In vitro repositioning therapy with olaparib, temozolomide and oxaliplatin in glioblastoma cell lines: U118, U87, U251, H4 and human fibroblasts

Abstract

Background

Central nervous system (CNS) tumors, including gliomas, are among the most aggressive cancers, with glioblastoma multiforme (GBM) being the most common and lethal. This study explores the potential of multidrug repositioning as a modern chemotherapy strategy for GBM cell lines. It combines the standard GBM chemotherapeutic temozolomide (TMZ) with olaparib (OLA) and oxaliplatin (OXA), both repurposed from other cancer types. Most experimental drug therapy studies focus on just one or two selected high-grade GBM cell lines, but in this study, four such cell lines were used.

Methods

Glioblastoma (GBM) cell lines U118 MG, H4, U251 MG and U87 MG were treated for 72 h with oxaliplatin (OXA, 50–200 µM), olaparib (OLA, 1–100 µM), or temozolomide (TMZ, 10–100 µM). Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) assay. Half-maximal inhibitory concentration (IC₅₀) values were calculated using GraphPad Prism 8. A human fibroblast line (hFib) from a healthy donor was used as a control. The type of cell death following the above treatments was analysed using a fluorescence-based Apoptotic, Necrotic & Healthy Cells Quantification Kit.

Results

The combination of OLA, OXA, and TMZ significantly reduced cell viability and survival, inducing apoptosis/necrosis more effectively than TMZ alone. These synergistic effects alter glioblastoma metabolism, promote apoptosis, and enhance antitumor activity in vitro.

Conclusions

The proposed multidrug repositioning chemotherapy produced a therapeutic effect at lower doses, suggesting that it is potentially a safer and more effective treatment option.

Supplementary Information

The online version contains supplementary material available at 10.1007/s43440-025-00783-w.

Introduction

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor, with a high mortality rate and a five-year survival of only 7.2% [1, 2]. Treatment is challenging due to GBM genetic heterogeneity, rapid proliferation, and resistance to therapy [1, 2]. Standard treatment includes surgical removal followed by radiotherapy (RT) with temozolomide (TMZ); however, its limited effectiveness highlights the need for more effective therapeutic strategies [3, 4]. The last studies indicate that the presence of glioma stem-like cells (GSCs) is a key factor in GBM resistance, which exhibits high self-renewal, metastasis potential, and DNA repair efficiency. GSCs overexpress DNA damage response proteins, including checkpoint kinase 1 (CHK1), and poly(ADP-ribose) polymerase 1 (PARP1), making them highly resistant to chemotherapy and radiotherapy [5–7]. Targeting these repair mechanisms could improve treatment outcomes.

TMZ, a first-line chemotherapy drug [8], exerts cytotoxic effects via DNA methylation, creating lesions such as O6-methylguanine (O6-MeG), which induces DNA double-strand breaks (DSBs) and tumor cell death through the mismatch repair (MMR) pathway [3, 8, 9]. However, GBM frequently acquires resistance through MMR loss or overexpression of O6methylguanine-DNA methyltransferase (MGMT), which repairs O6-MeG lesions. Nearly 50% of GBM cases lack MGMT due to promoter hypermethylation, initially responding to TMZ but later developing resistance. This leads to tumor relapse and high mortality, as TMZ also accelerates tumor mutation rates, increasing genomic instability [3, 9–11].

Clinical studies show that recurrent GBM is highly resistant to TMZ and other alkylating agents, such as cisplatin and carboplatin [12, 13]. Oxaliplatin (OXA), a third-generation platinum compound, induces DNA cross-links and DSBs while causing fewer side effects than earlier platinum-based drugs [12–14]. However, tumors may still develop resistance by activating homologous recombination and nonhomologous end-joining repair [15, 16].

PARP enzymes play a crucial role in DNA repair and cancer cell survival [3, 15, 17]. Olaparib (OLA), a PARP inhibitor approved for treating BRCA1/2-mutated ovarian and metastatic breast cancer, prevents DNA repair by trapping the PARP-DNA complex, leading to the accumulation of DSBs and cell death. Thus, OLA may be a promising chemotherapy drug for GBM with homologous recombination deficiency (HRD), which impairs the repair of DNA DSBs [3, 17, 18], particularly in isocitrate dehydrogenase (IDH) mutant variants [3, 18, 19]. Additionally, even in the absence of HRD, PARP inhibitors can enhance the effects of alkylating agents like TMZ and OXA by increasing DNA damage and disrupting repair pathways [3, 17, 18].

