analysis of hydroxyurea oral solutions. Journal of Pediatric Hematology/Oncology, 26(3), 179\u0026ndash;184. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/00043426-200403000-00007\u003c/span\u003e\u003cspan address=\"10.1097/00043426-200403000-00007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJada, S. R., Matthews, C., Saad, M. S., Hamzah, A. S., Lajis, N. H., Stevens, M. F. G., \u0026amp; Stanslas, J. (2008). Benzylidene derivatives of andrographolide inhibit growth of breast and colon cancer cells in vitro by inducing G1 arrest and apoptosis. British Journal of Pharmacology, 155(5), 641\u0026ndash;654. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/bjp.2008.368\u003c/span\u003e\u003cspan address=\"10.1038/bjp.2008.368\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJada, S. R., Subur, G. S., Matthews, C., Hamzah, A. S., Lajis, N. H., Saad, M. S., Stevens, M. F. G., \u0026amp; Stanslas, J. (2007). Semisynthesis and in vitro anticancer activities of andrographolide analogues. Phytochemistry, 68(6), 904\u0026ndash;912. DOI:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.phytochem.2006.11.031\u003c/span\u003e\u003cspan address=\"10.1016/j.phytochem.2006.11.031\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKo\u0026ccedil;, A., Wheeler, L. J., Mathews, C. K., \u0026amp; Merrill, G. F. (2004). Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools. Journal of Biological Chemistry, 279(1), 223\u0026ndash;230. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1074/jbc.M303952200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M303952200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLato, M. W., Przysucha, A., Grosman, S., Zawitkowska, J., \u0026amp; Lejman, M. (2021). The new therapeutic strategies in pediatric t-cell acute lymphoblastic leukemia. International Journal of Molecular Sciences, 22(9), 4502. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms22094502\u003c/span\u003e\u003cspan address=\"10.3390/ijms22094502\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadaan, K., Kaushik, D., \u0026amp; Verma, T. (2012). Hydroxyurea: a key player in cancer chemotherapy. Expert Review of Anticancer Therapy, 12(1), 19\u0026ndash;29. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1586/era.11.175\u003c/span\u003e\u003cspan address=\"10.1586/era.11.175\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatthews, H., Deakin, J., Rajab, M., Idris-Usman, M., \u0026amp; Nirmalan, N. J. (2017). Investigating antimalarial drug interactions of emetine dihydrochloride hydrate using CalcuSyn-based interactivity calculations. PloS one, 12(3), e0173303.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNautiyal, J., Kanwar, S. S., Yu, Y., \u0026amp; Majumdar, A. P. N. (2011). Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells. Journal of Molecular Signaling, 6(1), 1\u0026ndash;11. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1750-2187-6-7\u003c/span\u003e\u003cspan address=\"10.1186/1750-2187-6-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOffenbacher, A. R., \u0026amp; Barry, B. A. (2020). A proton wire mediates proton coupled electron transfer from hydroxyurea and other hydroxamic acids to tyrosyl radical in class Ia ribonucleotide reductase. The Journal of Physical Chemistry B, 124(2), 345\u0026ndash;354. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acs.jpcb.9b08587\u003c/span\u003e\u003cspan address=\"10.1021/acs.jpcb.9b08587\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel, A. A., Thomas, J., Rojek, A. E., \u0026amp; Stock, W. (2020). Biology and treatment paradigms in T cell acute lymphoblastic leukemia in older adolescents and adults. Current Treatment Options in Oncology, 21(7), 1\u0026ndash;21. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11864-020-00757-5\u003c/span\u003e\u003cspan address=\"10.1007/s11864-020-00757-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuah, S. Y., Wong, C. C., Wong, H. C., Ho, K. L., Manan, N. A., Deb, P. K., Sagineedu, S. R., \u0026amp; Stanslas, J. (2021). Microarray-based identification of differentially expressed genes associated with andrographolide derivatives-induced resistance in colon and prostate cancer cell lines. Toxicology and Applied Pharmacology, 425, 115605. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.taap.2021.115605\u003c/span\u003e\u003cspan address=\"10.1016/j.taap.2021.115605\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSelvarajoo, N., Sagineedu, S. R., Lim, W., Chee, J., Ho, K. L., \u0026amp; Stanslas, J. (2022). Interplay Between Autophagy And Apoptosis In A Pancreatic Cancer Cell Line Treated With SRJ23, Gemcitabine Or Combination Of Both Agents. Malaysian Journal of Medicine \u0026amp; Health Sciences, 18. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11523-013-0278-5\u003c/span\u003e\u003cspan address=\"10.1007/s11523-013-0278-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh, A., \u0026amp; Xu, Y.-J. (2016). The cell killing mechanisms of hydroxyurea. Genes, 7(11), 99. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/genes7110099\u003c/span\u003e\u003cspan address=\"10.3390/genes7110099\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpivak, J. L., \u0026amp; Hasselbalch, H. (2011). Hydroxycarbamide: a user\u0026rsquo;s guide for chronic myeloproliferative disorders. Expert Review of Anticancer Therapy, 11(3), 403\u0026ndash;414. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1586/era.11.10\u003c/span\u003e\u003cspan address=\"10.1586/era.11.10\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStearns, B., Losee, K. A., \u0026amp; Bernstein, J. (1963). Hydroxyurea. A new type of potential antitumor Agent1. Journal of Medicinal Chemistry, 6(2), 201. