Andrographolide and Berbamine Synergy in Glioblastoma Treatment: An Insight into the Pathways Assimilating Proteomics and Metabolomics

Andrographolide and Berbamine Synergy in Glioblastoma Treatment: An Insight into the Pathways Assimilating Proteomics and Metabolomics | 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 Andrographolide and Berbamine Synergy in Glioblastoma Treatment: An Insight into the Pathways Assimilating Proteomics and Metabolomics Vijeta Prakash, Manjula Kalia, reema gabrani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7585131/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Glioblastoma (GB) is one of the most aggressive and invasive cancers, characterized by poor survival rates and high recurrence. Standard treatments concerns about toxicity and long-term safety have led to growing interest in natural alternatives, such as phytotherapeutics. The study explores the effects of two phytocompounds: andrographolide, a diterpenoid, and berbamine, an alkaloid, on GB cells. Their cytotoxic effects were tested on GB cell lines LN229 and U-87 MG and HEK 293 cells to assess safety. Additionally, the impact of these compounds on wound healing, apoptosis (via Annexin V), cell cycle progression, oxidative stress, and mitochondrial dysfunction was evaluated in GB cells. The DSC, mass spectrometry and NMR along with ontology analysis were used to study the changes in protein structure and levels leading to changes in metabolism. The endothermic and exothermic events, highlighted shifts in thermal stability between treated and control cell lines. While, several proteins showed significant change in levels in response to the combination treatment of chosen phytocompound combination. Finally, the treatment with the combination caused significant changes in the metabolic profile of glioblastoma cells. The results highlight the strong potential of phytocompounds combination to target GB by targeting multiple pathways for GB. Statement of significance of the study Glioblastoma (GB) is one of the deadliest brain cancers. It has few treatment options and a poor prognosis because of tumor recurrence, resistance to therapy, and the toxicity of standard treatments. This study offers new insights into the potential of two plant compounds, andrographolide and berbamine, as a combination treatment for GB. Our results show that the combination produces stronger effects, increases cell death, raises mitochondrial oxidative stress, and causes cell cycle arrest, which reduces GB cell growth and movement. In addition, analysis through computer simulation revealed disruptions in tumor-related signaling pathways and metabolic processes. Proteomic and metabolomic profiling showed notable changes in energy metabolism, amino acid turnover, and lipid production. These findings suggest that the combination of these plant compounds targets several key features of glioblastoma. Moreover, it could disrupt the metabolism enough to overcome resistance to standard therapies. Importantly, safety tests on HEK293 cells indicate that the treatment selectively harms GB cells. Overall, this research highlights the importance of using natural compounds in multi-target treatment strategies. It points to andrographolide and berbamine as promising candidates for treating glioblastoma, which calls for more investigation. Apoptosis Cytotoxicity Metabolic reprogramming Mitochondrial dysfunction Oxidative stress Phytocompounds Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introduction Glioblastoma (GB), formerly known as glioblastoma multiforme has a very poor prognosis and lacks in efficient treatment options. The interventional therapies include treatments with temozolomide (TMZ) [ 1 ], nitrosoureas including lomustine and carmustine and bevacizumab in certain recurring cases [ 2 ]. The side effects of existing chemotherapy for GB pose a major matter of concern. For instance, TMZ is associated with adverse effects such as pneumocystis pneumonia [ 3 ], fetal harm and hepatotoxicity [ 4 ]. Therefore, a study on alternative therapies with fewer side effects must be studied. The therapeutic gap in treating glioblastoma (GB) can be bridged by utilizing phytocompound combinations, which may help overcome resistance and reduce the adverse effects commonly related to conventional chemotherapy combinations. Phytochemicals, which are natural compounds obtained from plant sources, have gathered significant consideration recently, because of their likely therapeutic applications in various diseases, including cancer [ 5 ]. To overcome the selective targeting of single phytocompounds, the use of combination phytotherapy in cancer treatment can potentially enhance anticancer efficacy and overcome drug resistance. Rather than targeting a single alteration in cancer cells, combination therapy aims to inhibit or modulate multiple relevant targets simultaneously [ 6 ]. There has been evidence of effective combination therapy of phytocompounds with TMZ such as α-pinene, where the combination effectively enhanced the cytotoxic effect of TMZ on GB cell lines and helped combat resistance [ 7 ]. The combined treatment of D-limonene and temozolomide demonstrated a synergistic effect on glioblastoma cells by targeting the proliferation of cells and inducing apoptosis, causing arrest during G0/G1 stages of the cell cycle, and suppressing cell migration [ 8 ]. It was also shown that the combined effects of bromelain and temozolomide showed synergistic effects by inhibiting cell growth, colony formation, and migration while triggering early apoptosis and arrest of GB cells during the G0/G1 phase [ 9 ]. Recently, a combination of 5-fluorouracil and thymoquinone have shown strong cytotoxicity against U-251 MG cell lines. A pair of phenolic acids, irinotekan and ellagic acid, led to cadherin inhibition, thereby promoting an anti-angiogenic process in C6 glioma cells [ 10 ]. Andrographolide and berberine are two specific phytochemicals that have shown promising effects in cancer. Andrographolide, a diterpenoid compound, is derived from a medicinal plant, named Andrographis paniculata [ 11 ]. Andrographolide has been found to exhibit potent anti-cancer properties, such as apoptosis induction, tumor growth suppression, and metastasis in the cell lines of gastric cancer and breast cancer [ 12 , 13 ]. On the other hand, berbamine is an alkaloid compound found in various medicinal plants like Berberis species. It exhibits broad anti-cancer properties, with its mechanism linked to modulating gene and protein expression, while inhibiting tumor growth and metastasis [ 14 – 16 ]. Differential scanning calorimetry (DSC) is used to assess the impact of the treatments on the thermal stability of the glioblastoma cell lines [ 17 ]. DSC provides information on structural and functional integrity of the cellular components, such as proteins and membranes, under different treatment conditions [ 18 ]. The observed thermal transitions reflect changes in the stability of cellular constituents, offering clues about the extent of molecular damage and stress induced by the treatment. Advances in mass spectrometry-based proteomics allow the identification of key protein biomarkers and can shed light on mapping of protein interactions within GB cells [ 19 ]. These studies have uncovered critical oncogenic pathways, such as those involving the EGFR [ 20 ], VEGF [ 21 ], and PI3K [ 22 ], all of which are frequently dysregulated in GB. By analyzing these proteins, researchers can also identify potential drug targets, paving the way for more precise, targeted treatments.. The use of mass spectrometry enables the detailed characterization of the proteome, highlighting key proteins involved in critical processes such as cell cycle regulation, apoptosis, and metabolic reprogramming [ 23 ]. Metabolomics, on the other hand, offers a complementary perspective by analyzing the global metabolic changes occurring in response to treatment [ 23 ]. Alongside proteomics, metabolomics studies small molecules and metabolites provide additional insights into GB’s biology [ 24 ]. Together, these approaches offer a comprehensive, system-wide view of glioblastoma at both molecular and functional levels. The current study has assessed the effectiveness of andrographolide and berbamine, both individually and in combination, on LN229 and U-87 MG cell lines of GB, focusing on proliferation and apoptosis of cells. Additionally, the safety of both the compounds and their combination has been tested on the HEK 293 cell line. Further, a combination of DSC, proteomics and metabolomics techniques were employed to gain a deeper understanding of the molecular and metabolic alterations induced by the compounds’ treatment in GB cells. Materials and Methods Cells and Reagent LN229, U-87 MG and HEK 293 cell lines were procured from NCCS, Pune, India. Mammalian cells were incubated under standard conditions (5% CO 2, 37°C). The cell lines were supplemented with Dulbecco’s modified Eagle medium (DMEM) with 10% FBS, along with penicillin, streptomycin, and gentamycin. Measurement of cytotoxicity The cytotoxicity of phytocompounds (andrographolide, berbamine) on chosen cell lines (LN229 and U-87 MG) was checked through 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay MTT assay as described before by Gautam et al. The cells (1 X 10 5 cells per mL) were treated with the test compounds (3.1–50 µM) in a 96-well microtiter plate. MTT solution (5mg/mL) was added and the plate was kept in the incubator (37°C, 5% CO 2 ) for 4 h. After incubation, the media was decanted, and each well was filled with DMSO (200 µL) to dissolve the formazan dye [ 6 ]. The reading of optical density was taken at 570nm, and the percentage of growth inhibition upon treatment with phytocompounds concerning control cells was determined using the equation $$\:\text{V}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}\:\text{o}\text{f}\:\text{c}\text{e}\text{l}\text{l}\text{s}\:\left(\text{\%}\right)=\frac{\text{T}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}\:\text{c}\text{e}\text{l}\text{l}\text{s}-\text{b}\text{l}\text{a}\text{n}\text{k}\:}{\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:\text{c}\text{e}\text{l}\text{l}\text{s}-\text{b}\text{l}\text{a}\text{n}\text{k}}\:\text{X}\:100$$ Combination index calculation Using CompuSyn software, the Chou and Talalay approach [ 17 ] was used to identify combination index (CI) values. The andrographolide and berbamine were combined in a set concentration ratio according to their IC 50 values, and the viability of LN229 and U-87 MG cells was studied through the MTT assay. The treatment was considered synergistic when the CI value came out to be less than 1[ 17 ]. The dose-reduction index, or DRI, was calculated, wherein, its value exceeding one signifies a reduction in the total concentration of the drug [ 17 ]. Cell safety of the combination The safety of the concentration of compounds on the non-transformed cell line was studied for the single compounds and combination on HEK 293 cell line. The phytocompounds andrographolide and berbamine were individually tested on HEK 293 cells at concentrations ranging from 156.25 to 5000 µM. Additionally, the compounds were combined at their IC 50 values, serially diluted, and tested on HEK 293 cells to assess toxicity using the MTT assay [ 25 ]. Wound closure capability The migratory capacity of LN229 and U-87 MG cells was assessed by the wound healing assay. Cells were incubated at a density of 2 X 10 5 per mL in 6-well plates for 24 h. The media was aspirated, and a straight-line scratch was made using a sterile tip. The wells were then washed with PBS to remove any debris [ 26 ]. The cells were treated with respective IC 50 values from a single or combination of andrographolide and berbamine for 48 h under standard conditions. The wells were photographed at 0, 24, and 48 hours using field microscopy to estimate the migrated area and analyze gap closure. The distance of the wound was measured and calculated by ImageJ software in both treated and untreated cells. Annexin V assay The LN229 and U-87 MG cell lines were plated at a density of 5 x 10 5 cells per well in a 6-well plate. The cells were exposed to varying concentrations of andrographolide, berbamine, and their combination, and then incubated for 48 hours. The cells incubated without treatment were considered as controls. Next, the cells were trypsinized, and 1 × 10 6 density of cells were treated with Annexin V & Dead Cell Reagent, followed by a 20-minute incubation at RT. Apoptosis was then assessed using Muse® Cell Analyzer [ 27 ]. Caspase3/7 To evaluate caspase 3/7 activity, firstly cell suspension was prepared by centrifuging trypsinized cells. After this, 1X Assay Buffer BA was added to each sample as per manufacturer’s instructions. Subsequently, the working solution of Muse™ Caspase-3/7 Reagent was supplied to each tube and mixed thoroughly. The tubes were incubated for 30 mins (37°C, 5% CO 2 ) with the working solution of Muse™ Caspase 7-AAD. Finally, the tubes were incubated at RT, shielded from light, for 5 mins before being analyzed with the Muse™ Cell Analyzer. Examination of Oxidative stress MitoSox The LN229 cells were subjected to cell cytometry after incubation with Mitosox dye. Reactive oxygen species are known to oxidize the dye, which subsequently binds to DNA, eventually producing fluorescence. To assess the ROS production in cells post-treatment, the control and treated cells were incubated in 6-well plates for 48 h. The cells were given a wash with PBS and MitoSOX dye (5µM) was administered in the wells for 30 mins. The cells were trypsinized, resuspended in PBS, and emission was measured at 580nm through BD FACSVerse™ Cell Analyzer [ 28 ]. Rhodamine 123 To evaluate the effects of phytocompound treatment on cellular oxidative stress, control and treated cells were incubated in a 6-well plate for 48 h. Later, the cells were washed with PBS, and rhodamine 123 dye (5 µg/mL) was added to each well, followed by incubation for 30 mins. Afterward, the cells were washed with PBS, trypsinized, resuspended in PBS, and analyzed by BD FACS Verse for fluorescence at 535nm of wavelength [ 29 ]. Cell Cycle Assay The Muse™ cell cycle assay was performed after a 48-hour incubation period post-treatment. Cells (LN229) were seeded at 2 x 10 4 cells/well density and incubated in complete media at 37°C with 5% CO 2 . Cells were treated with andrographolide and berbamine singly as well as in combination at IC 50 concentrations for 48 hours, washed with PBS, and centrifuged. The pellet was resuspended in PBS and fixed in 70% ethanol at -20°C until analysis. After centrifugation, 200 µL of cell cycle reagent was added, incubated for 30 minutes, and analyzed via Muse™ Analyzer [ 30 ]. Statistical analysis The Tukey Honestly Significant Difference (HSD) post hoc test calculator was utilized to perform one-way ANOVA statistical analysis ( http://www.astatsa.com ). A statistical comparison was conducted between the GB cells treated with the combination of andrographolide and berbamine, and control or single compound-treated cells for all the assays. The differences in treatments were statistically significant, with p-values less than 0.01 and 0.05. Swiss Target Prediction Analysis The Swiss Target Prediction tool was used to predict target proteins based on their similarity to known bioactive agents [ 31 ]. The chemical structures of andrographolide and berbamine were