This study aimed to evaluate multidrug repositioning by studying OLA, OXA, and TMZ combinations as a modern chemotherapy strategy for GBM cell lines that served as preclinical models. Notably, to our best knowledge, this study presents the first in vitro data on the combination of chemotherapeutics (OXA + OLA, OXA + TMZ, or OXA + OLA + TMZ). Furthermore, there is a lack of pharmacodynamic data and reliable biomarkers to predict the sensitivity of GBM to multidrug chemotherapy. Therefore, the OLA synthesis pathway and a detailed view of its binding site were visualized. Also, pharmacodynamic data, like the half maximal inhibitory concentration (IC₅₀) (for all studied drugs), were assessed and presented.

Materials and methods

Cell lines and cell culture

The following GBM cell lines were used as preclinical models [20, 21]: U118 MG (Cat. No. HTB-15), H4 (Cat. No. HTB-148), and U87 MG (Cat. No. HTB-14) obtained from the American Type Culture Collection (ATCC), as well as U-251 MG (Cat. No. 9063001) from Sigma-Aldrich.

A healthy donor human fibroblast line (hFib) served as a control. Before the fragment of skin was taken, the participant signed written informed consent; the study protocol was reviewed and approved by the Human Bioethical Committee of the Regional Medical Board in Kraków (No. 163/KBL/OIL/2023). Details on cell lines and culturing are provided in the supplementary information (SI).

Chemistry and in silico study

Chemotherapeutics and their chemical synthesis

TMZ (Cat. No. T2577) and OXA (Cat. No. PHR1528) were obtained from Sigma-Aldrich. The synthesis of OLA was conducted following the literature data [22, 23], with minor modifications, which are described in detail in the SI. The starting substrate for OLA synthesis, 2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid, was commercially available (Cat. No. A330791, Angene). All three drugs were dissolved in dimethyl sulfoxide (DMSO) (Cat. 3176, Tocris) to create a stock solution of 10 mM. In the cellular experiments, the starting concentration was OLA 1µM, TMZ 10 µM, and OXA 50 µM.

In silico study

The interaction of OLA with the catalytic domain of human PARP1 was modeled using the crystal structure reported by Ryan et al. (PDB ID: 7KK4) [24]. The protein was prepared for analysis via the Protein Preparation Wizard tool within the Maestro program (Schrödinger Release 2021-2: Maestro, Schrödinger, LLC, New York, US). The visualizations were created with PyMOL (PyMOL Molecular Graphics System, Version 2.5, Schrödinger, LLC, New York, US).

IC₅₀ and cell viability assessment of OLA, TMZ, OXA, and their combination

The cytotoxicity tests of TMZ, OLA, and OXA on cell lines seeded in 96-well plates (4 × 10³ cells/well) were conducted by treating with increasing concentrations of OLA, TMZ, and OXA alone or in combination as provided in the SI. The cell viability was measured by Multiskan SkyHigh Microplate Spectrophotometer (ThermoFisher Scientific) after 72 h with the MTS test (detailed in the SI).

Cell death analysis

Cell death was analyzed by fluorescent Zeiss Axio Imager Z2 microscopy (MetaSystems™, Altlussheim, Germany) using Apoptotic, Necrotic & Healthy Cells Quantification Kit (Cat 37-30018-1, Biotium) following the manufacturer’s protocol, with U118, H4, U87, and hFib cells incubated under various drug concentrations for 72 h. The ISIS software (MetaSystems™) for each cell was used to capture and assess early/late apoptosis, and necrosis (more details see SI). Generally, 4–6 representative fields of at least 100 cells per drug and their combinations were analysed separately from coded slides, blinded to the scorer. Data on early and late apoptosis, necrosis, and live cells were presented as percentages [%] after 72 h for each cell line.

