The Effectiveness of Multidrug Chemotherapy with Olaparib, Temozolomide and Oxaliplatin Compared to Radiotherapy in Glioblastoma Multiforme in Vitro Models and Human Fibroblasts

The Effectiveness of Multidrug Chemotherapy with Olaparib, Temozolomide and Oxaliplatin Compared to Radiotherapy in Glioblastoma Multiforme in Vitro Models and Human Fibroblasts | 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 The Effectiveness of Multidrug Chemotherapy with Olaparib, Temozolomide and Oxaliplatin Compared to Radiotherapy in Glioblastoma Multiforme in Vitro Models and Human Fibroblasts Anna Zajac-Grabiec, Anna Czopek, Paula Ajersch, Filip Michałkiewicz, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9193345/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Introduction : Glioblastoma multiforme (GBM) remains one of the most treatment-resistant central nervous system (CNS) tumors. This study evaluated the efficacy of multidrug repositioning combining oxaliplatin (OXA), olaparib (OLA), and temozolomide (TMZ), administered alone or in combination with photon radiotherapy, in GBM cell lines as in vitro models. Methods U118 MG and U87 MG cells, as well as control human fibroblasts (hFib) from a healthy donor, were treated with OXA (50–200 µM), OLA (1-100 µM), and TMZ (10–100 µM), alone and in combination. Cell viability was assessed after 72 hours using the MTS assay, and apoptosis/necrosis was quantified using a fluorescent apoptosis, necrosis, and healthy cell quantification kit. Treatments were tested with (2 Gy, 4 Gy) and without irradiation. Results Radiotherapy significantly enhanced the cytotoxic effects of all drugs, with the strongest reductions in viability observed for multidrug combinations. The addition of 2 Gy and 4 Gy irradiation markedly enhanced the activity of OLA + TMZ, OXA + TMZ, and OLA + OXA, leading to substantial loss of GBM cell viability. The greatest synergistic response has been observed with the triple drug combination (OLA + OXA + TMZ) plus radiotherapy, which produced extensive necrosis across all GBM models used. Conclusions The combination of OXA, OLA, and TMZ, especially when combined with photon radiotherapy, demonstrates potential synergistic cytotoxicity in GBM while sparing healthy fibroblasts. These results support the use of multidrug combination chemotherapy with radiotherapy as a promising and potentially safer treatment strategy for GBM. Olaparib Temozolomide Oxaliplatin Glioblastoma Multiforme Radiotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor, with a high mortality rate and a five-year survival of only 7.2% [ 1 , 2 ]. Standard management includes surgical resection followed by radiotherapy (RT) combined with temozolomide (TMZ), yet its limited efficacy underscores the need for more effective therapeutic strategies [ 3 , 4 ]. Glioma stem-like cells (GSCs) have been identified as a key factor in GBM resistance, exhibiting high self-renewal, metastatic potential, and efficient DNA repair. These cells overexpress DNA damage response proteins, such as checkpoint kinase 1 (CHK1) and poly(ADP-ribose) polymerase 1 (PARP-1), which contribute to chemoresistance and radioresistance [ 1 , 3 , 4 ]. TMZ, a first-line chemotherapy agent for GBM, induces cytotoxicity via DNA methylation, creating lesions such as O6-methylguanine (O6-MeG), which trigger DNA double-strand breaks (DSBs) and tumor cell death through the mismatch repair (MMR) pathway [ 1 , 3 ]. GBM frequently acquires resistance via MMR deficiency or overexpression of O6-methylguanine-DNA methyltransferase (MGMT), which repairs O6-MeG lesions [ 5 ]. Other chemotherapeutic agents, such as oxaliplatin (OXA), a third-generation platinum compound, induce DNA cross-links and DSBs while presenting lower systemic toxicity compared to earlier platinum drugs [ 6 , 7 ]. However, tumor cells can evade cytotoxicity via homologous recombination and non-homologous end-joining repair pathways [ 8 , 9 ]. PARP inhibitors, such as olaparib (OLA), interfere with DNA repair by trapping the PARP-DNA complex, leading to accumulation of DSBs and cell death. OLA has proven efficacy in tumors with homologous recombination deficiency (HRD), including certain isocitrate dehydrogenase (IDH) mutant GBM variants, and can enhance the cytotoxicity of alkylating agents even in HR-proficient cells [ 10 , 11 ]. Oxaliplatin has been primarily studied as a radiosensitizer in combination with radiotherapy in glioblastoma multiforme models, where it has been shown to potentiate radiation-induced cytotoxicity [ 12 , 13 ]. Combination including radiotherapy and drugs, such as TMZ or PARP inhibitors, without including OXA in multi-agent radiotherapy regimens has already been studied [ 10 , 14 ]. In this research, OXA has been combined with repositioning chemotherapy (TMZ and OLA) at lower doses and radiotherapy. While other studies show only combinations of OXA with drugs or OXA alone with radiotherapy [ 1 , 12 ]. Radiotherapy remains a cornerstone of glioblastoma (GBM) management. In this study, in vitro irradiation was performed using a Cobalt-60 (Co-60) source [ 15 ]. GBM cells were exposed to clinically relevant doses of 2 Gy and 4 Gy [ 16 ]. This experimental framework allowed further evaluation of potential synergistic interactions between radiotherapy and chemotherapy, including whether combined treatments enhance tumor cell killing while reducing toxicity compared to monotherapies [ 10 , 11 , 15 , 16 ]. MATERIALS AND METHODS 1. Cell lines and cell culture The following GBM cell lines were used as preclinical in vitro models [ 17 , 18 ]. U118 MG (Cat. No. HTB-15), and U87 MG (Cat. No. HTB-14) were obtained from the American Type Culture Collection (ATCC). A healthy donor human fibroblast line (hFib) served as a control [ 1 ]. Before the fragment of skin was taken, the participant signed written informed consent; the study protocol was reviewed and approved by the Human Bioethical Committee of the Regional Medical Board in Kraków (No. 163/KBL/OIL/2023). Details on cell lines and culturing are provided in the Supplementary Information (SI). 2. Chemiotherapeutics TMZ (Cat. No. T2577) and OXA (Cat. No. PHR1528) were obtained from Sigma-Aldrich. The following synthesis of OLA was conducted following the literature data [ 19 , 20 ], with minor modifications, which are described in detail in the SI. 3. Irradiation with a Cobalt-60 (Co-60) beam and dosimetry The irradiation was performed using a Cobalt-60 (Co-60) source (Theratron 780E, Best Theratronics; activity 236.7 TBq on November 2, 2018) at the Institute of Nuclear Physics, Polish Academy of Sciences (IFJ PAN), Krakow, Poland. Photon beam dosimetry was conducted using a UNIDOS Webline electrometer (PTW-Freiburg, T10021) in combination with a Farmer-type ionization chamber (PTW-Freiburg, TM30010-1). Details on irradiation with a Co-60 beam are provided in the SI. 4. Cell Viability Assessment of OLA, TMZ, OXA, and their Combination Cell viability of TMZ, OLA, and OXA and their combinations with radiotherapy was evaluated using GBM cell lines seeded in 96-well plates at a density of 4 × 10³ cells per well. Cells were treated with increasing concentrations of each drug individually or in combination with radiotherapy or alone as detailed in the SI. 5. Cell death analysis Cells (U118, U87, and human fibroblasts) were seeded in 6-well plates and following attachment, exposed to chemotherapeutic agents at various concentrations are provided in the SI. Immediately after drug addition, the plates were exposed to radiation using a cobalt-60 source (Theratron 780E, Best Theratronics), according to the manufacturer’s certificate. Irradiation was delivered as single doses of 2 Gy and 4 Gy. Detailed procedures and representative images are provided in the SI. 6. Statistical analysis Cells were subjected to drug treatment, radiotherapy (at single doses of 2 Gy or 4 Gy), or combined treatments. Cell viability was assessed 72 hours after treatment using the MTS assay, with absorbance measured at 490 nm. Statistical analyses were conducted using GraphPad Prism version 10.6.1 (892) for Windows (GraphPad Software, Boston, Massachusetts, USA). The data are presented as mean ± standard deviation (SD) of at least three replicates. All data were tested for the assumptions of normality (the Shapiro-Wilk test). Two-way ANOVA of the variance was performed by the Tukey test. Statistical significance is indicated by asterisks (* p < 0.05; **p < 0.01; *** p < 0.001; **** p < 0.0001). Error bars in all graphs represent the standard deviation of at least three replicates. Both chemotherapeutic treatments and irradiation doses (2 Gy and 4 Gy) were included in the statistical comparisons, allowing assessment of monotherapy and combined effects. RESULTS 1. Chemistry OLA was resynthesized using literature-based conditions with slight modifications [ 19 ]. Briefly, the carboxylic acid component was first activated with O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) and then coupled with Boc-protected piperazine. Subsequent Boc deprotection afforded compound 2 , which was finally acylated with cyclopropanecarbonyl chloride to yield OLA. This synthetic sequence provided isolated yields of 60–91%. The structure and purity of the final compound were confirmed by chromatographic and spectroscopic analyses, as detailed in the SI (Fig. 1 ). 2. Cell viability To assess the effects of OXA, OLA, TMZ, and their combinations on cell viability, two human GBM cell lines were used: U87 (grade IV), U118 (grade III/IV). As a non-cancerous control, normal human fibroblasts (hFib) were used, obtained from a healthy donor. Details on cell lines and culturing are provided in SI (Fig. 2 ). A two-way ANOVA using Type III sums of squares was conducted to examine the effects of radiation doses and treatment groups on cell viability. Unequal sample sizes were handled using Type III SS. The main effects of radiation were statistically significant, while treatment group as well as interaction between OLA 1 µM and OLA 100 µM or OXA 50 µM and OXA 100 µM or TMZ 10 µM and TMZ 100 µM were non-significant. Treatment group was analyzed as a categorical factor due to non-equivalent drug concentrations between compounds. The interaction between radiation and treatment group was not significant (F( 2, 30 ) = 0.5599, p = 0.5771), indicating that the effect of radiation on cell viability was consistent across treatment conditions. There was a significant main effect of radiation dose (F( 2, 30 ) = 11.85, p = 0.0002), demonstrating that radiation significantly altered U87 cell viability. In contrast, the main effect of treatment group was not significant (F( 1, 30 ) = 2.348, p = 0.1360), suggesting that drug treatment alone did not significantly affect cell viability under the tested conditions. Post hoc Tukey comparison was performer separately for each drug to examine the interaction between various drug concentrations and radiation doses. For OLA, radiation significantly reduced cell viability at higher concentration (100 µM), while lower concentration (1 µM) showed non-significant effect. For OXA, significant differences were