uploaded into the tool, and the predicted target proteins were analyzed for their potential involvement in the therapeutic effects exerted by andrographolide and berbamine. STRING Analysis The protein-protein interactions of the predicted targets for andrographolide and berbamine were explored using the STRING database. The database focuses on protein-protein interaction networks, considering direct and indirect interactions. A high confidence score threshold of 0.9 was applied, and the potential functional roles and molecular mechanisms of the probable proteins involved in interaction with andrographolide and berbamine were determined [ 32 ]. WEB-based Gene Set Analysis Tool Analysis The WEB-based Gene Set Analysis Tool (Web Gestalt) was used to understand the involvement of the targets determined for andrographolide and berbamine inhibitory action in specific cellular pathways. The list of proteins was subjected to WebGestalt to analyse GO terms (biological processes, cellular components, and molecular function) and study KEGG pathways. The predicted target proteins were uploaded into WebGestalt, and the KEGG pathway analysis revealed the biological processes and molecular interactions associated with the interacting proteins of andrographolide and berbamine [ 33 ]. Differential Scanning Calorimetry The Hitachi DSC67000X, differential scanning calorimeter (DSC) was used to examine how the andrographolide-berbamine combination treatment affected the LN229 cell line. Following a 48-hour treatment with the combination, the LN229 cells were isolated and lysed based on the freeze-thaw method (5 incubation cycles of -80°C and 37°C for 10 mins each) then resuspended in PBS and analyzed [ 34 ]. Thermal transitions, which can reveal information on the stability and structural changes of biological constituents, including proteins and lipids, in response to therapy, were measured using the DSC67000X. After loading cell samples into DSC pans, temperature scans were carried out in a nitrogen atmosphere under regulated conditions, ranging from low to high temperatures. The heat flow, endothermic and exothermic events, which represent modifications in the thermal characteristics of the cells after treatment, were recorded. In order to identify any notable variations in thermal stability between the control and treated cell lines, the generated thermograms were examined. Proteomics analysis Sample Preparation Cells (2 X 10 7 ) (control and treated) were grown in a T-75 flask and treated with the andrographolide-berbamine combination. After 48 hrs of treatment, the flask was rinsed with 10 mL PBS. The cells were scraped using a cell scraper and spun at 1500 rpm at 4°C. The pellet was then subjected to RIPA lysis buffer. Protein samples were separated using SDS-PAGE with a 15% separating gel and a 4% stacking gel. The gel was stained overnight and destained to remove the background and eventually visualised for the separation of bands. Excised protein bands were cut into ~ 1x1 mm pieces and washed three times with 50% acetonitrile in 50 mM ammonium bicarbonate to remove the stain. Gel pieces were then dehydrated in 100% acetonitrile, reduced with 5 mM DTT, and alkylated with 20 mM iodoacetic acid. Trypsin digestion was carried out using sequencing-grade trypsin (1 µg of trypsin per 50 µg of protein) overnight at 37°C. Peptides were extracted twice with 60% acetonitrile and 0.1% formic acid, pooled, and dried using a centrifugal evaporator. Desalting was performed using Zip-Tip C18 as per standard procedures [ 35 ]. Mass Spectrometry Dried peptides, obtained after trypsin digestion, were reconstituted in 2% acetonitrile and 0.1% formic acid and injected into a TripleTOF® 5600 + mass spectrometer (ABsciex) with a ChromeXP C18 column. The peptides were eluted using a dual solvent system: Reservoir A (98% water, 2% acetonitrile, 0.1% formic acid) and Reservoir B (98% acetonitrile, 2% water, 0.1% formic acid). The flow rate was set to 5 µL/min, and the total run time was 87 minutes. Mass spectra were acquired in the range of 350–1250 m/z for precursor ions, followed by MS/MS scanning of fragment ions in the range of 150–1600 m/z. Instrument settings included an ion spray voltage of 5500 V, nebulization gas (GS1) at 25 psi, and curtain gas (CUR) at 30 psi, ensuring stable ionization and data acquisition [ 35 ]. Mass Spectrometry Data Processing and Analysis MS Data Processing and Analysis (version 2.7.0, Matrix Science) was used to process the raw data obtained from the mass spectrometer. The software compared the data to known protein sequences listed in the Swiss-Prot database (check it out at https://www.uniprot.org/ ). The possibility that the enzyme used to prepare the samples might have missed cleaving the protein at up to two spots was also considered. The change in abundance of protein was investigated in the treated group in comparison to the control group using a trial version of a software program called Ingenuity Pathway Analysis (IPA) from QIAGEN (USA). A list of identified proteins was uploaded for analysis using their unique Swiss-Prot IDs. The results indicated whether the abundance of each protein increased or decreased in the extract-treated group compared to the control group. IPA constructed networks of interacting proteins highlighting the most significant biological pathways. Additionally, IPA revealed how networks differed between the control and combination-treated groups. Finally, the specific interactions between proteins within these networks were examined with ontology analysis [ 35 ]. NMR Intracellular Metabolite Extraction for Metabolomics Analysis A dual-phase methanol/chloroform extraction method separated aqueous and non-polar metabolites from cell proteins. Dried methanol-quenched cell pellet samples were kept on ice throughout the extraction process. To each cell pellet (obtained by centrifuging 1 X 10 7 cells per mL), 300 µl of a chloroform/methanol mixture in a 2:1 ratio was added and thoroughly mixed by vortexing. Following this, 300 µl of HPLC/UPLC-grade water was introduced to the sample, which was again mixed by vortex and then centrifuged at 16,000 g for 5 minutes to facilitate phase separation. Culture medium samples were stored at -80°C immediately after harvesting. A volume of 550 µl of the thawed medium sample was transferred to a fresh eppendorf tube, to which 50 µl of internal standard DSA (4,4-Dimethyl-4-silapentane-1-ammonium trifluoroacetate) in D₂O (11.6 mM) was added as a quantitative reference. The mixture was then transferred to a standard 5 mm NMR tube for analysis. Following the methanol/chloroform/water dual-phase extraction, the aqueous fractions were dried using a freeze-dryer. The dried samples were then reconstituted in 600 µl of phosphate buffer, composed of 0.2 M Na₂HPO₄, 0.043 M NaH₂PO₄, 100 µM TSP, and 3 mM NaN₃ in 100% D₂O. After reconstitution, the samples were centrifuged at 16,000 g for 5 minutes to remove any insoluble material and supernatant was transferred to a clean 5 mm NMR tubes for further analysis [ 36 ]. ¹H NMR Experiment Acquisition and Metabolite Identification: High-resolution ¹H NMR spectra were acquired using either a 5 mm broadband-inverse probe or a 5 mm cryoprobe, both mounted on a 14.1 T Bruker AVANCE 600 spectrometer (Bruker Biospin). A 3-second relaxation delay was incorporated into the acquisition, and gradient shimming was performed before all acquisitions to ensure optimal magnetic field homogeneity [ 36 ]. Metabolites were identified based on signal peaks observed in the spectra, with reference to databases such as the Human Metabolome Database (HMDB) and the Biological Magnetic Resonance Bank (BMRB), or through comparisons with previously published literature [ 36 ]. Results and Discussion Cytotoxic effect of andrographolide and berbamine The effect of andrographolide and berbamine were studied alone and in combination against the GB cell lines. The IC 50 values resulting from the study in LN229 cells were 11.4 µM for andrographolide and 6.3 µM for berbamine (Table 1), whereas their IC 50 values were higher in U-87 MG cells (Table 1). The CI plot and dose-effect curve revealed a better impact with the combination of andrographolide and berbamine on GB cells, revealing a synergistic action compared to the individual administration. The CI plot revealed the graphs having values < 1 in case of both cell lines, implying that the combination of compounds is synergistic in its inhibitory effect on GB cells (Fig. 1 (A-D)). A synergistic phytocompound combination can maintain efficacy and safety by lowering the dosage of the phytocompounds in treatment. The DRI values indicated a dose reduction for both compounds when used in combination, as the DRI values were greater than 1 (Fig. 1E and F). As observed in our data for LN229 cell line, the DRI values of andrographolide ranged from 3.6 to 6.9 whereas that of berbamine ranged from 4.0 to 6.7. The DRI values in our experiment demonstrated the significant advantages of combining andrographolide with berbamine [ 37 – 39 ] since DRI values greater than 1 depict successful dose reduction of the phytocompounds as demonstrated by Fu et al., 2016 [ 40 ].To the best of our knowledge, andrographolide and berbamine combination has not been reported on GB cells previously. Yang et al., reported that the cytotoxicity of C6 glioma upon treating with andrographolide resulted in IC 50 of approximately 15 µM, which strongly supports our finding indicating that andrographolide exhibits anticancer properties in GB treatment by reducing cell viability [ 41 ]. The choice of berbamine as an inhibitory compound is substantiated by its anti-angiogenic property on Human Umbilical Vein Endothelial Cells (HUVEC) [ 42 ]. Moreover, it has been studied iwith arcyriaflavin A against C6-drived glioblastoma stem-like cells [ 43 ]. The safety of compounds was studied on HEK 293 cell lines, and the IC 50 obtained for andrographolide and berbamine were 616µM and 380 µM, respectively. Nonetheless, the IC 90 concentration of compounds in combination obtained in LN229 and U-87 MG did not show cell cytotoxicity on HEK 293 cell line as depicted in Fig. 2. Table 1 Cytotoxic effect of andrographolide and berbamine on LN229 and U-87 MG cell line Cell line Value Andrographolide (µM) Berbamine (µM) Combination of Andrographolide and Berbamine Combination Index (CI) value Andrographolide (µM) Berbamine (µM) LN229 IC 50 11.4 6.3 3.2 1.6 0.53 IC 75 27.1 14.3 6.3 3.1 0.45 IC 90 64.2 32.4 12.5 6.2 0.38 U-87 MG IC 50 92.6 95.1 30.8 15.4 0.49 IC 75 198.4 717.7 82.7 41.4 0.47 IC 90 425.1 5415.0 222.0 111.0 0.54 Wound Healing The migratory effect of andrographolide and berbamine through wound closure property, was analysed in both the GB cell lines (Fig. 3 and Fig. 4). The andrographolide-berbamine combination resulted in 16% and 44% wound closure in the case of LN229 and U-87 MG cells, respectively, after the treatment (Fig. 5(B)). The migratory capacity of cells is a major cause of concern while studying the treatment of tumors. In case of GB, the cells function as single cells, further leading to invasion through the mesenchymal mode. The migration of cells is a major cause of recurrence in tumor cases even after treatment or resection [ 44 ]. The difference in migration inhibition by the combination of andrographolide and berbamine was statistically significant compared to individual compound treatment and control. Annexin V assay The apoptotic effect of andrographolide and berbamine combination in LN229 and U-87 MG cells was tested by MUSE Annexin V assay kit. The percentage of live cells in the control was 94.5% and it decreased to 69.6%, 72.2% and 48% after treatment with andrographolide, berbamine and the combination at their respective IC 50 concentrations, respectively, for LN229 cells as shown in Fig. 6 (A, C-F). Similarly, live cells were least in percentage after treatment with andrographolide and berbamine combination in U-87 MG cells as shown in Fig. 6 (B, G-J). The dead/late apoptotic cell population was 50.1% and 54.5% for combinatorial treatment in LN229 and U-87 MG cells, respectively. Apoptosis occurs due to biological and morphological alterations, including condensation of chromatin and subsequent DNA degradation [ 45 ]. In the later stages of apoptosis, there is a notable increase in the caspase-3/7 activation, poly (ADP-ribose) polymerase (PARP) cleavage, fragmentation of DNA, and cell membrane disruption. Following treatment with the combination of phytocompounds, there is a rise in the number of cells undergoing late-stage apoptosis, clearly indicating DNA cleavage and damage resulting from cellular dysfunction [ 46 ]. Because some metabolic enzymes likely remain active during the initial stages of apoptosis, the effects observed in cells at an early stage of apoptosis may be reversible and potentially recoverable [ 47 ]. Therefore, a prominent rise in late apoptotic phase cells imply better targeting of GB cells with the combination of compounds [ 48 ]. For instance, Lee et al., revealed through Annexin V that podophyllotoxin mediated apoptosis in cells of colorectal cancer in humans by p38 signalling [ 49 ]. Similarly, venetoclax led to apoptosis of human breast cancer cell lines through Bcl-2 inhibition [ 50 ]. The combination of berbamine and arcyriaflavin has been demonstrated to activate the apoptosis pathway due to the cytochrome c release (a pro-apoptotic factor), from the mitochondria [ 51 ]. Caspase 3/7 Caspases are key regulators of apoptosis, initiating and executing the process through the action of protease enzymes [ 46 ]. Since the pattern of apoptosis results using Annexin V was similar for both cell lines, we focused on confirming the role of caspases in apoptosis by treating only LN229 cells with the IC 50 values of the compounds and their combination. The apoptotic population of LN229 cells increased from 0% in control to 37% after andrographolide-berbamine treatment, while it was 2% and 1% when treated singularly with andrographolide and berbamine, respectively (Fig. 7). A berbamine derivative has been shown to exhibit apoptosis via activation of the caspase-3 cascade [ 47 ]. Andrographolide also triggered apoptosis in C6 glioma cells by PARP-dependent caspase 7 signalling [ 48 ]. This strongly indicates that caspase-dependent apoptosis would be characteristic of andrographolide-berbamine treatment. Executioner caspases, such as caspase-3, -6, and − 7, play a pivotal role in the terminal stages of apoptosis by dismantling cellular structures and ensuring cell death. In cancer, dysregulation or inhibition of these caspases allows cells to evade apoptosis, leading to uncontrolled proliferation and tumor growth. Oxidative stress analysis Mitochondrial oxidative stress analysis The role of oxidative stress in inducing cell death in LN229 cells upon treatment with andrographolide-berbamine was studied through Mitosox. The flow cytometry results, as shown in Fig. 8 depict a shift of the histogram confirming the mitochondrial ROS increased from 4.5% to 14.7% upon treatment with the combination of andrographolide-berbamine [ 49 ]. The results depict that the treatment has led to an increase in oxidative stress in GBM cells. Mitochondria serve as a marker for tumor cell survival due to their production of ROS. ROS are pivotal in regulating cellular physiology, while low levels are crucial for processes such as redox signaling that support normal cell function [ 50 ]. Wen et al., reported that