Statistical analysis

The results of at least three replicates are presented as the mean ± SD (p < 0.05; one-way ANOVA followed by Tukey’s HSD test). Cells were subjected to drug treatment, and then, after 72 h, the MTS cell viability assay was performed and measured at 490 nm. The error bars represent the standard deviation (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

GraphPad Prism version 10.3.1 (509) for Windows, GraphPad Software, Boston, Massachusetts, USA, www.graphpad.com, was used in all statistical analyses. The data are presented as mean ± standard deviation (SD) of at least three replicates. All data were tested for the assumptions of normality (the Shapiro-Wilk test). One-way ANOVA of the variance was performed by the Tukey test. Statistical significance is indicated by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Results

Chemistry and in silico study

OLA was resynthesized using literature-based conditions with slight modifications [22, 23]. Compound 2 was synthesized by coupling 2-fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (1) with N-boc-piperazine under basic conditions, employing various coupling agents (Fig. 1A). Among them, O-(benzotriazol-1-yl)-N, N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU) achieved the highest conversion rate, as determined by – high-performance liquid chromatography (HPLC), and was chosen for the reaction (see Table 1). Next, the Boc protecting group was removed using hydrochloric acid (HCl), and the resulting intermediate (3) reacted with cyclopropanecarbonyl chloride to yield OLA. This method enabled an efficient synthesis with yields ranging from 60 to 91%. The identity and purity of the final compound were confirmed by chromatographic and spectroscopic analyses, as detailed in the SI.

Fig. 1.

OLA synthesis pathway and a detailed view of its binding site. (A) Resynthesis of OLA and optimization of the first step of synthesis using different coupling agents, with conversion rates evaluated by HPLC. Reagents and conditions: i: N-boc-piperazine, TBTU, TEA, DMF, 24 h, room temperature, ii: HCl (aq), ethanol, iii: cyclopropanecarbonyl chloride, TEA, DCM, 30 min., 0 °C. 2-Fluoro-5-((4-oxo-3,4-dihydrophthalazin-1-yl)methyl)benzoic acid (1) was commercially available. (B) A detailed view of the OLA binding site and its interactions with key amino acids within the human PARP1 catalytic domain (PDB: 7KK4). The figure uses yellow dashed lines to indicate hydrogen bonds and green dashed lines to show aromatic interactions. OLA – olaparib; TBTU – 2-(1 H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate; TEA – triethylamine; DMF – dimethylformamide; DCM – dichloromethane

Table 1.

Product conversion during the first step of OLA synthesis using different coupling agents

Coupling agent	% of conversion (HPLC)
CDI	93.43
DCC	95.05
DIC	85.99
HBTU	92.80
HATU	80.28
TBTU	99.45

OLA – olaparib; HPLC – high performance liquid chromatography; CDI – carbonyldiimidazole; DCC – N, N’-dicyclo-hexylcarbodiimide; DIC – N, N’-diisopropylcarbodiimide; HBTU – O-benzotriazole-N, N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate; HATU – ahexafluorophosphate azabenzotriazole tetra-methyl uronium; TBTU – O-(benzotriazol-1-yl)-N, N,N’,N’-tetramethyluronium tetrafluoroborate

The PARP1 catalytic domain (Fig. 1B) includes helical (HD) and ADP-ribosyltransferase (ART) subdomains. The ART subdomain contains a NAD⁺ binding site with nicotinamide (NI), phosphate (PH), and adenine-ribose (AD) [23]. OLA, like other similar inhibitors, interacts with the highly conserved NI site where Gly863 and Ser904 form hydrogen bonds. Tyr907’s aromatic interactions are also important. It also interacts with Tyr896 and reaches into the AD pocket.

Cell viability assessment

To assess the effects of OXA, OLA, TMZ, and their combinations on cell viability, three human glioma cell lines were used: U87 (grade IV), U118 (grade III/IV), and H4 (grade III). All glioma cell lines were obtained from commercial sources. As a non-cancerous control, normal human fibroblasts (hFib) were used, obtained from a healthy donor. Details on cell lines and culturing are provided in the supplementary information (SI).

Cell viability after OXA

One-way ANOVA revealed a significant effect of OXA on IC₅₀ in the three cell lines: U87, F_6,27 = 74.47, p < 0.0001; U118, F_6,30 = 17.97, p < 0.0001; H4, F_6,27 = 72.70, p < 0.0001 (Fig. 2A). The IC₅₀ values were: 79.87 µM for U87, 63.80 µM for U118, and 45.18 µM for H4. Post hoc analysis revealed that the increased OXA concentrations caused a statistically significant difference between the control groups in each line. For individual drug doses, the decrease in viability was statistically significant (Fig. 2A).