observed between radiation doses at 50 µM. Last drug, TMZ at each of the concentrations showed non-significant effect with tendency to decrease of viability. These analyses demonstrate that the response to radiation depends on drug concentration within each compound (Fig. 3 ). Cell viability of the U118 cell line was analyzed as a function of radiation doses and treatment groups using a two-way ANOVA with Type III sums of squares. All three effects – the main effects of radiation doses and treatment groups, as well as their interaction – were statistically significant. A significant interaction between radiation and treatment group was observed (F( 14, 187 ) = 13.25, p < 0.0001), indicating that the response to radiation differed across treatment conditions. There was a significant main effect of radiation (F( 2, 187 ) = 247.9, p < 0.0001), with increasing radiation doses significantly reducing cell viability. A significant main effect of treatment groups were also observed (F( 7, 187 ) = 55.15, p < 0.0001), demonstrating that different treatment conditions significantly influenced cell viability. Treatment groups were analyzed as a categorical factor due to non-equivalent drug concentrations between compounds. Post hoc Tukey comparisons were performed separately for each drug to examine the interaction between drug concentration and radiation dose. For OLA, radiation significantly reduced cell viability at both concentrations (100 µM and1 µM). For OXA, radiation significantly reduces cell viability at lower concentrations (50 µM and 100 µM), while at higher concentration no changes were observed. For TMZ, reduction of cell viability with radiation were observed at both concentrations (10 µM and 100µM) (Fig. 4 ). A two-way ANOVA using Type III sums of squares was conducted to examine the effects of radiation doses and treatment groups, and their interaction on fibroblast viability. All three effects – the main effects of radiation and treatment group, as well as their interaction – were statistically significant. Treatment groups were analyzed as a categorical factor due to non-equivalent drug concentrations between compounds. A significant interaction between radiation doses and treatment groups was observed (F( 14, 48 ) = 4.837, p < 0.0001), indicating that the response to radiation differed across treatment conditions. There was a significant main effect of radiation (F( 2, 48 ) = 156.7, p < 0.0001), with higher radiation doses significantly reducing fibroblast viability. The main effect of treatment groups were also significant (F( 7, 48) = 18.79, p < 0.0001), demonstrating that different treatment conditions significantly affected cell viability. Post hoc Tukey comparisons were performed separately for each drug to examine the interaction between drug concentrations and radiation doses. For OLA, radiation significantly reduced cell viability at both concentrations (1 µM and 100 µM). Second drug, OXA showed significant decrease in cell viability between radiation doses in concentration 50 µM, while in 100 µM and 200 µM no differences were observed. For TMZ, radiation significantly reduced cell viability at both concentrations (10 µM and 100 µM) (Fig. 5 ). Cell viability in U87 cells was analyzed using a two-way ANOVA with radiation doses and drug treatment groups as factors (Type III sums of squares). No significant interaction between radiation doses and drug treatments was observed (F( 6, 34 ) = 2.012, p = 0.0911), indicating that the effect of radiation on cell viability was largely independent of drug treatment groups. The main effect of radiation dose did not reach statistical significance (F( 2, 34 ) = 3.141, p = 0.0560), although a trend toward reduced viability with increasing radiation dose was observed. In contrast, a significant main effect of drug treatment was detected (F( 3, 34 ) = 3.146, p = 0.0376). Post hoc test followed by Tukey was conducted and indicate significant differences between radiation doses in group with triple combinations of drugs. Cell viability in U118 cells was examined using a two-way ANOVA with radiation dose and drug treatment as factors. Interaction between radiation dose and drug treatment was not statistically significant (F( 6, 24 ) = 1.262, p = 0.3112). A significant main effect of radiation dose was observed (F( 2, 24 ) = 8.389, p = 0.0017), as well as drug treatment (F( 3, 24 ) = 6.707, p = 0.0019). In fibroblast group, cell viability was analyzed using a two-way ANOVA with radiation dose and drug treatment as factors. No significant interaction between radiation and drug treatment was detected (F( 6, 33 ) = 1.329, p = 0.2721). Radiation doses have significant main effect on fibroblast viability (F( 2, 33 ) = 22.36, p < 0.0001) as well as drug treatment gropus(F( 3, 33 ) = 6.762, p = 0.0011). Post hoc Tukey comparisons were performed and showed significant differences between radiation doses within the group treated with 50 µM OXA + 50 µM OLA and in group 50 µM OXA + 50 µM OLA + 50 µM TMZ. 3. Cell death analysis Two-way ANOVA revealed a significant interaction between drug treatment (OXA, OLA, TMZ) and radiation (2 Gy, 4 Gy) for all tested cell lines, indicating that the cellular response (viability and death profiles) significantly differed between drug-only and combined treatment groups (Fig. 6 ). Cells were subjected to drug (OXA, OLA, TMZ) treatment, radiotherapy (single doses of 2 Gy or 4 Gy) or combined treatments after 72 hours. The impact of TMZ, OXA, and/or OLA with radiotherapy on the type of cell death was assessed in two commercially available GBM cell lines (U118MG, U87MG). The human fibroblast line (hFib) obtained from a healthy donor was used as controls. The results for the compared groups were as follows: hFib, F70,216 = 408.5.0, p < 0.0001; U118, F70,216 = 787.9, p < 0.0001; and U87, F70,216 = 1224, p < 0.0001 (Table 1 ). Table 1 Effect of TMZ, OXA, and/or OLA on the type of cell death in glioblastoma cell lines (U118 MG, U87 MG – ATCC) and human fibroblasts (hFib, control, from a healthy donor). Anti-cancer drug Early and late apoptosis, necrosis and lid live cells presented as [%] after 72 hours for each cell line Type of cells hFib U118 U87 Radiotherapy [Gy] 0 2 4 0 2 4 0 2 4 Control, without anti-cancer drug live cells 100 0 0 98 0 0 100 5 10 Early apoptosis 0 91 0 2 64 32 0 18 19 Late apoptosis 0 9 28 0 34 52 0 31 35 necrosis 0 0 72 0 2 16 0 46 36 OLA 100µM Live cells 8 15 0 0 0 0 0 0 0 Early apoptosis 47 45 22 98 0 0 13 20 0 Late apoptosis 45 40 78 2 25 0 87 63 72 Necrosis 0 0 0 0 75 00 0 17 28 TMZ 100µM Live cells 0 0 0 32 0 0 0 0 0 Early apoptosis 54 0 0 34 0 0 48 0 0 Late apoptosis 46 100 10 34 100 50 52 6 0 Necrosis 0 0 90 0 0 50 0 94 100 OXA 50µM Live cells 74 0 0 0 0 0 0 0 0 Early apoptosis 24 0 0 32 0 0 46 0 0 Late apoptosis 2 100 100 68 20 0 54 0 0 Necrosis 0 0 0 0 80 100 0 100 100 100µM Live cells 45 0 0 0 0 0 0 0 0 Early apoptosis 55 0 0 0 0 0 0 0 0 Late apoptosis 0 60 0 55 10 5 100 0 0 Necrosis 0 40 100 45 90 95 0 100 100 200µM Live cells 0 0 0 0 0 0 0 0 0 Early apoptosis 20 0 0 2 0 0 5 0 0 Late apoptosis 51 32 0 59 0 0 90 0 0 Necrosis 29 68 100 39 100 100 5 100 100 OLA 50µM + TMZ 50µM Live cells 42 10 0 0 0 0 0 0 0 Early apoptosis 41 38 0 14 0 0 5 0 0 Late apoptosis 17 52 100 86 0 0 95 11 0 Necrosis 0 0 0 0 100 100 0 89 100 OLA 50µM + OXA 50µM Live cells 60 10 0 0 0 0 0 0 0 Early apoptosis 22 35 13 5 0 0 5 63 0 Late apoptosis 18 55 87 95 5 0 95 37 30 Necrosis 0 0 0 0 95 100 0 0 70 OLA 50µM + OXA 50µM + TMZ 50µM Live cells 57 0 0 9 0 0 0 0 0 Early apoptosis 20 0 0 46 0 0 24 0 0 Late apoptosis 23 50 35 45 20 0 76 0 0 Necrosis 0 50 65 0 80 100 0 100 100 Treatment with 100 µM OLA induced apoptosis in hFib (early apoptosis: 47%, 45%, 22% vs. late apoptosis: 45%, 40%, 78%, respectively) without evidence of necrosis. In contrast, GBM cell lines showed no viable cells; necrosis predominated in U118 cells at 2 and 4 Gy (75%, 100%) and in U87 cell line at 2 and 4 Gy (17%, 28%), (see Table 1 ). In contrast, treatment with 100 µM TMZ caused 90% necrosis in hFib cells at 4 Gy, 50% in U118 and 100% in U87 cell lines. At 2 Gy, necrosis was observed only in the U87 cell line (94%) (see Table 1 ). Treatment with 50 µM OXA without radiation induced early and late apoptosis in hFib cells (24% and 2%, respectively), with no necrosis observed. However, at 200 µM OXA, hFib cells exhibited necrosis (29%) and late apoptosis (51%), while U118 and U87 cells showed necrosis (39% and 5%, respectively) without radiotherapy. In the presence of 2 Gy and 4 Gy radiotherapy, the following effects were observed: after 72 hours of treatment with 200 µM OXA, complete necrosis (100%) were detected in both U118 and U87 cells, at both radiation doses. Combination treatments with 50 µM OLA and 50 µM TMZ or 50 µM OLA and 50 µM OXA at 2 Gy and 4 Gy predominantly induced apoptosis without necrosis in hFib cell line. At both doses: 2 Gy and 4 Gy, the combination of 50 µM OLA and 50 µM TMZ led to necrosis in U118 (100%, 100%, respectively) and in U87 (89%, 100%, respectively). A similar pattern was observed for combination 50 µM OLA and 50 µM OXA in U118 cell line (95%, 100% necrosis at 2 Gy and 4 Gy). In contrast, hFib and U87 cells treated with 50 µM OLA and 50 µM OXA showed early and late apoptosis following irradiation at 2 Gy. In hFib cells, early and late apoptosis exhibited in 35% and 55% respectively whereas in U87 cells these values were 63% and 39%, respectively (see Table 1 .). The three-drug combination 50 µM OLA, 50 µM TMZ, and 50 µM OXA resulted in early and late apoptosis without necrosis in all cell lines (20% and 23% in hFib, 46% and 45% in U118, 24% and 76% in U87). Following irradiation, necrosis was observed in GBM cells lines and in hFib cells, at 2 and 4 Gy, necrosis reached 50% and 65% in hFib cells, 80% and 100% in U118 cells, and 100% at both doses in U87 cells (see Table 1 ). DISCUSSION In this study, the cytotoxic effects of alkylating drugs OXA with TMZ administered alone or in combination with the PARP inhibitor OLA were assessed in GBM cell lines and human fibroblasts in combination with photon radiotherapy [ 1 , 21 , 22 ]. The results showed that increasing therapeutic complexity through multidrug combinations and concomitant ionizing radiation was associated with a progressive decrease in GBM cell viability compared to hFib [ 1 , 15 , 22 , 23 ]. Similar responses of U118 and U87 cell lines to drugs and radiotherapy combination were visualized in Figs. 2 , 3 , 5 , and illustrating the modulatory role of MGMT expression in drug resistance. U118 cells, characterized by high MGMT expressions, showed limited sensitivity to monotherapies but clearly responded significantly to combination therapies including OLA [ 17 , 18 , 24 , 25 ]. In contrast, U87 cells showed greater sensitivity to OLA + TMZ and to the triplet OLA + TMZ + OLA without RT [ 1 ]. Zajac-Grabiec et al. [ 1 ] also reported a synergistic effect of OLA with TMZ in the GBM models, especially in the U87, H4, and U118 cell lines [ 1 ]. OLA enhances the effect of TMZ, but not OXA, in glioma cell lines irrespective of MGMT status, as shown in previous reports [ 1 , 2 , 4 ]. In the present study, the drugs used alone and in combinations: OLA and TMZ or OXA and TMZ or OXA and OLA, as well as the triplet preparation of OXA, OLA and TMZ administrated with radiotherapy (2Gy, 4Gy), showed synergistic effects. In Fig. 5 , enhanced activity is observed for the combination regimens, with higher cytotoxicity being caused by OLA + TMZ, OXA + TMZ, and OLA + OXA following radiotherapy compared to monotherapy, while the most pronounced reduction in cell viability is observed with the triple combination (OLA + OXA + TMZ) in all GBM models. These findings support the conclusion to confirm that synergistic accumulation of cytotoxic DNA damage results from the simultaneous inhibition of multiple DNA repair pathways. Specifically, DNA alkylation is induced by TMZ with OXA, whereas OLA, thereby preventing the removal of single-strand breaks and promoting their conversion into lethal double-strand breaks (DSBs) during replication [ 10 , 25 ]. This impairment of DNA repair is considered to underline the enhanced efficacy of multidrug regimens following photon therapy [ 11 , 26 ]. Moreover, the efficacy of all drugs OLA, TMZ, and OXA, alone was enhanced by radiotherapy. As shown in Fig. 2 – 4 (single drug) and Fig. 5 (drug combinations), both 2 Gy and 4 Gy increased cytotoxicity. This effect is likely attributable to increased susceptibility of tumor cells to radiation-induced DSBs when DNA repair pathways are pharmacologically impaired. The synergic interaction between chemotherapy and radiotherapy was found to be significantly stronger in U118 and U87 cells than in hFib cells, supporting the notion that GBM-specific genomic instability is associated with an increased dependence on DNA repair mechanisms that are selectively targeted by OLA, OXA, and TMZ [ 1 , 15 , 16 ]. The results of this study demonstrated a differential response between hFib cells as a control and glioblastoma multiforme cell lines (U118 and U87) to treatment with OLA, TMZ, OXA, their combinations, and radiotherapy. In particular, in this study, hFib cells predominantly underwent apoptosis, whereas GBM cells responded mainly with necrosis, especially when combined with ionizing radiation (2 Gy, 4 Gy). These findings underscore the sensitivity of GBM cells to combined cytotoxic stress and the relative resistance of hFib cells to necrotic cell death (see Table 1 ). Treatment with 100 µM OLA, particularly after irradiation, induced an apoptotic response in hFib cells, with no evidence of necrosis. In contrast, GBM cells subjected to the same treatment did not exhibit viability or early apoptosis, and necrosis predominated the cell death profile (see Table 1 ). Furthermore, treatment with 100 µM TMZ caused significant necrosis of hFib cells only at the higher radiation dose (4 Gy). GBM cell lines showed greater susceptibility at the 2 Gy dose, particularly U87 cells, which showed 94% necrosis. The increased sensitivity of GBM cells demonstrated a synergistic interaction between DNA alkylation by TMZ and radiation-induced DNA damage, which impairs DNA repair pathways frequently mutated in GBM [ 15 , 16 , 23 ]. OXA treatment showed a dose-dependent response in hFib cells, with 50 µM predominantly inducing apoptosis without necrosis. In contrast, treatment with 100 and 200 µM OXA resulted in both necrosis and late apoptosis, particularly following irradiation. Notably, 200 µM OXA combined with 2 or 4 Gy caused 100% necrosis in both GBM cells. These findings support the observation that radiotherapy enhances the cytotoxic effects of OXA, particularly in tumor cells. Furthermore, when hFib cells were exposed to 50 µM OLA and TMZ combined with irradiation, apoptosis remained the dominant mechanism of cell death, with no necrosis detected. This indicates that at lower drug concentrations, hFib cells maintained controlled cell death pathways, even under combined chemotherapy and photon irradiation [ 21 , 26 ]. In contrast, complete necrosis was observed in GBM cells under the same treatment conditions, indicating a higher sensitivity to combined chemotherapy and radiotherapy [ 15 , 16 ]. In this study, OLA combined with TMZ or OXA induced apoptosis without necrosis in hFib cells, even following irradiation. In U118 and U87 cell lines, the same combinations caused extensive necrosis at doses of 2 Gy and 4 Gy, with U118 cells exhibiting almost complete necrosis and U87 cells displaying a mixed apoptotic-necrotic profile during chemotherapy and radiotherapy. These results indicate that hFib cells retained their capacity for programmed cell death after multiple drug administration, whereas GBM cells lost this regulatory control after exposure to combined stressors [ 1 , 11 ]. The three-drug combination (OLA, TMZ, OXA) induced apoptosis without evidence of necrosis in all cell lines in the absence of photon irradiation. However, following irradiation necrosis occurred in all cell types. Moreover, hFib as a control group demonstrated a different viability profile, as shown in Fig. 2 , 3 , 4 , 5 , and Table 1 . Despite a moderate reduction in viability, cytotoxicity was significantly lower than that observed in GBM cell lines, even under multidrug treatment or in the presence of radiotherapy. [ 27 , 28 ]. These findings highlight the potential of multidrug repositioning strategies (OXA, OLA) in combination with TMZ and photon therapy may bring a breakthrough, which will ultimately enable more effective and safer pharmacotherapy for patients [ 27 , 29 ]. Although our results are preliminary, the observed synergistic effect of alkylating agents with combination of OLA and radiotherapy supports the proposal of a novel chemotherapy strategy for GBM. Further investigation of the synergistic effects of repositioning drugs (OXA, OLA) combined with TMZ or modern radiotherapy, such as FLASH, may lead to therapeutic breakthroughs that will ultimately enable more effective and safer pharmacotherapy for patients. Therefore, in vitro and in vivo molecular studies of OXA and OLA combined with TMZ and various types of radiotherapy in GBM in different models (i.e., 2D and 3D) are necessary and will be conducted in the next part of this ongoing project. Abbreviations CHK1 checkpoint kinase 1 CNS central nervous system DCM dichloromethane DMF dimethylformamide DMSO dimethyl sulfoxide DSBs double–strand breaks GBM glioblastoma multiforme GSCs glioma stem–like cells HATU hexafluorophosphate azabenzotriazole tetramethyl uronium HBTU O–benzotriazole–N,N,N',N'–tetramethyl–uronium–hexafluoro–phosphate HCL hydrochloric acid HD helical hFib human fibroblast line HPLC high–performance liquid chromatography HRD homologous recombination deficiency IC 50 half maximal inhibitory concentration IDH isocitrate dehydrogenase MGMT O6–methylguanine–DNA methyltransferase MMR mismatch repair MTS 3–(4,5–dimethylthiazol–2–yl)–5–(3–carboxymethoxyphenyl)–2–(4–sulfophenyl)–2H–tetrazolium NI nicotinamide OLA olaparib OXA oxaliplatin O6 MeG –O6–methylguanine PARP1 poly(ADP–ribose) polymerase 1 PH phosphate RT radiotherapy SI supplementary information TBTU O–(benzotriazol–1–yl)–N,N,N',N'–tetramethyluronium tetrafluoroborate TEA triethylamine TMZ temozolomide Declarations Funding statement: These studies were performed at Cyclotron Centre Bronowice IFJ PAN under the project Miniatura 8, No. 2024/08/X/NZ7/00625, funded by the National Science Centre, Poland, and partially funded from the state budget under the Ministry of Education and Science (Poland) program entitled Science for Society II, No. NdS-II/SP/0295/2023/01, total project amount 1 mln PLN. The synthesis and in silico study were financially funded by JU MC Funds (N42/DBS/000412). Conflict of interest statement: The authors declare no conflicts of interest. Author contributions: Anna Zając-Grabiec*, conceptualization, data curation, funding acquisition, investigation, resources, methodology, roles/writing - original draft, writing - review & editing. Anna Czopek*, investigation, data curation, visualization, methodology, funding acquisition, roles/writing - original draft, writing - review & editing. Filip Michałkiewicz, software, formal analysis, validation, visualization. Tomasz Skóra, investigation. Beata Biesaga, supervision. Monika Bania, investigation. Krzysztof Łukowicz, investigation Marta Bałamut, investigation, methodology Dawid Krzempek, investigation, methodology Dominik Wiśniewski, investigation, methodology. Paula Ajersch, formal analysis, validation, visualization. Justyna Miszczyk, funding acquisition Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Human Bioethical Committee of the Regional Medical Board in Kraków (No. 163/KBL/OIL/2023, 06.07.2023). Written informed consent was obtained from the participant involved in the study before the fragment of skin was taken. Ethics declarations Ethics Approval and Consent to Participate. The study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Human Bioethical Committee of the Regional Medical Board in Kraków (Approval No. 163/KBL/OIL/2023, dated 06.07.2023). Written informed consent was obtained from the participant prior to the collection of skin fragments. Consent for publication Written informed consent for publication was obtained from the participant. Data available with manuscript or supplementary information: The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request. References Zając-Grabiec A, Czopek A, Pazdan K, Jończyk J, Michałkiewicz F, Skóra T, et al. In vitro repositioning therapy with olaparib, temozolomide and oxaliplatin in glioblastoma cell lines: U118, U87, U251, H4 and human fibroblasts. Pharmacol Rep. 2025;77(6):1716–26. https://doi.org/10.1007/s43440-025-00783-w . Epub 2025 Sep 3. PMID: 40900233; PMCID: PMC12647329. Wu W, Klockow JL, Zhang M, Lafortune F, Chang E, Jin L, et al. Glioblastoma multiforme (GBM): An overview of current therapies and mechanisms of resistance. Pharmacol Res. 2021;171:105780. https://doi.org/10.1016/j.phrs.2021.105780 . Epub 2021 Jul 21. PMID: 34302977; PMCID: PMC8384724. 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Combination Olaparib and Temozolomide for the Treatment of Glioma: A Retrospective Case Series. Neurology. 2022;99(17):750–55. https://doi.org/10.1212/WNL.0000000000201203 . 't Hart E, Bianco J, Bruin MAC, Derieppe M, Besse HC, Berkhout K, et al. Radiosensitisation by olaparib through focused ultrasound delivery in a diffuse midline glioma model. J Control Release. 2023;357:287–98. https://doi.org/10.1016/j.jconrel.2023.03.058 . Benzina S, Debomy F, Bergerat JP, Denis JM, Gueulette J, Dufour P, et al. The cytotoxicity of high-linear energy transfer radiation is reinforced by oxaliplatin in human glioblastoma cells. Cancer Lett. 2007;28(1):54–62. https://doi.org/10.1016/j.canlet.2007.02.001 . Charest G, Sanche L, Fortin D, Mathieu D, Paquette B. Glioblastoma treatment: bypassing the toxicity of platinum compounds by using liposomal formulation and increasing treatment efficiency with concomitant radiotherapy. 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Poly-(ADP-ribose)-polymerase inhibitors as radiosensitizers: a systematic review of pre-clinical and clinical human studies. Oncotarget. 