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/jm00338a026\u003c/span\u003e\u003cspan address=\"10.1021/jm00338a026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong, H. C., Wong, C. C., Sagineedu, S. R., Loke, S. C., Lajis, N. H., \u0026amp; Stanslas, J. (2014). SRJ23, a new semisynthetic andrographolide derivative: in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis in prostate cancer cells. Cell Biology and Toxicology, 30(5), 269\u0026ndash;288. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10565-014-9282-5\u003c/span\u003e\u003cspan address=\"10.1007/s10565-014-9282-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu, Y., Singh, A., \u0026amp; Alter, G. M. (2016). Hydroxyurea induces cytokinesis arrest in cells expressing a mutated sterol-14α-demethylase in the ergosterol biosynthesis pathway. Genetics, 204(3), 959\u0026ndash;973. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1534/genetics.116.191536\u003c/span\u003e\u003cspan address=\"10.1534/genetics.116.191536\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Medicine](https://link.springer.com/journal/44337)","snPcode":"44337","submissionUrl":"https://submission.springernature.com/new-submission/44337/3","title":"Discover Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"T-ALL, jurkat cell lines, hydroxyurea, SRJ23, growth inhibition assay, synergism, combination index, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-5419120/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5419120/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eT-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive blood cancer associated with high relapse rates, affecting about 15% of pediatric and 25% of adult cases. Initial management typically involves hydroxyurea (HU) to reduce elevated white blood cell counts before intensive chemotherapy. However, despite this approach, recurrence remains a substantial challenge, even with stem cell transplants. A promising therapeutic strategy for T-ALL might involve combining HU with an additional compound that can enhance cancer cell inhibition. SRJ23, a semi-synthetic andrographolide derivative, is a potential candidate due to its unique mechanism of inhibiting the GDP-GTP exchange, targeting the oncogenic K-Ras mutation pathway implicated in T-ALL progression. This study investigates the potential of HU and SRJ23 as a combined treatment to enhance apoptotic induction in T-ALL cells.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eJCLs is an immortalized lines of human T lymphocyte cells that are used to study T-ALL. They were cultured and treated with varying concentrations of HU (7.8\u0026ndash;1000 \u0026micro;M) and SRJ23 (0.1\u0026ndash;100 \u0026micro;M), individually and in combination, for 96 hours. The cell viability was then assessed using the MTT assay. The drug interactions were calculated using the CompuSyn software, which runs the Chou-Talalay method to calculate the combination index (CI) and generate isobolograms, allowing for the quantitative, determination of synergistic, additive, or antagonistic effects between the two compounds. We used the FITC Annexin V/PI Apoptosis Detection Kit to analyse the treated cells with BD FACS LSRFortressa\u0026trade; Cell Analyser and FACSDiva\u0026trade; Software to quantify early and late apoptotic populations.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur study shows a significant decline in the JCLs viability resulting from the exposure of HU and SRJ23, especially when in both drugs were in combination. A synergistic effect was observed at two occasions that is when 10\u0026micro;M SRJ23 combined with 125\u0026micro;M HU and 10\u0026micro;M SRJ23 combined with 250\u0026micro;M of HU on the dose-response curves. These combination treatment induced apoptosis confirmed by Annexin V-FITC staining.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eTo our knowledge, our study is the first to prove that the combination of HU and SRJ23 exerts a synergistic effect against JCLs and has a significant inhibitory effect in vitro with a remarkable ability to induce apoptosis. Further in-vitro and animal research is deemed required to thoroughly understand the detailed impact of this novel HU and SRJ23 synergism before it can be embarked as a potential novel targeted T-ALL chemotherapy.\u003c/p\u003e","manuscriptTitle":"Synergistic Antileukemic Effects of Hydroxyurea and SRJ23 in T-cell Acute Lymphoblastic Leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 12:58:57","doi":"10.21203/rs.3.rs-5419120/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-26T08:54:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-03T10:16:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-02T11:23:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337973354522617997180695855178517307621","date":"2024-11-28T13:54:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81697711033025951100539209823355651671","date":"2024-11-28T10:33:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-26T09:51:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-22T07:04:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-21T13:52:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Medicine","date":"2024-11-09T00:10:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Medicine](https://link.springer.com/journal/44337)","snPcode":"44337","submissionUrl":"https://submission.springernature.com/new-submission/44337/3","title":"Discover Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"86386377-a2f3-475a-a500-357ea62140b8","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-02-03T04:53:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-19 12:58:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5419120","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5419120","identity":"rs-5419120","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}