Fig. 2.

Half-maximal inhibitory concentration (IC₅₀) values were determined in GBM cell lines U118 MG, H4, U87 MG, and U-251 MG. Cells were incubated with OXA (A), OLA (C), and TMZ (E). GBM cell viability following treatment with OXA (B), OLA (D), and TMZ (F) was also assessed. Culture cells were incubated with the indicated range of concentrations of the chemotherapeutics for 72 h. IC₅₀ was calculated using GraphPad Prism 8 software. Cell viability was measured with the MTS assay. A human fibroblast line (hFib) obtained from a healthy donor was used as a control system. The results are presented as mean ± SD from three independent experiments (n = 3). One-way ANOVA followed by Tukey’s post hoc test. The error bars represent the standard deviation (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). TMZ – temozolomide; OLA – olaparib; OXA – oxaliplatin; CTR – control; GBM – glioblastoma multiforme; hFib – human fibroblasts; n – number of replicates

OXA significantly affected cell viability in each of the three cell lines tested (U118, F₃,₁₁ = 471.7, p < 0.0001, U87, F₃,₁₁ = 511.5, p < 0.0001, and H4, F₃,₁₁ = 2965.0, p < 0.0001) apart from control (hFib, F₂,₁₁ = 10.80, p = 0.0035) (Fig. 2B). Cell viability was significantly reduced in U118, U87, H4 group, with increasing drug dose compared to the control, p < 0.0001 (Fig. 2B).

Cell viability after OLA

One-way analysis of variance (ANOVA) showed that OLA had no significant effect on IC₅₀ in the U118, F_6,36 = 7.740, p = 0.0002, U251, F_5,21 = 22.24, p < 0.0001 cell lines apart from H4, F_9,37 = 14.18, p < 0.0001. Post hoc analysis showed significant differences between H4 cell lines compared to U118 and U251 cell lines after OLA. IC₅₀ for H4, 27.71 µM, IC₅₀ for U118, 282.85 µM, and IC₅₀ for U251, 412.94 µM (Fig. 2C).

Two concentrations of OLA: 1 µM, 100 µM had no significant effect on cell viability in the lines: U118, F_2,8 = 70.70, p < 0.0001, U87, F_2,8 = 31.75, p = 0.0006, U251, F_2,8 = 10.27, p = 0.0115 compared to the control group hFib, F_2,8 = 71.54, p < 0.0001. Olaparib decreased the viability for the line H4, F_2,8 = 27867.00, p < 0.0001, compared to the control group hFib, F_2,8 = 71.54, p < 0.0001 (one-way ANOVA followed by Tukey’s test) (Fig. 2D).

Cell viability after TMZ

One-way ANOVA revealed that TMZ affected IC₅₀ in two cell lines: U87, F_6,23 = 2895.00, p < 0.0001, and H4, F_6,31 = 758.5, p < 0.001, and differed compared to U118, F_6,17 = 339.1, p < 0.0001 (Fig. 2E). Post hoc analysis showed statistically significant differences between the control groups in each line. IC₅₀ of TMZ was similar in the two cell lines: 95.12 µM for U87, 98.07 µM for H4, but it was higher in U118, 305.14 µM (Fig. 2E).

TMZ did not significantly decrease viability in three cell lines: U118, F_2,8 = 28.32, p = 0.0009, U87, F_2,8 = 23.09, p = 0.0015; and U251, F_2,8 = 8.712, p = 0.0168, compared to hFib, F_2,8 = 1.364, p = 0.3249, apart from the H4, F_2,8 = 24499.00, p < 0.0001. The difference in cell viability between the 10 µM TMZ and the 100 µM TMZ was not statistically significant for all cell models besides the H4 cell line, p < 0,0001 (one-way ANOVA followed by Tukey´s test) (Fig. 2F).