2017;8(40):69105–124. https://doi.org/10.18632/oncotarget.19079 . Gradwohl G, Ménissier de Murcia JM, Molinete M, Simonin F, Koken M, Hoeijmakers JH, et al. The second zinc-finger domain of poly(ADP-ribose) polymerase determines specificity for single-stranded breaks in DNA. Proc Natl Acad Sci U S A. 1990;87(8):2990–4. https://doi.org/10.1073/pnas.87.8.2990 . Menear KA, Adcock C, Boulter R, Cockcroft XL, Copsey L, Cranston A, et al. 4-[3-(4-cyclopropanecarbonylpiperazine-1-carbonyl)-4-fluorobenzyl]-2H-phthalazin-1-one: a novel bioavailable inhibitor of poly(ADP-ribose) polymerase-1. J Med Chem. 2008;51(20):6581–91. https://doi.org/10.1021/jm8001263 . Xu Q, Chen J, Wang Z, Zang Y, Li G, Zhu F, et al. Two-step flow synthesis of Olaparib in microreactor: Route design, process development and kinetics research. Chem Eng J. 2023;471(3):144304. https://doi.org/10.1016/j.cej.2023.144304 . Bian X, Piipponen M, Liu Z, Luo L, Geara J, Chen Y, et al. Epigenetic memory of radiotherapy in dermal fibroblasts impairs wound repair capacity in cancer survivors. Nat Commun. 2024;15(1):9286. https://doi.org/10.1038/s41467-024-53295-1 . Hill RP, Kaspler P, Griffin AM, O'Sullivan B, Catton C, Alasti H, et al. Studies of the in vivo radiosensitivity of human skin fibroblasts. Radiother Oncol. 2007;84(1):75–83. https://doi.org/10.1016/j.radonc.2007.05.025 . Tokuyama Y, Mori K, Isobe M, Terato H. Comparison of mutation spectra induced by gamma-rays and carbon ion beams. J Radiat Res. 2024;65(4):491–9. https://doi.org/10.1093/jrr/rrae050 . PMID: 38940734; PMCID: PMC11262859. Helleday T, Petermann E, Lundin C, Hodgson B, Sharma RA. DNA repair pathways as targets for cancer therapy. Nat Rev Cancer. 2008;8(3):193–204. https://doi.org/10.1038/nrc2342 . Xu K, Chen Z, Cui Y, Qin C, He Y, Song X. Combined olaparib and oxaliplatin inhibits tumor proliferation and induces G2/M arrest and γ-H2AX foci formation in colorectal cancer. Onco Targets Ther. 2015;8:3047–54. https://doi.org/10.2147/OTT.S89154 . Nickoloff JA, Taylor L, Sharma N, Kato TA. Exploiting DNA repair pathways for tumor sensitization, mitigation of resistance, and normal tissue protection in radiotherapy. Cancer Drug Resist. 2021;4(2):244–63. https://doi.org/10.20517/cdr.2020.89 . Epub 2021 Jun 19. PMID: 34337349; PMCID: PMC8323830. Shaw R, Basu M, Karmakar S, Ghosh MK. MGMT in TMZ-based glioma therapy: Multifaceted insights and clinical trial perspectives. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2024;187(3):119673. https://doi.org/10.1016/j.bbamcr.2024.119673 Liu J, Jiang J, Hui X, Wang W, Fang D, Ding L. Mir-758-5p Suppresses Glioblastoma Proliferation, Migration and Invasion by Targeting ZBTB20. Cell Physiol Biochem. 2018;48(5):2074–83. https://doi.org/10.1159/000492545 . Schatz J, Ladinig A, Fietkau R, Putz F, Gaipl US, Frey B, et al. Normofractionated irradiation and not temozolomide modulates the immunogenic and oncogenic phenotype of human glioblastoma cell lines. Strahlenther Onkol. 2023;199(12):1140–51. https://doi.org/10.1007/s00066-022-02028-8 . Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 18 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers invited by journal 07 Apr, 2026 Editor assigned by journal 26 Mar, 2026 Submission checks completed at journal 26 Mar, 2026 First submitted to journal 22 Mar, 2026 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. 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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-9193345","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":621691814,"identity":"f6f83c16-ea65-464d-92d6-3fa7702ec62e","order_by":0,"name":"Anna 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Poland","correspondingAuthor":false,"prefix":"","firstName":"Krzysztof","middleName":"","lastName":"Łukowicz","suffix":""},{"id":621691823,"identity":"b707c355-1deb-4e22-9c6e-7784513a1fba","order_by":9,"name":"Tomasz Skóra","email":"","orcid":"","institution":"Maria Skłodowska-Curie National Research Institute of Oncology, Radiotherapy Department,","correspondingAuthor":false,"prefix":"","firstName":"Tomasz","middleName":"","lastName":"Skóra","suffix":""},{"id":621691824,"identity":"bc87993d-3b88-4f9d-b635-845526c51481","order_by":10,"name":"Beata Biesaga","email":"","orcid":"","institution":"Department of Tumor Pathology, Maria Skłodowska-Curie National Research Institute of Oncology, Cracow Branch,","correspondingAuthor":false,"prefix":"","firstName":"Beata","middleName":"","lastName":"Biesaga","suffix":""},{"id":621691825,"identity":"753e3ff1-a33d-4f96-be11-284214216ac3","order_by":11,"name":"Justyna Miszczyk","email":"","orcid":"","institution":"Department of Medical Physics, Institute of Nuclear Physics, Polish Academy of Sciences, Radzikowskiego 152, 31-342 Kraków, Poland","correspondingAuthor":false,"prefix":"","firstName":"Justyna","middleName":"","lastName":"Miszczyk","suffix":""}],"badges":[],"createdAt":"2026-03-22 19:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9193345/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9193345/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106960317,"identity":"dfd6d744-beb3-4bcf-ad4a-1e5bf573a009","added_by":"auto","created_at":"2026-04-15 09:20:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":90596,"visible":true,"origin":"","legend":"\u003cp\u003eResynthesis of Olaparib. Reagents and conditions; a: N-boc-piperazine, TBTU, TEA, DMF, 24 h, room temperature, b: HCl (aq), ethanol, c: cyclopropanecarbonyl chloride, TEA, DCM, 30 min., 0 ºC. 2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (\u003cstrong\u003e1\u003c/strong\u003e) was commercially available.\u003c/p\u003e\n\u003cp\u003eTMZ and OXA were commercially available and used as received for biological studies.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-9193345/v1/04ecbbbdbdfd6d6b716fa18f.png"},{"id":106960034,"identity":"3e418a6b-b4ea-4e80-9a12-c689b5157925","added_by":"auto","created_at":"2026-04-15 09:18:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":441712,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of the U87 cell line with different drug concentrations with different doses of photon radiation. Results are expressed as mean ± SD. * p\u0026lt;0.05; **p\u0026lt;0.01; *** p\u0026lt;0.001; **** p\u0026lt;0.0001 denote statistically significant group differences. C – control; TMZ – temozolomide; OLA – olaparib; OXA – oxaliplatin; Gy – Grey.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-9193345/v1/404a780fab1e0ca1967100d4.png"},{"id":107480522,"identity":"605707de-a57c-41c8-a759-444e679a15aa","added_by":"auto","created_at":"2026-04-22 02:11:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":480891,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of the U118 cell line with drug at different concentrations with different doses of radiation. Results are expressed as mean ± SD. * p\u0026lt;0.05; *** p\u0026lt;0.001; **** p\u0026lt;0.0001 denote statistically significant group differences. C – control; TMZ – temozolomide; OLA – olaparib; OXA – oxaliplatin; Gy – Grey.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-9193345/v1/7eb6c657c70c89987b7649b5.png"},{"id":106960197,"identity":"cbb7da92-1270-453c-844c-3e2461d11a23","added_by":"auto","created_at":"2026-04-15 09:19:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":501740,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of fibroblast cell line with different drug concentrations with different doses of radiation. Results are expressed as mean ± SD. * p\u0026lt;0.05; **p\u0026lt;0.01; *** p\u0026lt;0.001; **** p\u0026lt;0.0001 denote statistically significant group differences. C – control; TMZ – temozolomide; OLA – olaparib; OXA – oxaliplatin; Gy – Grey.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-9193345/v1/f3e33658c7ada7c3d930f94e.png"},{"id":106960582,"identity":"b0e97cbc-db52-45e2-b6db-7d8966cad44e","added_by":"auto","created_at":"2026-04-15 09:21:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":588028,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of three cell lines: U 118, U 87, and fibroblast, using drugs in the following combinations: 50 μM OLA with 50 μM TMZ, or 50 μM OXA with 50 μM TMZ, or 50 μM OXA with 50 μM OLA, and in a triplet 50 μM OXA+50 μM OLA+50 μM TMZ in combination with different radiation doses (0, 2, 4 Gy). Results are expressed as mean ± SD. C – control; TMZ – temozolomide; OLA – olaparib; OXA – oxaliplatin; Gy – Grey.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-9193345/v1/eb5a3ff39e9abb4d23a89a6e.png"},{"id":106785782,"identity":"abe32d70-ff3f-45b2-b3eb-fa22664f2296","added_by":"auto","created_at":"2026-04-13 12:34:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5830825,"visible":true,"origin":"","legend":"\u003cp\u003eType of cell death of three cell lines: U118, U87, and hFib. Fluorescence microscopy analysis of apoptotic and necrotic cells after 72 h of treatment.\u003cbr\u003e\nCells were stained with Annexin V-FITC and Ethidium Homodimer III (Biotium) and analyzed using a Zeiss Axio Imager Z2 microscope. Fig. 1A- hFib live cells, Fig. 1B- hFib apoptosis, Fig.1C- hFib necrosis, Fig. 2A- U87 GBM live cells, Fig. 2B- U87 GBM apoptosis, Fig. 2C- U87 GBM necrosis, Fig. 3A- U118 GBM live cells, Fig. 3B- U118 GBM apoptosis, Fig. 3C- U118 GBM necrosis.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-9193345/v1/89b2e42cc95da3222b31e99c.png"},{"id":107483315,"identity":"2eae374b-c627-42e1-8721-f915251c7378","added_by":"auto","created_at":"2026-04-22 02:27:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7599111,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9193345/v1/2f8e73d5-ad55-46be-b65c-64ec372b135c.pdf"},{"id":106785777,"identity":"d636aef3-904a-424e-b987-81841d3d2aa7","added_by":"auto","created_at":"2026-04-13 12:34:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":394956,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-9193345/v1/1e501357a1743b6d638579ac.