high levels of mitochondrial ROS can induce cytotoxic effects, which is suggestive of impairment of glioblastoma cell survival and tumor growth. Wen et al., also utilised MitoSox assay to report the mitochondrial ROS production, where they found that ROS in mitochondria significantly increased after treating GB cells with a compound isoaaptamine [ 51 ]. Mitochondrial Membrane Potential assay The impact of the andrographolide-berbamine combination on mitochondrial membrane potential (MMP) of LN229 via rhodamine assay is depicted in Fig. 9. The treatment resulted in a substantial lowering of mitochondrial membrane potential in LN229 cells, evident by the shift in the peak due to the treated sample. During cell apoptosis, the intracellular fluorescence intensity of rhodamine 123 decreases due to increased mitochondrial membrane permeability. This results in reduced mitochondrial membrane potential (MMP) and decreased uptake of Rhodamine 123. MMP, a key component and a hallmark of cell apoptosis, decreases in the early stages of programmed cell death. One of the primary causes of the decline in MMP is the opening of mitochondrial permeability transition pore (MPTP) [ 52 ]. ROS is known to use a positive feedback loop to start and speed up the opening of MPTP. It causes pro-apoptotic components to be released into the cytoplasm, leading to an irreversible decline in MMP as they exit the mitochondrial matrix leading to apoptosis [ 53 ]. Cell cycle assay The effect of the combinatorial treatment of andrographolide-berbamine on the LN229 cell line revealed arrest of the cell cycle in both G0/G1 and S phases. The treatment led to restriction on GBM cell growth, resulting in cell death, which could be related to arrest in different phases of cell cycle, as shown in Fig. 10. The cells in S phase rose from 16% in the case of control to 34% upon combinatorial treatment. The results presented in this paper suggest that the treatment, particularly the combination of andrographolide-berbamine, inhibited the cell cycle before they enter into the replication and division phases, thereby effectively preventing cell proliferation. Huang et al. observed a marked S phase cell cycle arrest in the neuronal cells after aflatoxin treatment, which was related to the upregulation of certain types of cyclin-dependent kinases [ 54 ]. Moreover, Song et al. reported arrest of HepG2 liver cancer cells in S phase leading to cell death, which was related to the upregulation of Bax, cytochrome C, and p53, and the downregulation of Bcl-2 [ 55 ]. In silico analysis of effect of phytocompounds on cell lines Target identification through Swiss Target prediction The possible mechanism of action of andrographolide-berbamine was investigated by Swiss Target prediction tool [ 56 ]. Figure 11 shows the interaction of andrographolide and berbamine with several classes of proteins. The results depict that the highest proportion of proteins involved with andrographolide actions are kinases, which encompass 20% of all the protein classes (Fig. 9A). The expression levels of CDKs fluctuate cyclically throughout the cell cycle, influencing various cellular processes. Numerous cyclins and the CDKs 2, 4, and 6 progress the cell towards the end stage of the cell cycle and consequently encourage cell growth. GB tumors have higher levels of CDK2 expression than normal brain tissue, leading to tumour proliferation and poor prognosis [ 57 ]. MAP kinases, including ERKs, JNKs, and p38/SAPKs, are upregulated within protein kinase cascades that control the cell growth processes [ 58 ]. These kinases phosphorylate specific target proteins, leading to changes in cellular behavior. Numerous pathways, including Ras/MAPK triggered by EGFR, get activated to provide the ambient microenvironment for GB growth, which can be targeted for effective treatment [ 59 ]. In the context of apoptosis, kinases such as JNK and p38 MAPK are activated in response to stress stimuli [ 60 ]. They mediate the signaling pathways that induce cell death and control the balance between cell viability and programmed cell death [ 61 ]. A hypoxic environment can trigger SRC proto-oncogene non-receptor tyrosine kinase and lead to activation of pathways associated with radioresistance and invasion in glioblastoma. It underscores the role of hypoxia and ionizing radiation in driving tumor infiltration. SRC also promotes the invasiveness and malignancy of GB [ 62 ]. While, in case of berbamine, 60% of the proteins involved were of family G protein-coupled receptor (GPCR) (Fig. 9B). The involvement of GPCRs has been observed with phytochemical treatment in different cases [ 63 ]. GPCRs are pivotal in cell cycle regulation by activating checkpoint proteins like p53 in response to DNA damage or stress [ 64 ]. They can alter cyclin and CDK levels, leading to arrest of the cell in the G1 stage of the cell cycle by downregulation of a cyclin D1. GPCRs also increase CDK inhibitors (e.g., p21, p27), which leads to blocking cyclin-CDK complexes and stopping cell cycle progression [ 65 ]. Additionally, GPCRs influence growth factor receptors, impacting cellular proliferation and potentially inducing cell cycle arrest [ 66 ]. Moreover, GPCRs activate apoptotic pathways through MAPK/ERK, PI3K/Akt, and JNK signaling, regulating Bcl-2 family proteins to promote apoptosis [ 67 – 68 ]. The involvement of GPCRs has been observed to target GB cells through mast cell activation, which can influence angiogenesis and recruit other immune cells to increase the effectiveness of treatment [ 69 ]. Ontology analysis through Web Gestalt The list of protein interactors of andrographolide and berbamine was subjected to Venny 2.1 to find out the common proteins involved. The andrographolide and berbamine seemed to affect the cell communication and developmental processes the most. The cellular component organizations and localization seemed to be linked with 13 proteins (Fig. 12). The analysis revealed that cellular components such as vesicles, nuclei, cytosol, the endomembrane system, lumens, and the cytoskeleton may be significantly affected, thereby hampering their integrity. Analyses of the molecular functions revealed protein binding, nucleotide binding, transferase activity and ion binding were enriched by both the compounds. Changes in thermal properties of GB cells analysed through differential scanning calorimetry The DSC results (Fig. 13) revealed how glioblastoma cells responded to different treatments by analyzing their thermal behavior. In the untreated control cells, heat flow gradually decreased with rising temperature, indicating a steady absorption of energy without major structural disruptions until around 55°C. Andrographolide showed more pronounced heat flow decrease between 55°C and 60°C (Fig. 13 A). The data suggests moderate destabilization, likely due to the compound’s effects on proteins and membrane integrity. While the changes indicate some level of thermal stress, they are not drastic, implying that andrographolide alone may only partially compromise glioblastoma cell stability. This aligns with andrographolide’s known biological actions, including interference with cellular signaling and metabolism [ 70 – 71 ]. While berbamine treatment caused a more dramatic shift in the DSC profile, with a steep drop in heat flow beginning around 50°C. This graph indicates significant cellular destabilization, marked by early protein denaturation and membrane disruption. Berbamine’s potent apoptotic properties have been demonstrated through its activation of the p53-dependent apoptotic signaling pathway in colorectal cancer cells [ 72 ], as well as its ability to disrupt mitochondrial membrane potential and activate caspases in hepatoma cells [ 73 ]. These mechanisms are closely linked to its influence on calcium homeostasis, membrane stability, and intracellular signaling, contributing to its pronounced anticancer effects. The extensive structural damage observed suggests that berbamine exerts a much stronger destabilizing influence on glioblastoma cells than andrographolide alone. The combination of andrographolide and berbamine resulted in the most severe thermal destabilization (Fig. 13). Heat flow began to decline sharply as early as 45°C, signaling widespread molecular disruption. The pronounced endothermic dip around 55°C suggested extensive protein denaturation and membrane breakdown, likely due to the combined impact of both compounds on multiple cellular pathways. The synergistic effect of andrographolide and berbamine overwhelmed the cells’ ability to maintain structural integrity, making the combination treatment particularly effective at targeting glioblastoma cells. By tracking changes in heat flow as temperature increases, these results provide valuable insights into the structural and molecular stability of the cells, particularly about protein denaturation and membrane integrity [ 74 ]. Proteomics study through mass spectrometry The analysis of log2ratio values for various proteins has provided a comprehensive overview of changes in their expression levels, with a negative log2ratio signifying downregulation compared to control conditions [ 75 ]. The screened results revealed that certain key proteins (Table 2) involved in metabolic, structural, and stress-response pathways were significantly up/downregulated, suggesting widespread cellular alterations in response to treating andrographolide-berbamine combination. Table 2 The log2fold change of proteins expressed in treated vs that in control Protein name Short form log2fold Aldolase A ALDOA -1.13 Binding Immunoglobulin Protein BIP -1.05 Glucose Regulated Protein 75 GRP75 -1 Cathepsin D CATD -1 Transketolase TKT -0.82 Beta-Actin ACTB -0.5 Alpha-Enolase ENOA -0.49 Lamin A/C LMNA -0.42 Gelsolin GELS -0.26 Splicing Factor Proline-Glutamine Rich SFPQ -0.15 Ribosomal Protein S6 RS6 -0.14 Syntaxin-11 SYEP -0.09 Heat Shock Protein 70 HSP7C -0.05 The results of the enrichment analysis offer further insight into the biological pathways affected by the downregulated proteins (Fig. 14). KEGG pathway analysis highlights the Pentose Phosphate Pathway (PPP) as a major affected pathway. This pathway is crucial for maintaining cellular redox balance and supporting biosynthesis [ 76 – 77 ]. Hypoxia leads GSCs to shift metabolically toward glycolysis and decrease the production of PPP enzymes and as a result, glycolysis is activated, leading to more energy requirement [ 78 ]. The downregulation of TKT suggests that the cells' ability to generate NADPH is compromised, impairing their antioxidant defense mechanisms [ 79 ]. This could lead to an accumulation of ROS, which exacerbates cellular stress and potentially triggers apoptotic pathways. Moreover, reduced ribose-5-phosphate production could impair nucleotide synthesis, hindering DNA and RNA synthesis, thus affecting cell proliferation and growth [ 80 ]. The enrichment of pathways related to biosynthesis of amino acids further points to a broad metabolic shift in the cells. The downregulation of enzymes involved in these pathways could limit the biosynthetic capacity of the cells, affecting their ability to synthesize proteins and other vital biomolecules needed for growth and survival. Interestingly, multiple cardiomyopathy pathways such as arrhythmogenic right ventricular cardiomyopathy, hypertrophic cardiomyopathy, and dilated cardiomyopathy are enriched, indicating proteins like LMNA (Lamin A/C) in the observed changes. LMNA, a nuclear structural protein, is involved in maintaining the integrity of the nuclear envelope and regulating chromatin organization [ 81 ]. Its downregulation could compromise nuclear stability, leading to defects in cellular architecture and mechanical stress responses. Although these pathways are typically associated with cardiac muscle function, the downregulation of LMNA in this context suggests that similar structural vulnerabilities may occur in glioblastoma cells [ 82 ], potentially affecting their ability to withstand mechanical and proliferative stress. These structural changes could mirror those seen in cardiomyopathies, where weakened cellular integrity leads to dysfunction and disease progression. Overall, the enrichment of metabolic pathways, such as the PPP and amino acid biosynthesis, aligns with the observed downregulation of metabolic enzymes (e.g., ALDOA and TKT), indicating a reduction in biosynthetic and energy-producing pathways in response to cellular stress. The involvement of cardiomyopathy pathways and cytoskeletal processes underscores the importance of structural proteins like LMNA in maintaining cellular and tissue integrity, particularly in muscle cells. Additionally, the enrichment of nuclear structure-related processes (nuclear matrix, periphery) and chromatin remodeling complexes highlights potential disruptions in gene regulation and nuclear architecture, which could affect genome stability and transcriptional responses under stress. Metabolomic analysis by nuclear magnetic resonance The provided NMR data captures the metabolic profiles of control and treated LN229 glioblastoma cells, with the treated cells exposed to a combination of andrographolide and berbamine. This combination treatment is particularly interesting due to its reported anti-inflammatory, anti-tumor, and anti-proliferative effects, potentially leading to significant metabolic disturbances in cancer cells. Through a detailed metabolomics analysis, we explore the specific alterations induced by the treatment in key metabolic pathways, focusing on energy metabolism, amino acid utilization, and potential shifts in lipid and nucleotide synthesis. The application of NMR spectroscopy allows for the identification of metabolic perturbations in energy production, lipid synthesis, and nucleotide biosynthesis, which are essential for the survival and proliferation of glioblastoma cells [ 83 ]. In the control sample, prominent peaks between 9–11 ppm correspond to key energy metabolism intermediates, such as pyruvate and lactate, which play essential roles in glycolysis and the tricarboxylic acid (TCA) cycle. The metabolites are crucial for maintaining the high energy demands of rapidly proliferating glioblastoma cells. However, in the treated sample, a significant reduction in peak intensity around 8–9 ppm suggests a disruption in these glycolytic intermediates or TCA cycle components (Fig. 15). The decline indicates that the treatment may be interfering with key enzymes or transporters necessary for energy production, resulting in metabolic stress and limiting the cells’ ability to sustain growth [ 84 ] (Fig. 15). Additionally, andrographolide inhibits glucose transporters, which could explain the observed reduction in glycolytic intermediates, reinforcing the notion that the treatment effectively hampers energy metabolism in glioblastoma cells [ 85 ]. By interfering with calcium signaling, berbamine can trigger programmed cell death pathways, adding another layer of metabolic stress to the glioblastoma cells [ 86 ]. Beyond energy metabolism, the treatment appeared to have a profound impact on amino acid metabolism (Fig. 15). Peaks observed between 4–6 ppm in the control sample, likely corresponding to amino acids such as glutamine and glutamate, were significantly diminished in the treated sample. This reduction suggests a disruption in amino acid availability, which is particularly relevant given that glutamine serves as an essential nutrient for glioblastoma cells, fueling biosynthetic