Cell viability after multidrug combinations for selected cell lines

One-way ANOVA revealed that multidrug combinations reduced viability in three cell lines: U118, F_4,14 = 119.2, p < 0.0001; U87, F_4,14 = 11.71, p = 0.0009; H4, F_4,14 = 130.1, p < 0.0001, but had no significant effect on hFib, F_4,14 = 6.592, p = 0.0073 (Fig. 3).

Fig. 3.

Drug combinations for selected cell lines. The anti-tumor effects of two drug combinations: 50 µM OLA with 50 µM TMZ or 50 µM OXA with 50 µM TMZ or 50 µM OXA with 50 µM OLA, and in a triplet 50 µM OXA + 50 µM OLA + 50 µM TMZ were evaluated for their impact on the viability of GBM cell lines U118 MG, H4, U87 MG, and U-251 MG. A human fibroblast cell line (hFib), obtained from a healthy donor, was used as a non-cancerous control. Cell viability was measured with the MTS assay. The results are presented as mean ± SD from three independent experiments (n = 3). One-way ANOVA followed by Tukey’s post hoc test.The error bars represent the standard deviation (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). TMZ – temozolomide; OLA – olaparib; OXA – oxaliplatin; CTR – control; GBM – glioblastoma multiforme; hFib – human fibroblasts; n – number of replicates

Post hoc analysis revealed that the multidrug concentrations caused a statistically significant difference between the control groups in each cell line. For all combined with TMZ at a concentration of 50 µM significantly decreased the viability in U118, U87, and H4 cell lines compared to hFib (Fig. 3).

Cell death analysis

Statistical analysis was performed using one-way ANOVA. The impact of TMZ, OXA, and/or OLA on cell viability and the type of cell death was assessed in various commercially available GBM cell lines (U118MG, H4MG, and U87MG). The human fibroblast line hFib cell lines obtained from a healthy donor were used as controls. The results for the compared groups were as follows: Fib, F_41,84 = 528.0, p < 0.0001; U118, F_41,84 = 1454, p < 0.0001; H4, F_41,84 = 1107, p < 0.0001; and U87, F_41,84 = 1262, p < 0.0001 (Table 2).

Table 2.

Effect of TMZ, OXA, and/or OLA on cell viability and type of cell death in glioblastoma cell lines (U118 MG, H4, U87 MG – ATCC; U-251 MG – Sigma-Aldrich) and human fibroblasts (hFib, control, from a healthy donor)

Anti-cancer drug			Early and late apoptosis, necrosis and live cells presented as [%] after 72 h for each cell line
			hFib	U118	H4	U87
Control, without anti-cancer drug		Live cells	100	98	100	100
		Early apoptosis	0	2	0	0
OLA	1µM	Live cells	55	0	80	94
		Early apoptosis	45	38	14	3
		Late apoptosis	0	62	6	3
		Necrosis	0	0	0	0
	100µM	Live cells	8	0	0	0
		Early apoptosis	47	98	0	13
		Late apoptosis	45	2	72	87
		Necrosis	0	0	28	0
TMZ	10µM	Live cells	48	97	21	44
		Early apoptosis	52	3	79	47
		Late apoptosis	0	0	0	9
		Necrosis	0	0	0	0
	100µM	Live cells	0	32	0	0
		Early apoptosis	54	34	5	48
		Late apoptosis	46	34	95	52
		Necrosis	0	0	0	0
OXA	50µM	Live cells	74	0	17	0
		Early apoptosis	24	32	47	46
		Late apoptosis	2	68	36	54
		Necrosis	0	0	0	0
	100µM	Live cells	45	0	0	0
		Early apoptosis	55	0	8	0
		Late apoptosis	0	55	62	100
		Necrosis	0	45	30	0
	200µM	Live cells	0	0	0	0
		Early apoptosis	20	2	0	5
		Late apoptosis	51	59	46	90
		Necrosis	29	39	54	5
OLA 50µM + TMZ 50µM		Live cells	42	0	0	0
		Early apoptosis	41	14	5	5
		Late apoptosis	17	86	95	95
		Necrosis	0	0	0	0
OLA 50µM + OXA 50µM		Live cells	60	0	0	0
		Early apoptosis	22	5	0	5
		Late apoptosis	18	95	70	95
		Necrosis	0	0	30	0
OLA 50µM + OXA 50µM + TMZ 50µM		Live cells	57	9	0	0
		Early apoptosis	20	46	69	24
		Late apoptosis	23	45	31	76
		Necrosis	0	0	0	0