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eThe Effectiveness of Multidrug Chemotherapy with Olaparib, Temozolomide and Oxaliplatin Compared to Radiotherapy in Glioblastoma Multiforme in Vitro Models and Human Fibroblasts\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eGlioblastoma multiforme (GBM) is the most common and aggressive brain tumor, with a high mortality rate and a five-year survival of only 7.2% [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Standard management includes surgical resection followed by radiotherapy (RT) combined with temozolomide (TMZ), yet its limited efficacy underscores the need for more effective therapeutic strategies [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Glioma stem-like cells (GSCs) have been identified as a key factor in GBM resistance, exhibiting high self-renewal, metastatic potential, and efficient DNA repair. These cells overexpress DNA damage response proteins, such as checkpoint kinase 1 (CHK1) and poly(ADP-ribose) polymerase 1 (PARP-1), which contribute to chemoresistance and radioresistance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. TMZ, a first-line chemotherapy agent for GBM, induces cytotoxicity via DNA methylation, creating lesions such as O6-methylguanine (O6-MeG), which trigger DNA double-strand breaks (DSBs) and tumor cell death through the mismatch repair (MMR) pathway [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. GBM frequently acquires resistance via MMR deficiency or overexpression of O6-methylguanine-DNA methyltransferase (MGMT), which repairs O6-MeG lesions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOther chemotherapeutic agents, such as oxaliplatin (OXA), a third-generation platinum compound, induce DNA cross-links and DSBs while presenting lower systemic toxicity compared to earlier platinum drugs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, tumor cells can evade cytotoxicity via homologous recombination and non-homologous end-joining repair pathways [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. PARP inhibitors, such as olaparib (OLA), interfere with DNA repair by trapping the PARP-DNA complex, leading to accumulation of DSBs and cell death. OLA has proven efficacy in tumors with homologous recombination deficiency (HRD), including certain isocitrate dehydrogenase (IDH) mutant GBM variants, and can enhance the cytotoxicity of alkylating agents even in HR-proficient cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Oxaliplatin has been primarily studied as a radiosensitizer in combination with radiotherapy in glioblastoma multiforme models, where it has been shown to potentiate radiation-induced cytotoxicity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Combination including radiotherapy and drugs, such as TMZ or PARP inhibitors, without including OXA in multi-agent radiotherapy regimens has already been studied [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In this research, OXA has been combined with repositioning chemotherapy (TMZ and OLA) at lower doses and radiotherapy. While other studies show only combinations of OXA with drugs or OXA alone with radiotherapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRadiotherapy remains a cornerstone of glioblastoma (GBM) management. In this study, in vitro irradiation was performed using a Cobalt-60 (Co-60) source [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. GBM cells were exposed to clinically relevant doses of 2 Gy and 4 Gy [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This experimental framework allowed further evaluation of potential synergistic interactions between radiotherapy and chemotherapy, including whether combined treatments enhance tumor cell killing while reducing toxicity compared to monotherapies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\n\u003ch3\u003e1. Cell lines and cell culture\u003c/h3\u003e\n\u003cp\u003eThe following GBM cell lines were used as preclinical \u003cem\u003ein vitro\u003c/em\u003e models [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. U118 MG (Cat. No. HTB-15), and U87 MG (Cat. No. HTB-14) were obtained from the American Type Culture Collection (ATCC).\u003c/p\u003e \u003cp\u003eA healthy donor human fibroblast line (hFib) served as a control [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Before the fragment of skin was taken, the participant signed written informed consent; the study protocol was reviewed and approved by the Human Bioethical Committee of the Regional Medical Board in Krak\u0026oacute;w (No. 163/KBL/OIL/2023). Details on cell lines and culturing are provided in the Supplementary Information (SI).\u003c/p\u003e\n\u003ch3\u003e2. Chemiotherapeutics\u003c/h3\u003e\n\u003cp\u003eTMZ (Cat. No. T2577) and OXA (Cat. No. PHR1528) were obtained from Sigma-Aldrich. The following synthesis of OLA was conducted following the literature data [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], with minor modifications, which are described in detail in the SI.\u003c/p\u003e\n\u003ch3\u003e3. Irradiation with a Cobalt-60 (Co-60) beam and dosimetry\u003c/h3\u003e\n\u003cp\u003eThe irradiation was performed using a Cobalt-60 (Co-60) source (Theratron 780E, Best Theratronics; activity 236.7 TBq on November 2, 2018) at the Institute of Nuclear Physics, Polish Academy of Sciences (IFJ PAN), Krakow, Poland. Photon beam dosimetry was conducted using a UNIDOS Webline electrometer (PTW-Freiburg, T10021) in combination with a Farmer-type ionization chamber (PTW-Freiburg, TM30010-1). Details on irradiation with a Co-60 beam are provided in the SI.\u003c/p\u003e\n\u003ch3\u003e4. Cell Viability Assessment of OLA, TMZ, OXA, and their Combination\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCell viability of TMZ, OLA, and OXA and their combinations with radiotherapy was evaluated using GBM cell lines seeded in 96-well plates at a density of 4 \u0026times; 10\u0026sup3; cells per well. Cells were treated with increasing concentrations of each drug individually or in combination with radiotherapy or alone as detailed in the SI.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e5. Cell death analysis\u003c/h3\u003e\n\u003cp\u003eCells (U118, U87, and human fibroblasts) were seeded in 6-well plates and following attachment, exposed to chemotherapeutic agents at various concentrations are provided in the SI. Immediately after drug addition, the plates were exposed to radiation using a cobalt-60 source (Theratron 780E, Best Theratronics), according to the manufacturer\u0026rsquo;s certificate. Irradiation was delivered as single doses of 2 Gy and 4 Gy. Detailed procedures and representative images are provided in the SI.\u003c/p\u003e\n\u003ch3\u003e6. Statistical analysis\u003c/h3\u003e\n\u003cp\u003eCells were subjected to drug treatment, radiotherapy (at single doses of 2 Gy or 4 Gy), or combined treatments. Cell viability was assessed 72 hours after treatment using the MTS assay, with absorbance measured at 490 nm.\u003c/p\u003e \u003cp\u003eStatistical analyses were conducted using GraphPad Prism version 10.6.1 (892) for Windows (GraphPad Software, Boston, Massachusetts, USA). The data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of at least three replicates. All data were tested for the assumptions of normality (the Shapiro-Wilk test). Two-way ANOVA of the variance was performed by the Tukey test. Statistical significance is indicated by asterisks (* p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003eError bars in all graphs represent the standard deviation of at least three replicates. Both chemotherapeutic treatments and irradiation doses (2 Gy and 4 Gy) were included in the statistical comparisons, allowing assessment of monotherapy and combined effects.\u003c/p\u003e"},{"header":"RESULTS","content":"\n\u003ch3\u003e1. Chemistry\u003c/h3\u003e\n\u003cp\u003eOLA was resynthesized using literature-based conditions with slight modifications [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly, the carboxylic acid component was first activated with O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) and then coupled with Boc-protected piperazine. Subsequent Boc deprotection afforded compound \u003cb\u003e2\u003c/b\u003e, which was finally acylated with cyclopropanecarbonyl chloride to yield OLA. This synthetic sequence provided isolated yields of 60\u0026ndash;91%. The structure and purity of the final compound were confirmed by chromatographic and spectroscopic analyses, as detailed in the SI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e2. Cell viability\u003c/h3\u003e\n\u003cp\u003eTo assess the effects of OXA, OLA, TMZ, and their combinations on cell viability, two human GBM cell lines were used: U87 (grade IV), U118 (grade III/IV). As a non-cancerous control, normal human fibroblasts (hFib) were used, obtained from a healthy donor. Details on cell lines and culturing are provided in SI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA two-way ANOVA using Type III sums of squares was conducted to examine the effects of radiation doses and treatment groups on cell viability. Unequal sample sizes were handled using Type III SS. The main effects of radiation were statistically significant, while treatment group as well as interaction between OLA 1 \u0026micro;M and OLA 100 \u0026micro;M or OXA 50 \u0026micro;M and OXA 100 \u0026micro;M or TMZ 10 \u0026micro;M and TMZ 100 \u0026micro;M were non-significant. Treatment group was analyzed as a categorical factor due to non-equivalent drug concentrations between compounds.\u003c/p\u003e \u003cp\u003eThe interaction between radiation and treatment group was not significant (F(\u003csub\u003e2, 30\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;0.5599, p\u0026thinsp;=\u0026thinsp;0.5771), indicating that the effect of radiation on cell viability was consistent across treatment conditions. There was a significant main effect of radiation dose (F(\u003csub\u003e2, 30\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;11.85, p\u0026thinsp;=\u0026thinsp;0.0002), demonstrating that radiation significantly altered U87 cell viability. In contrast, the main effect of treatment group was not significant (F(\u003csub\u003e1, 30\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;2.348, p\u0026thinsp;=\u0026thinsp;0.1360), suggesting that drug treatment alone did not significantly affect cell viability under the tested conditions.\u003c/p\u003e \u003cp\u003ePost hoc Tukey comparison was performer separately for each drug to examine the interaction between various drug concentrations and radiation doses. For OLA, radiation significantly reduced cell viability at higher concentration (100 \u0026micro;M), while lower concentration (1 \u0026micro;M) showed non-significant effect. For OXA, significant differences were observed between radiation doses at 50 \u0026micro;M. Last drug, TMZ at each of the concentrations showed non-significant effect with tendency to decrease of viability. These analyses demonstrate that the response to radiation depends on drug concentration within each compound (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCell viability of the U118 cell line was analyzed as a function of radiation doses and treatment groups using a two-way ANOVA with Type III sums of squares. All three effects \u0026ndash; the main effects of radiation doses and treatment groups, as well as their interaction \u0026ndash; were statistically significant.\u003c/p\u003e \u003cp\u003eA significant interaction between radiation and treatment group was observed (F(\u003csub\u003e14, 187\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;13.25, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating that the response to radiation differed across treatment conditions. There was a significant main effect of radiation (F(\u003csub\u003e2, 187\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;247.9, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with increasing radiation doses significantly reducing cell viability. A significant main effect of treatment groups were also observed (F(\u003csub\u003e7, 187\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;55.15, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), demonstrating that different treatment conditions significantly influenced cell viability. Treatment groups were analyzed as a categorical factor due to non-equivalent drug concentrations between compounds.