processes and supporting redox balance. Risen glutamine metabolism is a characteristic of aggressive mesenchymal GB, leading to increased glutamine flux. Therefore, lowering glutamine uptake can aid in targeting GB [ 87 ]. Thus, treating cells with andrographolide-berbamine might be the reason for downregulating the glutamine uptake, eventually restricting the ability of glioblastoma cells to survive and proliferate. Further analysis of the treated sample reveals a downward shift in peaks around 8.5–8.8 ppm. This shift likely reflects altered levels of amino acids, which are essential for DNA synthesis and cell cycle progression. Together, the combination of andrographolide and berbamine appears to impose significant metabolic stress, disrupting pathways that sustain glioblastoma growth. The reduction in glycolysis and altered amino acid metabolism indicate that treated cells could not meet the bioenergetic and biosynthetic demands necessary for rapid proliferation. The shifts and broadening of NMR peaks suggest complex interactions between the two compounds, potentially limiting the metabolic flexibility of GB cells and impairing their ability to adapt to treatment-induced stress. Conclusion The research investigated how andrographolide and berbamine, both individually and in combination, influence the LN229 and U-87 MG glioblastoma cell lines. Both compounds exhibited dose-dependent cytotoxicity against GB cells. Their combination showed a synergistic effect, supported by combination index (CI) values below 1 and DRI values above 1. The combination also inhibited cell migration, indicating reduced tumor growth. Increased caspase 3/7 activity pointed to enhanced late apoptosis, while elevated mitochondrial ROS levels suggested oxidative stress, confirmed by Mitosox and MMP assays. Arrest of the cell cycle in G0/G1 and S stages further suppressed proliferation. In silico analysis identified tumor suppressor and GPCR signaling targets, supporting the combination's anti-glioblastoma potential. Overall, this study suggests that andrographolide and berbamine could serve as a promising treatment strategy for glioblastoma. The in silico results supported the hypothesis that combining andrographolide and berbamine enhanced the overall anti-tumor efficacy by inducing greater metabolic and structural stress than either compound alone. Additionally, the KEGG and GO enrichment analyses suggested that the downregulation of proteins in this dataset has significant effects on metabolism, cell structure, and gene regulation, with potential implications for cardiomyopathy and apoptosis. The NMR-based metabolomics analysis revealed that the combination of andrographolide and berbamine significantly altered the metabolic profile of LN229 glioblastoma cells. The key pathways, such as energy metabolism (glycolysis and the TCA cycle), amino acid metabolism, and possibly lipid biosynthesis, were disrupted by the treatment. These metabolic changes are consistent with the anti-proliferative effects observed with this combination treatment, suggesting that it could serve as a promising therapeutic approach for glioblastoma. Further investigations with more targeted metabolomics techniques will help to elucidate the exact nature of the metabolic alterations induced by this treatment. Declarations Conflict of interest disclosure The authors declare no conflict of interest. Acknowledgement The authors would like to acknowledge the Jaypee Institute of Information Technology for the necessary resources required for the work. Data availability statement The data supporting the findings of this study are available from the corresponding author upon reasonable request. References Glantz, M., Chamberlain, M., Liu, Q., Litofsky, N. S., & Recht, L. D. 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07:25:09","extension":"png","order_by":146,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":16655,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure8A.png","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/d9f56f617257db1c8c375ed2.png"},{"id":92638287,"identity":"a701fc80-fc1e-4536-9264-05a4e2eb76ff","added_by":"auto","created_at":"2025-10-02 07:09:02","extension":"png","order_by":147,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8646,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure8B.png","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/9001987109ecbd214a560f87.png"},{"id":92638330,"identity":"5a6d41e4-f6e9-433a-a9ff-8e94c7967cbf","added_by":"auto","created_at":"2025-10-02 07:09:04","extension":"png","order_by":148,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7149,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure9.png","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/727467e8a02da9be0eb7e5be.png"},{"id":92638580,"identity":"76c34354-0bde-4f74-b113-77ea26d6be24","added_by":"auto","created_at":"2025-10-02 07:17:09","extension":"xml","order_by":149,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":210060,"visible":true,"origin":"","legend":"","description":"","filename":"ABABD25029330structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/0ef22a62b38312716e97cc6e.xml"},{"id":92638557,"identity":"f98ce8a0-f2a0-46ff-9e90-7667813d648d","added_by":"auto","created_at":"2025-10-02 07:17:06","extension":"html","order_by":150,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":231682,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/5d370117b96abff9fcb08f56.html"},{"id":92638244,"identity":"0dce36eb-554c-4cb9-967a-83efdd46b449","added_by":"auto","created_at":"2025-10-02 07:08:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":295966,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of andrographolide and berbamine on proliferation of cell was assessed at various doses of andrographolide, berbamine alone, and their combination in GB cells. The dose-response curves for (A) LN229 and (B) U-87 MG cells are presented. Panels (C) and (D) display the Fa-CI plots for the andrographolide-berbamine combination in LN229 and U-87 MG cells, respectively. Additionally, panels (E) and (F) show the DRI plots for LN229 and U-87 MG cells, respectively.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/d7fd88a4b8554903010ef9aa.jpg"},{"id":92638241,"identity":"00b84e28-03bd-49e1-a1a4-86d8a453a8c8","added_by":"auto","created_at":"2025-10-02 07:08:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":185088,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of andrographolide and berbamine on cell proliferation was evaluated at various doses on HEK 293 cells: (A) Andrographolide alone, (B) Berbamine alone, (C) Andrographolide and berbamine combination at higher dosage, and (D) plot of the combined effect of both compounds at a specific dosage on GB cells.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/31e311ae3205be0368caa1b2.jpg"},{"id":92638510,"identity":"c0c89478-c27d-4121-880e-811b3920e6e9","added_by":"auto","created_at":"2025-10-02 07:16:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":131336,"visible":true,"origin":"","legend":"\u003cp\u003eMigratory potential of LN229 (Panel 1) control (Panel 2) andrographolide treated (Panel 3) berbamine treated and (Panel 4) andrographolide-berbamine treated LN229 cells, assessed through wound healing assay for (A) 0 hr (B) 24 hrs (C) 48 hrs\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/281cb0766bdd9ea8c38636e4.jpg"},{"id":92638261,"identity":"490cb907-6ce6-46fa-807c-15119d34d97a","added_by":"auto","created_at":"2025-10-02 07:08:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":179535,"visible":true,"origin":"","legend":"\u003cp\u003eMigratory potential of U-87 MG (Panel 1) control (Panel 2) andrographolide treated (Panel 3) berbamine treated and (Panel 4) andrographolide-berbamine treated LN229 cells, assessed through wound healing assay for (A) 0 hr (B) 24 hrs (C) 48 hrs\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/2f19b3d28dc60533b75866bb.jpg"},{"id":92638243,"identity":"bce916a5-20ee-417e-8605-1c271c687cf0","added_by":"auto","created_at":"2025-10-02 07:08:58","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":234643,"visible":true,"origin":"","legend":"\u003cp\u003eThe wound healing % in LN229 cells (Panel A); U-87 MG cells (Panel B). The markings ** denote statistical significance at P ≤ 0.01, concerning each group.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/32d2c41cef1709a21d058d31.jpg"},{"id":92638248,"identity":"8f478ab5-d9f5-4e33-af09-9e82b2b91453","added_by":"auto","created_at":"2025-10-02 07:08:59","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":573083,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of phytocompounds on apoptosis in glioblastoma cell lines (LN229 and U-87 MG) after 48 hours of treatment, analyzed using the MUSE Cell Analyzer.\u003cbr\u003e\n(A–D) Apoptosis profile of LN229 cells: (A) Control, (B) Andrographolide, (C) Berbamine, (D) Combination of andrographolide and berbamine.\u003cbr\u003e\n(E–H) Apoptosis profile of U-87 MG cells: (E) Control, (F) Andrographolide, (G) Berbamine, (H) Combination of andrographolide and berbamine.\u003c/p\u003e\n\u003cp\u003ePanels I and J depict the different phases of apoptosis in the LN229 and U-87 MG cell lines as stacked graphs, respectively. The markings ** denote substantial statistical difference with a p-value ≤ 0.01 compared to each group.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/535616025bec8874211ff9da.jpg"},{"id":92638970,"identity":"687c3a59-f79d-438d-9728-4c48db9580f1","added_by":"auto","created_at":"2025-10-02 07:24:59","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":386082,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of andrographolide and berbamine on GB Caspase 3/7 after 48 h treatment, as assessed by MUSE on LN229 cells (A) control (B) cells treated with andrographolide, (C) cells treated with berbamine, (D) cells treated with the combination.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/3ca6c063e1abc16623b3462a.jpg"},{"id":92638253,"identity":"9f5b9a13-8e92-4063-84a2-cc2fd53479c0","added_by":"auto","created_at":"2025-10-02 07:08:59","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":171838,"visible":true,"origin":"","legend":"\u003cp\u003ePanel (A) shows histogram of flow cytometry depicting unstained (US), control and treated with andrographolide-berbamine. The markings ** denote substantial statistical difference with a p-value ≤ 0.01 compared to each group. Panel (B) shows the percentage of Mitosox+ cells.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/c1233b4ba6f76d33812013c5.jpg"},{"id":92638274,"identity":"c3be368b-7428-488e-813b-027d73268672","added_by":"auto","created_at":"2025-10-02 07:09:00","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":11906,"visible":true,"origin":"","legend":"\u003cp\u003eDepiction of cell population with rhodamine 123 fluorescence of control (C3) and andrographolide-berbamine (AB3)\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/37361ce3316506033b803fea.jpg"},{"id":92638971,"identity":"dcc915b0-54b9-4a11-8fa1-c98bfbd1513e","added_by":"auto","created_at":"2025-10-02 07:24:59","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":298940,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of compounds on the LN229 cell cycle after 48 h treatment was evaluated using the MUSE system. The counts of LN229 cells in certain stages of the cell cycle are displayed for (A) the control, (B) andrographolide treated, (C) berbamine treated, and (D) combined andrographolide-berbamine treated. Panel (E) shows a stacked graph representing the percentage of cell population in various cell cycle stages. The markings ** denote statistical difference with a p-value ≤ 0.01 compared to each group.\u003c/p\u003e\n\u003cp\u003eA: Andrographolide; B: Berbamine, AB: combination of andrographolide and berbamine\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/113bd93586614814beab8c16.jpg"},{"id":92638285,"identity":"be1813c0-5103-4940-af35-d554892e5b16","added_by":"auto","created_at":"2025-10-02 07:09:01","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":140495,"visible":true,"origin":"","legend":"\u003cp\u003eThe pie chart represents the categorization of the classes of the predictive interactors for (A) Andrographolide and (B) Berbamine.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/293f434866fd8aa2f8044f9e.jpg"},{"id":92638245,"identity":"193ded78-426b-4355-af9f-a97109a74130","added_by":"auto","created_at":"2025-10-02 07:08:58","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":272696,"visible":true,"origin":"","legend":"\u003cp\u003eThe bar graphs represent gene ontology in different categories (A) biological process (B) Cellular component (C) Molecular function.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/89191642a1012c07f24725da.jpg"},{"id":92638339,"identity":"dcc382d5-81bf-4955-85a1-bb0a4c71d86e","added_by":"auto","created_at":"2025-10-02 07:09:05","extension":"jpg","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":285532,"visible":true,"origin":"","legend":"\u003cp\u003eThermogram of cells after treatment with phytocompounds. Orange line: only cells; blue line: treatment with andrographolide; grey line: treatment with berbamine; yellow line: combination treatment on LN229 cell line.\u003c/p\u003e","description":"","filename":"13.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/7b265463d6c56c618e98cbfb.jpg"},{"id":92638311,"identity":"59d04c06-1fd7-4cce-b604-fa3c15717115","added_by":"auto","created_at":"2025-10-02 07:09:03","extension":"jpg","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":216840,"visible":true,"origin":"","legend":"\u003cp\u003eEnrichment analysis of expressed proteins\u003c/p\u003e","description":"","filename":"14.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/17978e4bb7c4aba749f796bc.jpg"},{"id":92638555,"identity":"9d8887fa-702e-43dd-ac13-b547ff90aa2b","added_by":"auto","created_at":"2025-10-02 07:17:05","extension":"jpg","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":333247,"visible":true,"origin":"","legend":"\u003cp\u003eNMR spectra of glioblastoma LN229 cell line sample (A) control (B) after treatment with combination\u003c/p\u003e","description":"","filename":"15.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/bbf75d00310dea35acddbab3.jpg"},{"id":92639784,"identity":"e6cf242a-f103-4c8c-b91b-03d726634ed6","added_by":"auto","created_at":"2025-10-02 07:41:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5032125,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7585131/v1/d7584f7a-4f2b-4801-b492-ac390d8fee65.pdf"}],"financialInterests":"","formattedTitle":"Andrographolide and Berbamine Synergy in Glioblastoma Treatment: An Insight into the Pathways Assimilating Proteomics and Metabolomics","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlioblastoma (GB), formerly known as glioblastoma multiforme has a very poor prognosis and lacks in efficient treatment options. The interventional therapies include treatments with temozolomide (TMZ) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], nitrosoureas including lomustine and carmustine and bevacizumab in certain recurring cases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The side effects of existing chemotherapy for GB pose a major matter of concern. For instance, TMZ is associated with adverse effects such as pneumocystis pneumonia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], fetal harm and hepatotoxicity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, a study on alternative therapies with fewer side effects must be studied.