* Data from a fluorescence microscope: AXIO imager. Z2 from Zeiss with a connected CoolCube 1 camera from Metasystems for imaging samples in fluorescent light showed that the use of TMZ induces apoptosis, not necrosis. The use of OLA induces the most apoptosis, necrosis can be observed only in the H4 cell line at a concentration of OLA 100 μm, while the use of OXA induces the most apoptosis. In the FIB, U118MG, H4MG cell lines at OXA concentration of 200 μm we can observe necrosis, and in the U118MG, H4MG cell line at OXA concentration of 100 μm we also observe necrosis. The most interesting studies concern the combination of 50 µM OLA and 50 µM TMZ, which induces the most apoptosis in the U118MG, H4, U87 cell lines without necrosis at lower concentrations of these drugs than when they are administered separately. A similar situation occurred when we combined OXA with TMZ and OLA in the U118MG, U87 and H4 cell lines

Data are presented as mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. All comparisons were statistically significant (p < 0.05)

CTR – control; TMZ – temozolomide; OLA – olaparib; OXA – oxaliplatin; n-number of replicates

Treatment with 100 µM TMZ induced apoptosis in hFib, U118, H4, and U87 cells (early apoptosis: 54%, 34%, 5%, and 48% vs. late apoptosis 46%, 34%, 95%, 52%, respectively), without triggering necrosis in GBM or hFib cells. In contrast, 100 µM OLA caused 28% necrosis in H4 cells, while U118, U87, and hFib underwent early apoptosis (98%, 13%, 47%) without signs of necrosis (see Table 2). OXA at 200 µM induced necrosis in U118, H4, U87 cells, and hFib (39%, 54%, 5%, 29%), but no necrosis was seen at 50 µM. After 72 h of OXA and OLA treatment, necrosis (30%) and late apoptosis (70%) were observed only in H4 cells, whereas in U118, U87, and hFib cells, this treatment induced mainly late apoptosis (95%, 95%, and 18% respectively), with minimal early apoptosis (5%, 5%, and 22%, respectively). Combination treatments with 50 µM OLA and TMZ, as well as OXA with TMZ and OLA, led predominantly to apoptosis without necrosis in U118, U87, and H4 cells. In GBM (H4, U118, U87) and hFib, the three-drug combination induced only early or late apoptosis (69%, 46%, 24% and 20% or 31%, 45%, 76% and 23%), with over 50% viable cells in hFib (see Table 2).

Discussion

The MTS assay revealed that OXA in combination with OLA or TMZ, as well as the triple combination of these drugs, induced more profound antitumor activity in GBM cell line compared to hFib. These data indicate that OXA-based treatment enhances the antitumor effect, which may be related to MGMT, a “suicide” DNA repair enzyme whose expression level is an indicator of genome stability [25]. When MGMT is overexpressed, DNA alkylation caused by OXA and/ or TMZ can be efficiently repaired, resulting in high resistance [25]. However, PARP inhibitors such as OLA target al.ternative DNA repair pathways such as base excision repair (BER), which can sensitize MGMT-overexpressing GBM to OXA and TMZ. Based on the obtained results, PARP inhibitors like OLA can also enhance the efficacy of ΤMΖ and OXA in MGMT-expressing GBM, providing additional therapeutic benefits. These results suggest that combined therapy of multidrug repositioning with PARP inhibitors and alkylating drugs shows promising results in GBM treatment [26]. Typically, patients with MGMT-deficient tumors are treated with TMZ [27, 28]. Resistance of GBM cells to TMZ is primarily due to their robust repair mechanisms, including BER and MGMT. PARP1, which is inhibited by OLA, senses DNA damage and then activates signalling pathways that promote appropriate cellular DNA repair responses, including BER [27, 28]. The presented results are in accordance with a study performed by Xu et al. [14], which demonstrated that OLA modulated PARP1 expression and enhanced the sensitivity of colorectal cancer (CRC) cells to OXA. Moreover, OLA alone did not induce DSBs but enhanced the induction of DSBs by OXA [14]. Flow cytometry results performed by Xu et al. showed that cells exposed to the combined treatment had a higher percentage of cells in the G2/ M phase than the control. Furthermore, studies suggest that the combined treatment with OLA and OXA inhibited the growth of CRC [14, 29].