\u003c/p\u003e \u003cp\u003ePost hoc Tukey comparisons were performed separately for each drug to examine the interaction between drug concentration and radiation dose. For OLA, radiation significantly reduced cell viability at both concentrations (100 \u0026micro;M and1 \u0026micro;M). For OXA, radiation significantly reduces cell viability at lower concentrations (50 \u0026micro;M and 100 \u0026micro;M), while at higher concentration no changes were observed. For TMZ, reduction of cell viability with radiation were observed at both concentrations (10 \u0026micro;M and 100\u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA two-way ANOVA using Type III sums of squares was conducted to examine the effects of radiation doses and treatment groups, and their interaction on fibroblast viability. All three effects \u0026ndash; the main effects of radiation and treatment group, as well as their interaction \u0026ndash; were statistically significant. Treatment groups were analyzed as a categorical factor due to non-equivalent drug concentrations between compounds. A significant interaction between radiation doses and treatment groups was observed (F(\u003csub\u003e14, 48\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;4.837, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating that the response to radiation differed across treatment conditions. There was a significant main effect of radiation (F(\u003csub\u003e2, 48\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;156.7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with higher radiation doses significantly reducing fibroblast viability. The main effect of treatment groups were also significant (F(\u003csub\u003e7, 48)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;18.79, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), demonstrating that different treatment conditions significantly affected cell viability.\u003c/p\u003e \u003cp\u003ePost hoc Tukey comparisons were performed separately for each drug to examine the interaction between drug concentrations and radiation doses. For OLA, radiation significantly reduced cell viability at both concentrations (1 \u0026micro;M and 100 \u0026micro;M). Second drug, OXA showed significant decrease in cell viability between radiation doses in concentration 50 \u0026micro;M, while in 100 \u0026micro;M and 200 \u0026micro;M no differences were observed. For TMZ, radiation significantly reduced cell viability at both concentrations (10 \u0026micro;M and 100 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCell viability in U87 cells was analyzed using a two-way ANOVA with radiation doses and drug treatment groups as factors (Type III sums of squares). No significant interaction between radiation doses and drug treatments was observed (F(\u003csub\u003e6, 34\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;2.012, p\u0026thinsp;=\u0026thinsp;0.0911), indicating that the effect of radiation on cell viability was largely independent of drug treatment groups. The main effect of radiation dose did not reach statistical significance (F(\u003csub\u003e2, 34\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;3.141, p\u0026thinsp;=\u0026thinsp;0.0560), although a trend toward reduced viability with increasing radiation dose was observed. In contrast, a significant main effect of drug treatment was detected (F(\u003csub\u003e3, 34\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;3.146, p\u0026thinsp;=\u0026thinsp;0.0376). Post hoc test followed by Tukey was conducted and indicate significant differences between radiation doses in group with triple combinations of drugs.\u003c/p\u003e \u003cp\u003eCell viability in U118 cells was examined using a two-way ANOVA with radiation dose and drug treatment as factors. Interaction between radiation dose and drug treatment was not statistically significant (F(\u003csub\u003e6, 24\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;1.262, p\u0026thinsp;=\u0026thinsp;0.3112). A significant main effect of radiation dose was observed (F(\u003csub\u003e2, 24\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;8.389, p\u0026thinsp;=\u0026thinsp;0.0017), as well as drug treatment (F(\u003csub\u003e3, 24\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;6.707, p\u0026thinsp;=\u0026thinsp;0.0019).\u003c/p\u003e \u003cp\u003eIn fibroblast group, cell viability was analyzed using a two-way ANOVA with radiation dose and drug treatment as factors. No significant interaction between radiation and drug treatment was detected (F(\u003csub\u003e6, 33\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;1.329, p\u0026thinsp;=\u0026thinsp;0.2721). Radiation doses have significant main effect on fibroblast viability (F(\u003csub\u003e2, 33\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;22.36, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) as well as drug treatment gropus(F(\u003csub\u003e3, 33\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;6.762, p\u0026thinsp;=\u0026thinsp;0.0011). Post hoc Tukey comparisons were performed and showed significant differences between radiation doses within the group treated with 50 \u0026micro;M OXA\u0026thinsp;+\u0026thinsp;50 \u0026micro;M OLA and in group 50 \u0026micro;M OXA\u0026thinsp;+\u0026thinsp;50 \u0026micro;M OLA\u0026thinsp;+\u0026thinsp;50 \u0026micro;M TMZ.\u003c/p\u003e\n\u003ch3\u003e3. Cell death analysis\u003c/h3\u003e\n\u003cp\u003eTwo-way ANOVA revealed a significant interaction between drug treatment (OXA, OLA, TMZ) and radiation (2 Gy, 4 Gy) for all tested cell lines, indicating that the cellular response (viability and death profiles) significantly differed between drug-only and combined treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Cells were subjected to drug (OXA, OLA, TMZ) treatment, radiotherapy (single doses of 2 Gy or 4 Gy) or combined treatments after 72 hours. The impact of TMZ, OXA, and/or OLA with radiotherapy on the type of cell death was assessed in two commercially available GBM cell lines (U118MG, U87MG). The human fibroblast line (hFib) obtained from a healthy donor was used as controls. The results for the compared groups were as follows: hFib, F70,216\u0026thinsp;=\u0026thinsp;408.5.0, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; U118, F70,216\u0026thinsp;=\u0026thinsp;787.9, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; and U87, F70,216\u0026thinsp;=\u0026thinsp;1224, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of TMZ, OXA, and/or OLA on the type of cell death in glioblastoma cell lines (U118 MG, U87 MG \u0026ndash; ATCC) and human fibroblasts (hFib, control, from a healthy donor).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"12\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eAnti-cancer drug\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c12\" namest=\"c4\"\u003e \u003cp\u003eEarly and late apoptosis, necrosis and lid live cells presented as [%] after 72 hours for each cell line\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eType of cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003ehFib\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003eU118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c12\" namest=\"c10\"\u003e \u003cp\u003eU87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRadiotherapy [Gy]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"3\" nameend=\"c2\" namest=\"c1\" rowspan=\"4\"\u003e \u003cp\u003eControl, without anti-cancer drug\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003elive cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLate apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003enecrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eOLA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e100\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLive cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLate apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNecrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eTMZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e100\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLive cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLate apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNecrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"11\" rowspan=\"12\"\u003e \u003cp\u003eOXA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e50\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLive cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLate apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNecrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e100\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLive cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLate apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNecrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e200\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLive cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLate apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNecrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"3\" nameend=\"c2\" namest=\"c1\" rowspan=\"4\"\u003e \u003cp\u003eOLA 50\u0026micro;M\u0026thinsp;+\u0026thinsp;TMZ 50\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLive cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLate apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNecrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"3\" nameend=\"c2\" namest=\"c1\" rowspan=\"4\"\u003e \u003cp\u003eOLA 50\u0026micro;M\u0026thinsp;+\u0026thinsp;OXA 50\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLive cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLate apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNecrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"3\" nameend=\"c2\" namest=\"c1\" rowspan=\"4\"\u003e \u003cp\u003eOLA 50\u0026micro;M\u0026thinsp;+\u0026thinsp;OXA 50\u0026micro;M\u0026thinsp;+\u0026thinsp;TMZ 50\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLive cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarly apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLate apoptosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNecrosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTreatment with 100 \u0026micro;M OLA induced apoptosis in hFib (early apoptosis: 47%, 45%, 22% vs. late apoptosis: 45%, 40%, 78%, respectively) without evidence of necrosis. In contrast, GBM cell lines showed no viable cells; necrosis predominated in U118 cells at 2 and 4 Gy (75%, 100%) and in U87 cell line at 2 and 4 Gy (17%, 28%), (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast, treatment with 100 \u0026micro;M TMZ caused 90% necrosis in hFib cells at 4 Gy, 50% in U118 and 100% in U87 cell lines. At 2 Gy, necrosis was observed only in the U87 cell line (94%) (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Treatment with 50 \u0026micro;M OXA without radiation induced early and late apoptosis in hFib cells (24% and 2%, respectively), with no necrosis observed. However, at 200 \u0026micro;M OXA, hFib cells exhibited necrosis (29%) and late apoptosis (51%), while U118 and U87 cells showed necrosis (39% and 5%, respectively) without radiotherapy. In the presence of 2 Gy and 4 Gy radiotherapy, the following effects were observed: after 72 hours of treatment with 200 \u0026micro;M OXA, complete necrosis (100%) were detected in both U118 and U87 cells, at both radiation doses.