\u003c/p\u003e\u003cp\u003eThe therapeutic gap in treating glioblastoma (GB) can be bridged by utilizing phytocompound combinations, which may help overcome resistance and reduce the adverse effects commonly related to conventional chemotherapy combinations. Phytochemicals, which are natural compounds obtained from plant sources, have gathered significant consideration recently, because of their likely therapeutic applications in various diseases, including cancer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To overcome the selective targeting of single phytocompounds, the use of combination phytotherapy in cancer treatment can potentially enhance anticancer efficacy and overcome drug resistance. Rather than targeting a single alteration in cancer cells, combination therapy aims to inhibit or modulate multiple relevant targets simultaneously [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThere has been evidence of effective combination therapy of phytocompounds with TMZ such as α-pinene, where the combination effectively enhanced the cytotoxic effect of TMZ on GB cell lines and helped combat resistance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The combined treatment of D-limonene and temozolomide demonstrated a synergistic effect on glioblastoma cells by targeting the proliferation of cells and inducing apoptosis, causing arrest during G0/G1 stages of the cell cycle, and suppressing cell migration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It was also shown that the combined effects of bromelain and temozolomide showed synergistic effects by inhibiting cell growth, colony formation, and migration while triggering early apoptosis and arrest of GB cells during the G0/G1 phase [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recently, a combination of 5-fluorouracil and thymoquinone have shown strong cytotoxicity against U-251 MG cell lines. A pair of phenolic acids, irinotekan and ellagic acid, led to cadherin inhibition, thereby promoting an anti-angiogenic process in C6 glioma cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAndrographolide and berberine are two specific phytochemicals that have shown promising effects in cancer. Andrographolide, a diterpenoid compound, is derived from a medicinal plant, named \u003cem\u003eAndrographis paniculata\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Andrographolide has been found to exhibit potent anti-cancer properties, such as apoptosis induction, tumor growth suppression, and metastasis in the cell lines of gastric cancer and breast cancer [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. On the other hand, berbamine is an alkaloid compound found in various medicinal plants like \u003cem\u003eBerberis\u003c/em\u003e species. It exhibits broad anti-cancer properties, with its mechanism linked to modulating gene and protein expression, while inhibiting tumor growth and metastasis [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDifferential scanning calorimetry (DSC) is used to assess the impact of the treatments on the thermal stability of the glioblastoma cell lines [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. DSC provides information on structural and functional integrity of the cellular components, such as proteins and membranes, under different treatment conditions [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The observed thermal transitions reflect changes in the stability of cellular constituents, offering clues about the extent of molecular damage and stress induced by the treatment. Advances in mass spectrometry-based proteomics allow the identification of key protein biomarkers and can shed light on mapping of protein interactions within GB cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These studies have uncovered critical oncogenic pathways, such as those involving the EGFR [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], VEGF [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and PI3K [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], all of which are frequently dysregulated in GB. By analyzing these proteins, researchers can also identify potential drug targets, paving the way for more precise, targeted treatments.. The use of mass spectrometry enables the detailed characterization of the proteome, highlighting key proteins involved in critical processes such as cell cycle regulation, apoptosis, and metabolic reprogramming [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMetabolomics, on the other hand, offers a complementary perspective by analyzing the global metabolic changes occurring in response to treatment [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Alongside proteomics, metabolomics studies small molecules and metabolites provide additional insights into GB\u0026rsquo;s biology [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Together, these approaches offer a comprehensive, system-wide view of glioblastoma at both molecular and functional levels.\u003c/p\u003e\u003cp\u003eThe current study has assessed the effectiveness of andrographolide and berbamine, both individually and in combination, on LN229 and U-87 MG cell lines of GB, focusing on proliferation and apoptosis of cells. Additionally, the safety of both the compounds and their combination has been tested on the HEK 293 cell line. Further, a combination of DSC, proteomics and metabolomics techniques were employed to gain a deeper understanding of the molecular and metabolic alterations induced by the compounds\u0026rsquo; treatment in GB cells.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCells and Reagent\u003c/h2\u003e\u003cp\u003eLN229, U-87 MG and HEK 293 cell lines were procured from NCCS, Pune, India. Mammalian cells were incubated under standard conditions (5% CO\u003csub\u003e2,\u003c/sub\u003e 37\u0026deg;C). The cell lines were supplemented with Dulbecco\u0026rsquo;s modified Eagle medium (DMEM) with 10% FBS, along with penicillin, streptomycin, and gentamycin.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMeasurement of cytotoxicity\u003c/h3\u003e\n\u003cp\u003eThe cytotoxicity of phytocompounds (andrographolide, berbamine) on chosen cell lines (LN229 and U-87 MG) was checked through 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay MTT assay as described before by Gautam et al. The cells (1 X 10\u003csup\u003e5\u003c/sup\u003e cells per mL) were treated with the test compounds (3.1\u0026ndash;50 \u0026micro;M) in a 96-well microtiter plate. MTT solution (5mg/mL) was added and the plate was kept in the incubator (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e) for 4 h. After incubation, the media was decanted, and each well was filled with DMSO (200 \u0026micro;L) to dissolve the formazan dye [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The reading of optical density was taken at 570nm, and the percentage of growth inhibition upon treatment with phytocompounds concerning control cells was determined using the equation\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{V}\\text{i}\\text{a}\\text{b}\\text{i}\\text{l}\\text{i}\\text{t}\\text{y}\\:\\text{o}\\text{f}\\:\\text{c}\\text{e}\\text{l}\\text{l}\\text{s}\\:\\left(\\text{\\%}\\right)=\\frac{\\text{T}\\text{r}\\text{e}\\text{a}\\text{t}\\text{e}\\text{d}\\:\\text{c}\\text{e}\\text{l}\\text{l}\\text{s}-\\text{b}\\text{l}\\text{a}\\text{n}\\text{k}\\:}{\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\:\\text{c}\\text{e}\\text{l}\\text{l}\\text{s}-\\text{b}\\text{l}\\text{a}\\text{n}\\text{k}}\\:\\text{X}\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eCombination index calculation\u003c/h3\u003e\n\u003cp\u003eUsing CompuSyn software, the Chou and Talalay approach [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] was used to identify combination index (CI) values. The andrographolide and berbamine were combined in a set concentration ratio according to their IC\u003csub\u003e50\u003c/sub\u003e values, and the viability of LN229 and U-87 MG cells was studied through the MTT assay. The treatment was considered synergistic when the CI value came out to be less than 1[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The dose-reduction index, or DRI, was calculated, wherein, its value exceeding one signifies a reduction in the total concentration of the drug [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCell safety of the combination\u003c/h3\u003e\n\u003cp\u003eThe safety of the concentration of compounds on the non-transformed cell line was studied for the single compounds and combination on HEK 293 cell line. The phytocompounds andrographolide and berbamine were individually tested on HEK 293 cells at concentrations ranging from 156.25 to 5000 \u0026micro;M. Additionally, the compounds were combined at their IC\u003csub\u003e50\u003c/sub\u003e values, serially diluted, and tested on HEK 293 cells to assess toxicity using the MTT assay [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eWound closure capability\u003c/h3\u003e\n\u003cp\u003eThe migratory capacity of LN229 and U-87 MG cells was assessed by the wound healing assay. Cells were incubated at a density of 2 X 10\u003csup\u003e5\u003c/sup\u003e per mL in 6-well plates for 24 h. The media was aspirated, and a straight-line scratch was made using a sterile tip. The wells were then washed with PBS to remove any debris [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The cells were treated with respective IC\u003csub\u003e50\u003c/sub\u003e values from a single or combination of andrographolide and berbamine for 48 h under standard conditions. The wells were photographed at 0, 24, and 48 hours using field microscopy to estimate the migrated area and analyze gap closure. The distance of the wound was measured and calculated by ImageJ software in both treated and untreated cells.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAnnexin V assay\u003c/h2\u003e\u003cp\u003eThe LN229 and U-87 MG cell lines were plated at a density of 5 x 10\u003csup\u003e5\u003c/sup\u003e cells per well in a 6-well plate. The cells were exposed to varying concentrations of andrographolide, berbamine, and their combination, and then incubated for 48 hours. The cells incubated without treatment were considered as controls. Next, the cells were trypsinized, and 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e density of cells were treated with Annexin V \u0026amp; Dead Cell Reagent, followed by a 20-minute incubation at RT. Apoptosis was then assessed using Muse\u0026reg; Cell Analyzer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCaspase3/7\u003c/h3\u003e\n\u003cp\u003eTo evaluate caspase 3/7 activity, firstly cell suspension was prepared by centrifuging trypsinized cells. After this, 1X Assay Buffer BA was added to each sample as per manufacturer\u0026rsquo;s instructions. Subsequently, the working solution of Muse\u0026trade; Caspase-3/7 Reagent was supplied to each tube and mixed thoroughly. The tubes were incubated for 30 mins (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e) with the working solution of Muse\u0026trade; Caspase 7-AAD. Finally, the tubes were incubated at RT, shielded from light, for 5 mins before being analyzed with the Muse\u0026trade; Cell Analyzer.\u003c/p\u003e\n\u003ch3\u003eExamination of Oxidative stress\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMitoSox\u003c/h2\u003e\u003cp\u003eThe LN229 cells were subjected to cell cytometry after incubation with Mitosox dye. Reactive oxygen species are known to oxidize the dye, which subsequently binds to DNA, eventually producing fluorescence. To assess the ROS production in cells post-treatment, the control and treated cells were incubated in 6-well plates for 48 h. The cells were given a wash with PBS and MitoSOX dye (5\u0026micro;M) was administered in the wells for 30 mins. The cells were trypsinized, resuspended in PBS, and emission was measured at 580nm through BD FACSVerse\u0026trade; Cell Analyzer [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eRhodamine 123\u003c/h2\u003e\u003cp\u003eTo evaluate the effects of phytocompound treatment on cellular oxidative stress, control and treated cells were incubated in a 6-well plate for 48 h. Later, the cells were washed with PBS, and rhodamine 123 dye (5 \u0026micro;g/mL) was added to each well, followed by incubation for 30 mins. Afterward, the cells were washed with PBS, trypsinized, resuspended in PBS, and analyzed by BD FACS Verse for fluorescence at 535nm of wavelength [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCell Cycle Assay\u003c/h2\u003e\u003cp\u003eThe Muse\u0026trade; cell cycle assay was performed after a 48-hour incubation period post-treatment. Cells (LN229) were seeded at 2 x 10\u003csup\u003e4\u003c/sup\u003e cells/well density and incubated in complete media at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were treated with andrographolide and berbamine singly as well as in combination at IC\u003csub\u003e50\u003c/sub\u003e concentrations for 48 hours, washed with PBS, and centrifuged. The pellet was resuspended in PBS and fixed in 70% ethanol at -20\u0026deg;C until analysis. After centrifugation, 200 \u0026micro;L of cell cycle reagent was added, incubated for 30 minutes, and analyzed via Muse\u0026trade; Analyzer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe Tukey Honestly Significant Difference (HSD) post hoc test calculator was utilized to perform one-way ANOVA statistical analysis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.astatsa.com\u003c/span\u003e\u003cspan address=\"http://www.astatsa.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A statistical comparison was conducted between the GB cells treated with the combination of andrographolide and berbamine, and control or single compound-treated cells for all the assays. The differences in treatments were statistically significant, with p-values less than 0.01 and 0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eSwiss Target Prediction Analysis\u003c/h2\u003e\u003cp\u003eThe Swiss Target Prediction tool was used to predict target proteins based on their similarity to known bioactive agents [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The chemical structures of andrographolide and berbamine were uploaded into the tool, and the predicted target proteins were analyzed for their potential involvement in the therapeutic effects exerted by andrographolide and berbamine.