\u003c/p\u003e \u003cp\u003eCombination treatments with 50 \u0026micro;M OLA and 50 \u0026micro;M TMZ or 50 \u0026micro;M OLA and 50 \u0026micro;M OXA at 2 Gy and 4 Gy predominantly induced apoptosis without necrosis in hFib cell line. At both doses: 2 Gy and 4 Gy, the combination of 50 \u0026micro;M OLA and 50 \u0026micro;M TMZ led to necrosis in U118 (100%, 100%, respectively) and in U87 (89%, 100%, respectively). A similar pattern was observed for combination 50 \u0026micro;M OLA and 50 \u0026micro;M OXA in U118 cell line (95%, 100% necrosis at 2 Gy and 4 Gy). In contrast, hFib and U87 cells treated with 50 \u0026micro;M OLA and 50 \u0026micro;M OXA showed early and late apoptosis following irradiation at 2 Gy. In hFib cells, early and late apoptosis exhibited in 35% and 55% respectively whereas in U87 cells these values were 63% and 39%, respectively (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.).\u003c/p\u003e \u003cp\u003eThe three-drug combination 50 \u0026micro;M OLA, 50 \u0026micro;M TMZ, and 50 \u0026micro;M OXA resulted in early and late apoptosis without necrosis in all cell lines (20% and 23% in hFib, 46% and 45% in U118, 24% and 76% in U87). Following irradiation, necrosis was observed in GBM cells lines and in hFib cells, at 2 and 4 Gy, necrosis reached 50% and 65% in hFib cells, 80% and 100% in U118 cells, and 100% at both doses in U87 cells (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, the cytotoxic effects of alkylating drugs OXA with TMZ administered alone or in combination with the PARP inhibitor OLA were assessed in GBM cell lines and human fibroblasts in combination with photon radiotherapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The results showed that increasing therapeutic complexity through multidrug combinations and concomitant ionizing radiation was associated with a progressive decrease in GBM cell viability compared to hFib [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSimilar responses of U118 and U87 cell lines to drugs and radiotherapy combination were visualized in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, and illustrating the modulatory role of MGMT expression in drug resistance. U118 cells, characterized by high MGMT expressions, showed limited sensitivity to monotherapies but clearly responded significantly to combination therapies including OLA [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In contrast, U87 cells showed greater sensitivity to OLA\u0026thinsp;+\u0026thinsp;TMZ and to the triplet OLA\u0026thinsp;+\u0026thinsp;TMZ\u0026thinsp;+\u0026thinsp;OLA without RT [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Zajac-Grabiec et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] also reported a synergistic effect of OLA with TMZ in the GBM models, especially in the U87, H4, and U118 cell lines [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. OLA enhances the effect of TMZ, but not OXA, in glioma cell lines irrespective of MGMT status, as shown in previous reports [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In the present study, the drugs used alone and in combinations: OLA and TMZ or OXA and TMZ or OXA and OLA, as well as the triplet preparation of OXA, OLA and TMZ administrated with radiotherapy (2Gy, 4Gy), showed synergistic effects.\u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, enhanced activity is observed for the combination regimens, with higher cytotoxicity being caused by OLA\u0026thinsp;+\u0026thinsp;TMZ, OXA\u0026thinsp;+\u0026thinsp;TMZ, and OLA\u0026thinsp;+\u0026thinsp;OXA following radiotherapy compared to monotherapy, while the most pronounced reduction in cell viability is observed with the triple combination (OLA\u0026thinsp;+\u0026thinsp;OXA\u0026thinsp;+\u0026thinsp;TMZ) in all GBM models. These findings support the conclusion to confirm that synergistic accumulation of cytotoxic DNA damage results from the simultaneous inhibition of multiple DNA repair pathways. Specifically, DNA alkylation is induced by TMZ with OXA, whereas OLA, thereby preventing the removal of single-strand breaks and promoting their conversion into lethal double-strand breaks (DSBs) during replication [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This impairment of DNA repair is considered to underline the enhanced efficacy of multidrug regimens following photon therapy [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, the efficacy of all drugs OLA, TMZ, and OXA, alone was enhanced by radiotherapy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (single drug) and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e (drug combinations), both 2 Gy and 4 Gy increased cytotoxicity. This effect is likely attributable to increased susceptibility of tumor cells to radiation-induced DSBs when DNA repair pathways are pharmacologically impaired. The synergic interaction between chemotherapy and radiotherapy was found to be significantly stronger in U118 and U87 cells than in hFib cells, supporting the notion that GBM-specific genomic instability is associated with an increased dependence on DNA repair mechanisms that are selectively targeted by OLA, OXA, and TMZ [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of this study demonstrated a differential response between hFib cells as a control and glioblastoma multiforme cell lines (U118 and U87) to treatment with OLA, TMZ, OXA, their combinations, and radiotherapy. In particular, in this study, hFib cells predominantly underwent apoptosis, whereas GBM cells responded mainly with necrosis, especially when combined with ionizing radiation (2 Gy, 4 Gy). These findings underscore the sensitivity of GBM cells to combined cytotoxic stress and the relative resistance of hFib cells to necrotic cell death (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTreatment with 100 \u0026micro;M OLA, particularly after irradiation, induced an apoptotic response in hFib cells, with no evidence of necrosis. In contrast, GBM cells subjected to the same treatment did not exhibit viability or early apoptosis, and necrosis predominated the cell death profile (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, treatment with 100 \u0026micro;M TMZ caused significant necrosis of hFib cells only at the higher radiation dose (4 Gy). GBM cell lines showed greater susceptibility at the 2 Gy dose, particularly U87 cells, which showed 94% necrosis. The increased sensitivity of GBM cells demonstrated a synergistic interaction between DNA alkylation by TMZ and radiation-induced DNA damage, which impairs DNA repair pathways frequently mutated in GBM [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOXA treatment showed a dose-dependent response in hFib cells, with 50 \u0026micro;M predominantly inducing apoptosis without necrosis. In contrast, treatment with 100 and 200 \u0026micro;M OXA resulted in both necrosis and late apoptosis, particularly following irradiation. Notably, 200 \u0026micro;M OXA combined with 2 or 4 Gy caused 100% necrosis in both GBM cells. These findings support the observation that radiotherapy enhances the cytotoxic effects of OXA, particularly in tumor cells. Furthermore, when hFib cells were exposed to 50 \u0026micro;M OLA and TMZ combined with irradiation, apoptosis remained the dominant mechanism of cell death, with no necrosis detected. This indicates that at lower drug concentrations, hFib cells maintained controlled cell death pathways, even under combined chemotherapy and photon irradiation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In contrast, complete necrosis was observed in GBM cells under the same treatment conditions, indicating a higher sensitivity to combined chemotherapy and radiotherapy [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, OLA combined with TMZ or OXA induced apoptosis without necrosis in hFib cells, even following irradiation. In U118 and U87 cell lines, the same combinations caused extensive necrosis at doses of 2 Gy and 4 Gy, with U118 cells exhibiting almost complete necrosis and U87 cells displaying a mixed apoptotic-necrotic profile during chemotherapy and radiotherapy. These results indicate that hFib cells retained their capacity for programmed cell death after multiple drug administration, whereas GBM cells lost this regulatory control after exposure to combined stressors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe three-drug combination (OLA, TMZ, OXA) induced apoptosis without evidence of necrosis in all cell lines in the absence of photon irradiation. However, following irradiation necrosis occurred in all cell types. Moreover, hFib as a control group demonstrated a different viability profile, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Despite a moderate reduction in viability, cytotoxicity was significantly lower than that observed in GBM cell lines, even under multidrug treatment or in the presence of radiotherapy. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These findings highlight the potential of multidrug repositioning strategies (OXA, OLA) in combination with TMZ and photon therapy may bring a breakthrough, which will ultimately enable more effective and safer pharmacotherapy for patients [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough our results are preliminary, the observed synergistic effect of alkylating agents with combination of OLA and radiotherapy supports the proposal of a novel chemotherapy strategy for GBM. Further investigation of the synergistic effects of repositioning drugs (OXA, OLA) combined with TMZ or modern radiotherapy, such as FLASH, may lead to therapeutic breakthroughs that will ultimately enable more effective and safer pharmacotherapy for patients. Therefore, \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e molecular studies of OXA and OLA combined with TMZ and various types of radiotherapy in GBM in different models (i.e., 2D and 3D) are necessary and will be conducted in the next part of this ongoing project.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCHK1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echeckpoint kinase 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCNS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecentral nervous system\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDCM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edichloromethane\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDMF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edimethylformamide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDMSO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edimethyl sulfoxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDSBs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edouble\u0026ndash;strand breaks\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGBM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglioblastoma multiforme\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eGSCs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglioma stem\u0026ndash;like cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHATU\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehexafluorophosphate azabenzotriazole tetramethyl uronium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHBTU\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eO\u0026ndash;benzotriazole\u0026ndash;N,N,N',N'\u0026ndash;tetramethyl\u0026ndash;uronium\u0026ndash;hexafluoro\u0026ndash;phosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHCL\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehydrochloric acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehelical\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ehFib\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehuman fibroblast line\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHPLC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh\u0026ndash;performance liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHRD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehomologous recombination deficiency\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehalf maximal inhibitory concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIDH\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eisocitrate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMGMT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eO6\u0026ndash;methylguanine\u0026ndash;DNA methyltransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMMR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emismatch repair\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMTS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3\u0026ndash;(4,5\u0026ndash;dimethylthiazol\u0026ndash;2\u0026ndash;yl)\u0026ndash;5\u0026ndash;(3\u0026ndash;carboxymethoxyphenyl)\u0026ndash;2\u0026ndash;(4\u0026ndash;sulfophenyl)\u0026ndash;2H\u0026ndash;tetrazolium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enicotinamide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOLA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eolaparib\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOXA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoxaliplatin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eO6\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cb\u003eMeG\u003c/b\u003e\u0026ndash;O6\u0026ndash;methylguanine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePARP1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epoly(ADP\u0026ndash;ribose) polymerase 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePH\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eradiotherapy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esupplementary information\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTBTU\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eO\u0026ndash;(benzotriazol\u0026ndash;1\u0026ndash;yl)\u0026ndash;N,N,N',N'\u0026ndash;tetramethyluronium tetrafluoroborate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTEA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etriethylamine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTMZ\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etemozolomide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding statement:\u003c/strong\u003eThese studies were performed at Cyclotron Centre Bronowice IFJ PAN under the project Miniatura 8, No. 2024/08/X/NZ7/00625, funded by the National Science Centre, Poland, and partially funded from the state budget under the Ministry of Education and Science (Poland) program entitled Science for Society II, No. NdS-II/SP/0295/2023/01, total project amount 1 mln PLN. The synthesis and in silico study were financially funded by JU MC Funds (N42/DBS/000412).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest statement:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnna Zając-Grabiec*, conceptualization, data curation, funding acquisition, investigation, resources, methodology, roles/writing - original draft, writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eAnna Czopek*, investigation, data curation, visualization, methodology, funding acquisition, roles/writing - original draft, writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eFilip Michałkiewicz, software, formal analysis, validation, visualization.\u003c/p\u003e\n\u003cp\u003eTomasz Skóra, investigation.\u003c/p\u003e\n\u003cp\u003eBeata Biesaga, supervision.\u003c/p\u003e\n\u003cp\u003eMonika Bania, investigation.\u003c/p\u003e\n\u003cp\u003eKrzysztof Łukowicz, investigation\u003c/p\u003e\n\u003cp\u003eMarta Bałamut, investigation, methodology\u003c/p\u003e\n\u003cp\u003eDawid Krzempek, investigation, methodology\u003c/p\u003e\n\u003cp\u003eDominik Wiśniewski, investigation, methodology.\u003c/p\u003e\n\u003cp\u003ePaula Ajersch, formal analysis, validation, visualization.\u003c/p\u003e\n\u003cp\u003eJustyna Miszczyk, funding acquisition\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Human Bioethical Committee of the Regional Medical Board in Kraków (No. 163/KBL/OIL/2023, 06.07.2023). Written informed consent was obtained from the participant involved in the study before the fragment of skin was taken.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics Approval and Consent to Participate. The study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Human Bioethical Committee of the Regional Medical Board in Kraków (Approval No. 163/KBL/OIL/2023, dated 06.07.2023). Written informed consent was obtained from the participant prior to the collection of skin fragments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for publication was obtained from the participant.\u003c/p\u003e\n\u003cp\u003eData available with manuscript or supplementary information:\u003c/p\u003e\n\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZając-Grabiec A, Czopek A, Pazdan K, Jończyk J, Michałkiewicz F, Sk\u0026oacute;ra T, et al. 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Strahlenther Onkol. 2023;199(12):1140\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00066-022-02028-8\u003c/span\u003e\u003cspan address=\"10.1007/s00066-022-02028-8\" 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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pharmacological-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prep","sideBox":"Learn more about [Pharmacological Reports](https://link.springer.com/journal/43440)","snPcode":"43440","submissionUrl":"https://submission.springernature.com/new-submission/43440/3","title":"Pharmacological Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Olaparib, Temozolomide, Oxaliplatin, Glioblastoma Multiforme, Radiotherapy","lastPublishedDoi":"10.21203/rs.3.rs-9193345/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9193345/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction\u003c/h2\u003e \u003cp\u003e: Glioblastoma multiforme (GBM) remains one of the most treatment-resistant central nervous system (CNS) tumors. This study evaluated the efficacy of multidrug repositioning combining oxaliplatin (OXA), olaparib (OLA), and temozolomide (TMZ), administered alone or in combination with photon radiotherapy, in GBM cell lines as \u003cem\u003ein vitro\u003c/em\u003e models.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eU118 MG and U87 MG cells, as well as control human fibroblasts (hFib) from a healthy donor, were treated with OXA (50\u0026ndash;200 \u0026micro;M), OLA (1-100 \u0026micro;M), and TMZ (10\u0026ndash;100 \u0026micro;M), alone and in combination. Cell viability was assessed after 72 hours using the MTS assay, and apoptosis/necrosis was quantified using a fluorescent apoptosis, necrosis, and healthy cell quantification kit. Treatments were tested with (2 Gy, 4 Gy) and without irradiation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eRadiotherapy significantly enhanced the cytotoxic effects of all drugs, with the strongest reductions in viability observed for multidrug combinations. The addition of 2 Gy and 4 Gy irradiation markedly enhanced the activity of OLA\u0026thinsp;+\u0026thinsp;TMZ, OXA\u0026thinsp;+\u0026thinsp;TMZ, and OLA\u0026thinsp;+\u0026thinsp;OXA, leading to substantial loss of GBM cell viability. The greatest synergistic response has been observed with the triple drug combination (OLA\u0026thinsp;+\u0026thinsp;OXA\u0026thinsp;+\u0026thinsp;TMZ) plus radiotherapy, which produced extensive necrosis across all GBM models used.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe combination of OXA, OLA, and TMZ, especially when combined with photon radiotherapy, demonstrates potential synergistic cytotoxicity in GBM while sparing healthy fibroblasts. These results support the use of multidrug combination chemotherapy with radiotherapy as a promising and potentially safer treatment strategy for GBM.\u003c/p\u003e","manuscriptTitle":"The Effectiveness of Multidrug Chemotherapy with Olaparib, Temozolomide and Oxaliplatin Compared to Radiotherapy in Glioblastoma Multiforme in Vitro Models and Human Fibroblasts","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 12:34:03","doi":"10.21203/rs.3.rs-9193345/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-18T22:03:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92741989203534837439486360023725707254","date":"2026-05-07T16:12:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"134337719240747648941750179438513610649","date":"2026-05-07T01:39:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-07T06:53:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-26T17:24:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-26T12:44:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pharmacological Reports","date":"2026-03-22T19:11:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pharmacological-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prep","sideBox":"Learn more about [Pharmacological Reports](https://link.springer.com/journal/43440)","snPcode":"43440","submissionUrl":"https://submission.springernature.com/new-submission/43440/3","title":"Pharmacological Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d5759e56-0e1e-4a66-94ed-03d18ad0f224","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-18T22:03:35+00:00","index":196,"fulltext":""},{"type":"reviewerAgreed","content":"92741989203534837439486360023725707254","date":"2026-05-07T16:12:53+00:00","index":193,"fulltext":""},{"type":"reviewerAgreed","content":"134337719240747648941750179438513610649","date":"2026-05-07T01:39:07+00:00","index":188,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T12:34:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 12:34:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9193345","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9193345","identity":"rs-9193345","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}