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eSTRING Analysis\u003c/h2\u003e\u003cp\u003eThe protein-protein interactions of the predicted targets for andrographolide and berbamine were explored using the STRING database. The database focuses on protein-protein interaction networks, considering direct and indirect interactions. A high confidence score threshold of 0.9 was applied, and the potential functional roles and molecular mechanisms of the probable proteins involved in interaction with andrographolide and berbamine were determined [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eWEB-based Gene Set Analysis Tool Analysis\u003c/h2\u003e\u003cp\u003eThe WEB-based Gene Set Analysis Tool (Web Gestalt) was used to understand the involvement of the targets determined for andrographolide and berbamine inhibitory action in specific cellular pathways. The list of proteins was subjected to WebGestalt to analyse GO terms (biological processes, cellular components, and molecular function) and study KEGG pathways. The predicted target proteins were uploaded into WebGestalt, and the KEGG pathway analysis revealed the biological processes and molecular interactions associated with the interacting proteins of andrographolide and berbamine [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eDifferential Scanning Calorimetry\u003c/h2\u003e\u003cp\u003eThe Hitachi DSC67000X, differential scanning calorimeter (DSC) was used to examine how the andrographolide-berbamine combination treatment affected the LN229 cell line. Following a 48-hour treatment with the combination, the LN229 cells were isolated and lysed based on the freeze-thaw method (5 incubation cycles of -80\u0026deg;C and 37\u0026deg;C for 10 mins each) then resuspended in PBS and analyzed [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThermal transitions, which can reveal information on the stability and structural changes of biological constituents, including proteins and lipids, in response to therapy, were measured using the DSC67000X. After loading cell samples into DSC pans, temperature scans were carried out in a nitrogen atmosphere under regulated conditions, ranging from low to high temperatures. The heat flow, endothermic and exothermic events, which represent modifications in the thermal characteristics of the cells after treatment, were recorded. In order to identify any notable variations in thermal stability between the control and treated cell lines, the generated thermograms were examined.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eProteomics analysis\u003c/h2\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003eSample Preparation\u003c/h2\u003e\u003cp\u003eCells (2 X 10\u003csup\u003e7\u003c/sup\u003e) (control and treated) were grown in a T-75 flask and treated with the andrographolide-berbamine combination. After 48 hrs of treatment, the flask was rinsed with 10 mL PBS. The cells were scraped using a cell scraper and spun at 1500 rpm at 4\u0026deg;C. The pellet was then subjected to RIPA lysis buffer. Protein samples were separated using SDS-PAGE with a 15% separating gel and a 4% stacking gel. The gel was stained overnight and destained to remove the background and eventually visualised for the separation of bands. Excised protein bands were cut into ~\u0026thinsp;1x1 mm pieces and washed three times with 50% acetonitrile in 50 mM ammonium bicarbonate to remove the stain. Gel pieces were then dehydrated in 100% acetonitrile, reduced with 5 mM DTT, and alkylated with 20 mM iodoacetic acid. Trypsin digestion was carried out using sequencing-grade trypsin (1 \u0026micro;g of trypsin per 50 \u0026micro;g of protein) overnight at 37\u0026deg;C. Peptides were extracted twice with 60% acetonitrile and 0.1% formic acid, pooled, and dried using a centrifugal evaporator. Desalting was performed using Zip-Tip C18 as per standard procedures [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eMass Spectrometry\u003c/h2\u003e\u003cp\u003eDried peptides, obtained after trypsin digestion, were reconstituted in 2% acetonitrile and 0.1% formic acid and injected into a TripleTOF\u0026reg; 5600\u0026thinsp;+\u0026thinsp;mass spectrometer (ABsciex) with a ChromeXP C18 column. The peptides were eluted using a dual solvent system: Reservoir A (98% water, 2% acetonitrile, 0.1% formic acid) and Reservoir B (98% acetonitrile, 2% water, 0.1% formic acid). The flow rate was set to 5 \u0026micro;L/min, and the total run time was 87 minutes. Mass spectra were acquired in the range of 350\u0026ndash;1250 m/z for precursor ions, followed by MS/MS scanning of fragment ions in the range of 150\u0026ndash;1600 m/z. Instrument settings included an ion spray voltage of 5500 V, nebulization gas (GS1) at 25 psi, and curtain gas (CUR) at 30 psi, ensuring stable ionization and data acquisition [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eMass Spectrometry Data Processing and Analysis\u003c/h2\u003e\u003cp\u003eMS Data Processing and Analysis (version 2.7.0, Matrix Science) was used to process the raw data obtained from the mass spectrometer. The software compared the data to known protein sequences listed in the Swiss-Prot database (check it out at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The possibility that the enzyme used to prepare the samples might have missed cleaving the protein at up to two spots was also considered.\u003c/p\u003e\u003cp\u003eThe change in abundance of protein was investigated in the treated group in comparison to the control group using a trial version of a software program called Ingenuity Pathway Analysis (IPA) from QIAGEN (USA). A list of identified proteins was uploaded for analysis using their unique Swiss-Prot IDs. The results indicated whether the abundance of each protein increased or decreased in the extract-treated group compared to the control group. IPA constructed networks of interacting proteins highlighting the most significant biological pathways. Additionally, IPA revealed how networks differed between the control and combination-treated groups. Finally, the specific interactions between proteins within these networks were examined with ontology analysis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eNMR\u003c/h2\u003e\u003cdiv id=\"Sec24\" class=\"Section4\"\u003e\u003ch2\u003eIntracellular Metabolite Extraction for Metabolomics Analysis\u003c/h2\u003e\u003cp\u003eA dual-phase methanol/chloroform extraction method separated aqueous and non-polar metabolites from cell proteins. Dried methanol-quenched cell pellet samples were kept on ice throughout the extraction process. To each cell pellet (obtained by centrifuging 1 X 10\u003csup\u003e7\u003c/sup\u003e cells per mL), 300 \u0026micro;l of a chloroform/methanol mixture in a 2:1 ratio was added and thoroughly mixed by vortexing. Following this, 300 \u0026micro;l of HPLC/UPLC-grade water was introduced to the sample, which was again mixed by vortex and then centrifuged at 16,000 g for 5 minutes to facilitate phase separation. Culture medium samples were stored at -80\u0026deg;C immediately after harvesting. A volume of 550 \u0026micro;l of the thawed medium sample was transferred to a fresh eppendorf tube, to which 50 \u0026micro;l of internal standard DSA (4,4-Dimethyl-4-silapentane-1-ammonium trifluoroacetate) in D₂O (11.6 mM) was added as a quantitative reference. The mixture was then transferred to a standard 5 mm NMR tube for analysis. Following the methanol/chloroform/water dual-phase extraction, the aqueous fractions were dried using a freeze-dryer. The dried samples were then reconstituted in 600 \u0026micro;l of phosphate buffer, composed of 0.2 M Na₂HPO₄, 0.043 M NaH₂PO₄, 100 \u0026micro;M TSP, and 3 mM NaN₃ in 100% D₂O. After reconstitution, the samples were centrifuged at 16,000 g for 5 minutes to remove any insoluble material and supernatant was transferred to a clean 5 mm NMR tubes for further analysis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e\u0026sup1;H NMR Experiment Acquisition and Metabolite Identification:\u003c/h2\u003e\u003cp\u003eHigh-resolution \u0026sup1;H NMR spectra were acquired using either a 5 mm broadband-inverse probe or a 5 mm cryoprobe, both mounted on a 14.1 T Bruker AVANCE 600 spectrometer (Bruker Biospin). A 3-second relaxation delay was incorporated into the acquisition, and gradient shimming was performed before all acquisitions to ensure optimal magnetic field homogeneity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Metabolites were identified based on signal peaks observed in the spectra, with reference to databases such as the Human Metabolome Database (HMDB) and the Biological Magnetic Resonance Bank (BMRB), or through comparisons with previously published literature [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003cdiv id=\"Sec27\" class=\"Section4\"\u003e\u003ch2\u003eCytotoxic effect of andrographolide and berbamine\u003c/h2\u003e\u003cp\u003eThe effect of andrographolide and berbamine were studied alone and in combination against the GB cell lines. The IC\u003csub\u003e50\u003c/sub\u003e values resulting from the study in LN229 cells were 11.4 \u0026micro;M for andrographolide and 6.3 \u0026micro;M for berbamine (Table\u0026nbsp;1), whereas their IC\u003csub\u003e50\u003c/sub\u003e values were higher in U-87 MG cells (Table\u0026nbsp;1). The CI plot and dose-effect curve revealed a better impact with the combination of andrographolide and berbamine on GB cells, revealing a synergistic action compared to the individual administration. The CI plot revealed the graphs having values\u0026thinsp;\u0026lt;\u0026thinsp;1 in case of both cell lines, implying that the combination of compounds is synergistic in its inhibitory effect on GB cells (Fig.\u0026nbsp;1 (A-D)). A synergistic phytocompound combination can maintain efficacy and safety by lowering the dosage of the phytocompounds in treatment. The DRI values indicated a dose reduction for both compounds when used in combination, as the DRI values were greater than 1 (Fig.\u0026nbsp;1E and F). As observed in our data for LN229 cell line, the DRI values of andrographolide ranged from 3.6 to 6.9 whereas that of berbamine ranged from 4.0 to 6.7. The DRI values in our experiment demonstrated the significant advantages of combining andrographolide with berbamine [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] since DRI values greater than 1 depict successful dose reduction of the phytocompounds as demonstrated by Fu et al., 2016 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].To the best of our knowledge, andrographolide and berbamine combination has not been reported on GB cells previously. Yang et al., reported that the cytotoxicity of C6 glioma upon treating with andrographolide resulted in IC\u003csub\u003e50\u003c/sub\u003e of approximately 15 \u0026micro;M, which strongly supports our finding indicating that andrographolide exhibits anticancer properties in GB treatment by reducing cell viability [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The choice of berbamine as an inhibitory compound is substantiated by its anti-angiogenic property on Human Umbilical Vein Endothelial Cells (HUVEC) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Moreover, it has been studied iwith arcyriaflavin A against C6-drived glioblastoma stem-like cells [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe safety of compounds was studied on HEK 293 cell lines, and the IC\u003csub\u003e50\u003c/sub\u003e obtained for andrographolide and berbamine were 616\u0026micro;M and 380 \u0026micro;M, respectively. Nonetheless, the IC\u003csub\u003e90\u003c/sub\u003e concentration of compounds in combination obtained in LN229 and U-87 MG did not show cell cytotoxicity on HEK 293 cell line as depicted in Fig.\u0026nbsp;2.\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\u003eCytotoxic effect of andrographolide and berbamine on LN229 and U-87 MG cell line\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCell line\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eValue\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAndrographolide (\u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBerbamine (\u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eCombination of Andrographolide and Berbamine\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCombination Index (CI) value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAndrographolide (\u0026micro;M)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eBerbamine (\u0026micro;M)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eLN229\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"1\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003ccolgroup cols=\"1\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIC\u003csub\u003e75\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.45\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIC\u003csub\u003e90\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e64.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.38\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eU-87 MG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e92.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e95.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIC\u003csub\u003e75\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e198.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e717.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e82.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e41.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIC\u003csub\u003e90\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e425.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5415.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e222.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e111.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eWound Healing\u003c/h2\u003e\u003cp\u003eThe migratory effect of andrographolide and berbamine through wound closure property, was analysed in both the GB cell lines (Fig.\u0026nbsp;3 and Fig.\u0026nbsp;4). The andrographolide-berbamine combination resulted in 16% and 44% wound closure in the case of LN229 and U-87 MG cells, respectively, after the treatment (Fig.\u0026nbsp;5(B)). The migratory capacity of cells is a major cause of concern while studying the treatment of tumors. In case of GB, the cells function as single cells, further leading to invasion through the mesenchymal mode. The migration of cells is a major cause of recurrence in tumor cases even after treatment or resection [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The difference in migration inhibition by the combination of andrographolide and berbamine was statistically significant compared to individual compound treatment and control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eAnnexin V assay\u003c/h2\u003e\u003cp\u003eThe apoptotic effect of andrographolide and berbamine combination in LN229 and U-87 MG cells was tested by MUSE Annexin V assay kit. The percentage of live cells in the control was 94.5% and it decreased to 69.6%, 72.2% and 48% after treatment with andrographolide, berbamine and the combination at their respective IC\u003csub\u003e50\u003c/sub\u003e concentrations, respectively, for LN229 cells as shown in Fig.\u0026nbsp;6 (A, C-F). Similarly, live cells were least in percentage after treatment with andrographolide and berbamine combination in U-87 MG cells as shown in Fig.\u0026nbsp;6 (B, G-J). The dead/late apoptotic cell population was 50.1% and 54.5% for combinatorial treatment in LN229 and U-87 MG cells, respectively. Apoptosis occurs due to biological and morphological alterations, including condensation of chromatin and subsequent DNA degradation [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In the later stages of apoptosis, there is a notable increase in the caspase-3/7 activation, poly (ADP-ribose) polymerase (PARP) cleavage, fragmentation of DNA, and cell membrane disruption. Following treatment with the combination of phytocompounds, there is a rise in the number of cells undergoing late-stage apoptosis, clearly indicating DNA cleavage and damage resulting from cellular dysfunction [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Because some metabolic enzymes likely remain active during the initial stages of apoptosis, the effects observed in cells at an early stage of apoptosis may be reversible and potentially recoverable [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, a prominent rise in late apoptotic phase cells imply better targeting of GB cells with the combination of compounds [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. For instance, Lee et al., revealed through Annexin V that podophyllotoxin mediated apoptosis in cells of colorectal cancer in humans by p38 signalling [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Similarly, venetoclax led to apoptosis of human breast cancer cell lines through Bcl-2 inhibition [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The combination of berbamine and arcyriaflavin has been demonstrated to activate the apoptosis pathway due to the cytochrome c release (a pro-apoptotic factor), from the mitochondria [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCaspase 3/7\u003c/h3\u003e\n\u003cp\u003eCaspases are key regulators of apoptosis, initiating and executing the process through the action of protease enzymes [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Since the pattern of apoptosis results using Annexin V was similar for both cell lines, we focused on confirming the role of caspases in apoptosis by treating only LN229 cells with the IC\u003csub\u003e50\u003c/sub\u003e values of the compounds and their combination. The apoptotic population of LN229 cells increased from 0% in control to 37% after andrographolide-berbamine treatment, while it was 2% and 1% when treated singularly with andrographolide and berbamine, respectively (Fig.\u0026nbsp;7). A berbamine derivative has been shown to exhibit apoptosis via activation of the caspase-3 cascade [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Andrographolide also triggered apoptosis in C6 glioma cells by PARP-dependent caspase 7 signalling [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. This strongly indicates that caspase-dependent apoptosis would be characteristic of andrographolide-berbamine treatment. Executioner caspases, such as caspase-3, -6, and \u0026minus;\u0026thinsp;7, play a pivotal role in the terminal stages of apoptosis by dismantling cellular structures and ensuring cell death. In cancer, dysregulation or inhibition of these caspases allows cells to evade apoptosis, leading to uncontrolled proliferation and tumor growth.\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eOxidative stress analysis\u003c/h2\u003e\u003cdiv id=\"Sec32\" class=\"Section3\"\u003e\u003ch2\u003eMitochondrial oxidative stress analysis\u003c/h2\u003e\u003cp\u003eThe role of oxidative stress in inducing cell death in LN229 cells upon treatment with andrographolide-berbamine was studied through Mitosox. The flow cytometry results, as shown in Fig.\u0026nbsp;8 depict a shift of the histogram confirming the mitochondrial ROS increased from 4.5% to 14.7% upon treatment with the combination of andrographolide-berbamine [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The results depict that the treatment has led to an increase in oxidative stress in GBM cells. Mitochondria serve as a marker for tumor cell survival due to their production of ROS. ROS are pivotal in regulating cellular physiology, while low levels are crucial for processes such as redox signaling that support normal cell function [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Wen et al., reported that high levels of mitochondrial ROS can induce cytotoxic effects, which is suggestive of impairment of glioblastoma cell survival and tumor growth. Wen et al., also utilised MitoSox assay to report the mitochondrial ROS production, where they found that ROS in mitochondria significantly increased after treating GB cells with a compound isoaaptamine [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec33\" class=\"Section4\"\u003e\u003ch2\u003eMitochondrial Membrane Potential assay\u003c/h2\u003e\u003cp\u003eThe impact of the andrographolide-berbamine combination on mitochondrial membrane potential (MMP) of LN229 via rhodamine assay is depicted in Fig.\u0026nbsp;9. The treatment resulted in a substantial lowering of mitochondrial membrane potential in LN229 cells, evident by the shift in the peak due to the treated sample. During cell apoptosis, the intracellular fluorescence intensity of rhodamine 123 decreases due to increased mitochondrial membrane permeability. This results in reduced mitochondrial membrane potential (MMP) and decreased uptake of Rhodamine 123. MMP, a key component and a hallmark of cell apoptosis, decreases in the early stages of programmed cell death. One of the primary causes of the decline in MMP is the opening of mitochondrial permeability transition pore (MPTP) [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. ROS is known to use a positive feedback loop to start and speed up the opening of MPTP. It causes pro-apoptotic components to be released into the cytoplasm, leading to an irreversible decline in MMP as they exit the mitochondrial matrix leading to apoptosis [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\u003ch2\u003eCell cycle assay\u003c/h2\u003e\u003cp\u003eThe effect of the combinatorial treatment of andrographolide-berbamine on the LN229 cell line revealed arrest of the cell cycle in both G0/G1 and S phases. The treatment led to restriction on GBM cell growth, resulting in cell death, which could be related to arrest in different phases of cell cycle, as shown in Fig.\u0026nbsp;10. The cells in S phase rose from 16% in the case of control to 34% upon combinatorial treatment. The results presented in this paper suggest that the treatment, particularly the combination of andrographolide-berbamine, inhibited the cell cycle before they enter into the replication and division phases, thereby effectively preventing cell proliferation. Huang et al. observed a marked S phase cell cycle arrest in the neuronal cells after aflatoxin treatment, which was related to the upregulation of certain types of cyclin-dependent kinases [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Moreover, Song et al. reported arrest of HepG2 liver cancer cells in S phase leading to cell death, which was related to the upregulation of Bax, cytochrome C, and p53, and the downregulation of Bcl-2 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eIn silico analysis of effect of phytocompounds on cell lines\u003c/h3\u003e\n\u003cdiv id=\"Sec36\" class=\"Section2\"\u003e\u003ch2\u003eTarget identification through Swiss Target prediction\u003c/h2\u003e\u003cp\u003eThe possible mechanism of action of andrographolide-berbamine was investigated by Swiss Target prediction tool [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Figure\u0026nbsp;11 shows the interaction of andrographolide and berbamine with several classes of proteins. The results depict that the highest proportion of proteins involved with andrographolide actions are kinases, which encompass 20% of all the protein classes (Fig.\u0026nbsp;9A). The expression levels of CDKs fluctuate cyclically throughout the cell cycle, influencing various cellular processes. Numerous cyclins and the CDKs 2, 4, and 6 progress the cell towards the end stage of the cell cycle and consequently encourage cell growth. GB tumors have higher levels of CDK2 expression than normal brain tissue, leading to tumour proliferation and poor prognosis [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. MAP kinases, including ERKs, JNKs, and p38/SAPKs, are upregulated within protein kinase cascades that control the cell growth processes [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. These kinases phosphorylate specific target proteins, leading to changes in cellular behavior. Numerous pathways, including Ras/MAPK triggered by EGFR, get activated to provide the ambient microenvironment for GB growth, which can be targeted for effective treatment [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In the context of apoptosis, kinases such as JNK and p38 MAPK are activated in response to stress stimuli [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. They mediate the signaling pathways that induce cell death and control the balance between cell viability and programmed cell death [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. A hypoxic environment can trigger SRC proto-oncogene non-receptor tyrosine kinase and lead to activation of pathways associated with radioresistance and invasion in glioblastoma. It underscores the role of hypoxia and ionizing radiation in driving tumor infiltration. SRC also promotes the invasiveness and malignancy of GB [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile, in case of berbamine, 60% of the proteins involved were of family G protein-coupled receptor (GPCR) (Fig.\u0026nbsp;9B). The involvement of GPCRs has been observed with phytochemical treatment in different cases [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. GPCRs are pivotal in cell cycle regulation by activating checkpoint proteins like p53 in response to DNA damage or stress [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. They can alter cyclin and CDK levels, leading to arrest of the cell in the G1 stage of the cell cycle by downregulation of a cyclin D1. GPCRs also increase CDK inhibitors (e.g., p21, p27), which leads to blocking cyclin-CDK complexes and stopping cell cycle progression [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Additionally, GPCRs influence growth factor receptors, impacting cellular proliferation and potentially inducing cell cycle arrest [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Moreover, GPCRs activate apoptotic pathways through MAPK/ERK, PI3K/Akt, and JNK signaling, regulating Bcl-2 family proteins to promote apoptosis [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. The involvement of GPCRs has been observed to target GB cells through mast cell activation, which can influence angiogenesis and recruit other immune cells to increase the effectiveness of treatment [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\u003ch2\u003eOntology analysis through Web Gestalt\u003c/h2\u003e\u003cp\u003eThe list of protein interactors of andrographolide and berbamine was subjected to Venny 2.1 to find out the common proteins involved. The andrographolide and berbamine seemed to affect the cell communication and developmental processes the most. The cellular component organizations and localization seemed to be linked with 13 proteins (Fig.\u0026nbsp;12). The analysis revealed that cellular components such as vesicles, nuclei, cytosol, the endomembrane system, lumens, and the cytoskeleton may be significantly affected, thereby hampering their integrity. Analyses of the molecular functions revealed protein binding, nucleotide binding, transferase activity and ion binding were enriched by both the compounds.\u003c/p\u003e\u003cdiv id=\"Sec38\" class=\"Section3\"\u003e\u003ch2\u003eChanges in thermal properties of GB cells analysed through differential scanning calorimetry\u003c/h2\u003e\u003cp\u003eThe DSC results (Fig.\u0026nbsp;13) revealed how glioblastoma cells responded to different treatments by analyzing their thermal behavior. In the untreated control cells, heat flow gradually decreased with rising temperature, indicating a steady absorption of energy without major structural disruptions until around 55\u0026deg;C. Andrographolide showed more pronounced heat flow decrease between 55\u0026deg;C and 60\u0026deg;C (Fig.\u0026nbsp;13 A). The data suggests moderate destabilization, likely due to the compound\u0026rsquo;s effects on proteins and membrane integrity. While the changes indicate some level of thermal stress, they are not drastic, implying that andrographolide alone may only partially compromise glioblastoma cell stability. This aligns with andrographolide\u0026rsquo;s known biological actions, including interference with cellular signaling and metabolism [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. While berbamine treatment caused a more dramatic shift in the DSC profile, with a steep drop in heat flow beginning around 50\u0026deg;C. This graph indicates significant cellular destabilization, marked by early protein denaturation and membrane disruption. Berbamine\u0026rsquo;s potent apoptotic properties have been demonstrated through its activation of the p53-dependent apoptotic signaling pathway in colorectal cancer cells [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], as well as its ability to disrupt mitochondrial membrane potential and activate caspases in hepatoma cells [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. These mechanisms are closely linked to its influence on calcium homeostasis, membrane stability, and intracellular signaling, contributing to its pronounced anticancer effects. The extensive structural damage observed suggests that berbamine exerts a much stronger destabilizing influence on glioblastoma cells than andrographolide alone. The combination of andrographolide and berbamine resulted in the most severe thermal destabilization (Fig.\u0026nbsp;13). Heat flow began to decline sharply as early as 45\u0026deg;C, signaling widespread molecular disruption. The pronounced endothermic dip around 55\u0026deg;C suggested extensive protein denaturation and membrane breakdown, likely due to the combined impact of both compounds on multiple cellular pathways. The synergistic effect of andrographolide and berbamine overwhelmed the cells\u0026rsquo; ability to maintain structural integrity, making the combination treatment particularly effective at targeting glioblastoma cells.\u003c/p\u003e\u003cp\u003eBy tracking changes in heat flow as temperature increases, these results provide valuable insights into the structural and molecular stability of the cells, particularly about protein denaturation and membrane integrity [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec39\" class=\"Section2\"\u003e\u003ch2\u003eProteomics study through mass spectrometry\u003c/h2\u003e\u003cp\u003eThe analysis of log2ratio values for various proteins has provided a comprehensive overview of changes in their expression levels, with a negative log2ratio signifying downregulation compared to control conditions [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. The screened results revealed that certain key proteins (Table\u0026nbsp;2) involved in metabolic, structural, and stress-response pathways were significantly up/downregulated, suggesting widespread cellular alterations in response to treating andrographolide-berbamine combination.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe log2fold change of proteins expressed in treated vs that in control\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProtein name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eShort form\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003elog2fold\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAldolase A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eALDOA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-1.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBinding Immunoglobulin Protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBIP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-1.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGlucose Regulated Protein 75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGRP75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCathepsin D\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCATD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTransketolase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTKT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.82\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBeta-Actin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eACTB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlpha-Enolase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eENOA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.49\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLamin A/C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLMNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.42\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGelsolin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGELS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSplicing Factor Proline-Glutamine Rich\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSFPQ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRibosomal Protein S6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRS6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSyntaxin-11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSYEP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.09\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHeat Shock Protein 70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHSP7C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.05\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\u003eThe results of the enrichment analysis offer further insight into the biological pathways affected by the downregulated proteins (Fig.\u0026nbsp;14). KEGG pathway analysis highlights the Pentose Phosphate Pathway (PPP) as a major affected pathway. This pathway is crucial for maintaining cellular redox balance and supporting biosynthesis [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Hypoxia leads GSCs to shift metabolically toward glycolysis and decrease the production of PPP enzymes and as a result, glycolysis is activated, leading to more energy requirement [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. The downregulation of TKT suggests that the cells' ability to generate NADPH is compromised, impairing their antioxidant defense mechanisms [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. This could lead to an accumulation of ROS, which exacerbates cellular stress and potentially triggers apoptotic pathways. Moreover, reduced ribose-5-phosphate production could impair nucleotide synthesis, hindering DNA and RNA synthesis, thus affecting cell proliferation and growth [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. The enrichment of pathways related to biosynthesis of amino acids further points to a broad metabolic shift in the cells. The downregulation of enzymes involved in these pathways could limit the biosynthetic capacity of the cells, affecting their ability to synthesize proteins and other vital biomolecules needed for growth and survival.\u003c/p\u003e\u003cp\u003eInterestingly, multiple cardiomyopathy pathways such as arrhythmogenic right ventricular cardiomyopathy, hypertrophic cardiomyopathy, and dilated cardiomyopathy are enriched, indicating proteins like LMNA (Lamin A/C) in the observed changes. LMNA, a nuclear structural protein, is involved in maintaining the integrity of the nuclear envelope and regulating chromatin organization [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Its downregulation could compromise nuclear stability, leading to defects in cellular architecture and mechanical stress responses. Although these pathways are typically associated with cardiac muscle function, the downregulation of LMNA in this context suggests that similar structural vulnerabilities may occur in glioblastoma cells [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e], potentially affecting their ability to withstand mechanical and proliferative stress. These structural changes could mirror those seen in cardiomyopathies, where weakened cellular integrity leads to dysfunction and disease progression.\u003c/p\u003e\u003cp\u003eOverall, the enrichment of metabolic pathways, such as the PPP and amino acid biosynthesis, aligns with the observed downregulation of metabolic enzymes (e.g., ALDOA and TKT), indicating a reduction in biosynthetic and energy-producing pathways in response to cellular stress. The involvement of cardiomyopathy pathways and cytoskeletal processes underscores the importance of structural proteins like LMNA in maintaining cellular and tissue integrity, particularly in muscle cells. Additionally, the enrichment of nuclear structure-related processes (nuclear matrix, periphery) and chromatin remodeling complexes highlights potential disruptions in gene regulation and nuclear architecture, which could affect genome stability and transcriptional responses under stress.\u003c/p\u003e\u003cdiv id=\"Sec40\" class=\"Section3\"\u003e\u003ch2\u003eMetabolomic analysis by nuclear magnetic resonance\u003c/h2\u003e\u003cp\u003eThe provided NMR data captures the metabolic profiles of control and treated LN229 glioblastoma cells, with the treated cells exposed to a combination of andrographolide and berbamine. This combination treatment is particularly interesting due to its reported anti-inflammatory, anti-tumor, and anti-proliferative effects, potentially leading to significant metabolic disturbances in cancer cells. Through a detailed metabolomics analysis, we explore the specific alterations induced by the treatment in key metabolic pathways, focusing on energy metabolism, amino acid utilization, and potential shifts in lipid and nucleotide synthesis. The application of NMR spectroscopy allows for the identification of metabolic perturbations in energy production, lipid synthesis, and nucleotide biosynthesis, which are essential for the survival and proliferation of glioblastoma cells [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the control sample, prominent peaks between 9\u0026ndash;11 ppm correspond to key energy metabolism intermediates, such as pyruvate and lactate, which play essential roles in glycolysis and the tricarboxylic acid (TCA) cycle. The metabolites are crucial for maintaining the high energy demands of rapidly proliferating glioblastoma cells. However, in the treated sample, a significant reduction in peak intensity around 8\u0026ndash;9 ppm suggests a disruption in these glycolytic intermediates or TCA cycle components (Fig.\u0026nbsp;15). The decline indicates that the treatment may be interfering with key enzymes or transporters necessary for energy production, resulting in metabolic stress and limiting the cells\u0026rsquo; ability to sustain growth [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e] (Fig.\u0026nbsp;15). Additionally, andrographolide inhibits glucose transporters, which could explain the observed reduction in glycolytic intermediates, reinforcing the notion that the treatment effectively hampers energy metabolism in glioblastoma cells [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. By interfering with calcium signaling, berbamine can trigger programmed cell death pathways, adding another layer of metabolic stress to the glioblastoma cells [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBeyond energy metabolism, the treatment appeared to have a profound impact on amino acid metabolism (Fig.\u0026nbsp;15). Peaks observed between 4\u0026ndash;6 ppm in the control sample, likely corresponding to amino acids such as glutamine and glutamate, were significantly diminished in the treated sample. This reduction suggests a disruption in amino acid availability, which is particularly relevant given that glutamine serves as an essential nutrient for glioblastoma cells, fueling biosynthetic processes and supporting redox balance. Risen glutamine metabolism is a characteristic of aggressive mesenchymal GB, leading to increased glutamine flux. Therefore, lowering glutamine uptake can aid in targeting GB [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Thus, treating cells with andrographolide-berbamine might be the reason for downregulating the glutamine uptake, eventually restricting the ability of glioblastoma cells to survive and proliferate. Further analysis of the treated sample reveals a downward shift in peaks around 8.5\u0026ndash;8.8 ppm. This shift likely reflects altered levels of amino acids, which are essential for DNA synthesis and cell cycle progression. Together, the combination of andrographolide and berbamine appears to impose significant metabolic stress, disrupting pathways that sustain glioblastoma growth. The reduction in glycolysis and altered amino acid metabolism indicate that treated cells could not meet the bioenergetic and biosynthetic demands necessary for rapid proliferation. The shifts and broadening of NMR peaks suggest complex interactions between the two compounds, potentially limiting the metabolic flexibility of GB cells and impairing their ability to adapt to treatment-induced stress.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe research investigated how andrographolide and berbamine, both individually and in combination, influence the LN229 and U-87 MG glioblastoma cell lines. Both compounds exhibited dose-dependent cytotoxicity against GB cells. Their combination showed a synergistic effect, supported by combination index (CI) values below 1 and DRI values above 1. The combination also inhibited cell migration, indicating reduced tumor growth. Increased caspase 3/7 activity pointed to enhanced late apoptosis, while elevated mitochondrial ROS levels suggested oxidative stress, confirmed by Mitosox and MMP assays. Arrest of the cell cycle in G0/G1 and S stages further suppressed proliferation. In silico analysis identified tumor suppressor and GPCR signaling targets, supporting the combination's anti-glioblastoma potential. Overall, this study suggests that andrographolide and berbamine could serve as a promising treatment strategy for glioblastoma.\u003c/p\u003e\u003cp\u003eThe in silico results supported the hypothesis that combining andrographolide and berbamine enhanced the overall anti-tumor efficacy by inducing greater metabolic and structural stress than either compound alone. Additionally, the KEGG and GO enrichment analyses suggested that the downregulation of proteins in this dataset has significant effects on metabolism, cell structure, and gene regulation, with potential implications for cardiomyopathy and apoptosis. The NMR-based metabolomics analysis revealed that the combination of andrographolide and berbamine significantly altered the metabolic profile of LN229 glioblastoma cells. The key pathways, such as energy metabolism (glycolysis and the TCA cycle), amino acid metabolism, and possibly lipid biosynthesis, were disrupted by the treatment. These metabolic changes are consistent with the anti-proliferative effects observed with this combination treatment, suggesting that it could serve as a promising therapeutic approach for glioblastoma. Further investigations with more targeted metabolomics techniques will help to elucidate the exact nature of the metabolic alterations induced by this treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest disclosure\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to acknowledge the Jaypee Institute of Information Technology for the necessary resources required for the work.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGlantz, M., Chamberlain, M., Liu, Q., Litofsky, N. S., \u0026amp; Recht, L. D. (2003). 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Glutamine uptake and utilization of human mesenchymal glioblastoma in orthotopic mouse model. \u003cem\u003eCancer Metab\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e, 1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Apoptosis, Cytotoxicity, Metabolic reprogramming, Mitochondrial dysfunction, Oxidative stress, Phytocompounds","lastPublishedDoi":"10.21203/rs.3.rs-7585131/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7585131/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlioblastoma (GB) is one of the most aggressive and invasive cancers, characterized by poor survival rates and high recurrence. Standard treatments concerns about toxicity and long-term safety have led to growing interest in natural alternatives, such as phytotherapeutics. The study explores the effects of two phytocompounds: andrographolide, a diterpenoid, and berbamine, an alkaloid, on GB cells. Their cytotoxic effects were tested on GB cell lines LN229 and U-87 MG and HEK 293 cells to assess safety. Additionally, the impact of these compounds on wound healing, apoptosis (via Annexin V), cell cycle progression, oxidative stress, and mitochondrial dysfunction was evaluated in GB cells. The DSC, mass spectrometry and NMR along with ontology analysis were used to study the changes in protein structure and levels leading to changes in metabolism. The endothermic and exothermic events, highlighted shifts in thermal stability between treated and control cell lines. While, several proteins showed significant change in levels in response to the combination treatment of chosen phytocompound combination. Finally, the treatment with the combination caused significant changes in the metabolic profile of glioblastoma cells. The results highlight the strong potential of phytocompounds combination to target GB by targeting multiple pathways for GB.\u003c/p\u003e\u003cp\u003eStatement of significance of the study\u003c/p\u003e\u003cp\u003eGlioblastoma (GB) is one of the deadliest brain cancers. It has few treatment options and a poor prognosis because of tumor recurrence, resistance to therapy, and the toxicity of standard treatments. This study offers new insights into the potential of two plant compounds, andrographolide and berbamine, as a combination treatment for GB. Our results show that the combination produces stronger effects, increases cell death, raises mitochondrial oxidative stress, and causes cell cycle arrest, which reduces GB cell growth and movement. In addition, analysis through computer simulation revealed disruptions in tumor-related signaling pathways and metabolic processes. Proteomic and metabolomic profiling showed notable changes in energy metabolism, amino acid turnover, and lipid production. These findings suggest that the combination of these plant compounds targets several key features of glioblastoma. Moreover, it could disrupt the metabolism enough to overcome resistance to standard therapies. Importantly, safety tests on HEK293 cells indicate that the treatment selectively harms GB cells. Overall, this research highlights the importance of using natural compounds in multi-target treatment strategies. It points to andrographolide and berbamine as promising candidates for treating glioblastoma, which calls for more investigation.\u003c/p\u003e","manuscriptTitle":"Andrographolide and Berbamine Synergy in Glioblastoma Treatment: An Insight into the Pathways Assimilating Proteomics and Metabolomics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-02 07:08:52","doi":"10.21203/rs.3.rs-7585131/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1f218e91-8e4d-4c6d-84ae-c60d0c9e3519","owner":[],"postedDate":"October 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-02T07:08:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-02 07:08:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7585131","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7585131","identity":"rs-7585131","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}