In our in vitro studies, administering OLA increased the cell viability of both TMZ and OXA in GBM cells. All tested GBM cell lines were more sensitive to combinations of two (OLA + TMZ, OXA + TMZ, OXA + OLA) and three drugs (OLA + OXA + TMZ) than to TMZ and OLA alone at higher concentrations. Worthy to note that we used differentiated cell cultures (FBS-supported monolayers) instead of undifferentiated cell cultures (neurospheres) based on a previous study, which found that there were no significant differences between the two cell culture systems in TMZ-induced DNA damage [17].

Contrary to our speculations, we did not demonstrate a survival benefit for the combined treatment with OLA and OXA compared with OXA monotherapy in the in vitro study. The combination of OXA with OLA did not show a synergistic or additive effect at a concentration of 50 µM, as their combination caused approximately the same level of cell death as OXA alone (Figs. 2B and 3). However, in the study by Roberts et al. [16], OXA reduced the level of MGMT and increased the sensitivity to TMZ on KR158 and GL261 glioma cells [16, 29]. Nonetheless, we found no studies investigating the combination of OLA and OXA in GBM.

In a study conducted by Schaff et al. [18], OLA also proved to be a promising therapeutic drug for gliomas harboring an HRD variant of IDH, such as those observed in BRCA-mutated tumors [18, 30]. In our study, we observed that OLA can enhance the efficacy of the DNA alkylator, potentially sensitizing resistant H4 and U118 glioma cells to TMZ or OXA (H4, U118), even in the absence of HRD. The combination of TMZ or OXA with OLA can increase HRD in IDH-mutated tumors and is associated with higher mortality in glioma cell lines. Hwang et al. [3] also highlighted the synergistic effect of OLA with TMZ in the GBM model, especially in the T98, U87, and U251 cell lines [3]. The authors showed that OLA enhanced the effect of TMZ, but not OXA, in all glioma lines tested, regardless of MGMT status. The combination of OLA and TMZ resulted in a higher degree of cell death than OLA alone, suggesting an additive effect of OLA in combination with TMZ (Figs. 2F and 3).

The significant active involvement of OLA in TMZ- or OXA-induced DNA damage in GBM cells may prevent the activation of their systems by GSCs, making them more susceptible to TMZ and OXA. As a result, the viability of GBM cells is reduced when TMZ or OXA is used in combination with OLA (Fig. 3). A stronger chemotherapeutic effect was observed for the three-drug combination in cell lines (U118, U87) compared with the two-drug combination at the same concentration (OLA + TMZ, OXA + TMZ, OXA + OLA) (Fig. 3). The cell line H4 was the most sensitive to the combination of OLA and TMZ. The higher sensitivity to treatment of the H4 line may be due to lower MGMT expression compared to the U87, U118, and U251 glioma cell lines, which are characterized by higher MGMT expression and thus greater resistance to chemotherapy [14, 26–28]. No evidence of necrosis was observed in the above drug combinations. Therefore, the proposed multidrug repositioning is crucial not only for the drug’s efficacy but also might be important for minimizing side effects.

Based on the obtained results, concurrent treatment with OLA and TMZ or the triple combination of drugs (OXA + TMZ + OLA) induced apoptosis rather than necrosis in GBM cells. The clinically important results were observed in the normal cell line hFib, where more than 50% of cells remained viable after treatment with lower drug concentrations and showed early or late apoptosis without necrosis. The inclusion of appropriate amounts of OLA and/or OXA in the standard chemotherapy regimen for GBM, instead of frequent dose escalation of TMZ, may provide beneficial effects, especially in TMZ-resistant patients with advanced GBM. Once again, no evidence of necrosis was observed in the above drug combinations. These investigations were carried out as part of an examination of complex multidrug chemotherapy with radiotherapy (proton or photon) as a new strategy for the treatment of GBM. Human skin biopsies and then fibroblast culturing have become an established normal tissue response model after the mentioned types of therapies, especially radiotherapy [31, 32]. Therefore, in this study, the hFib serves as a control and elucidates the complexity of the GBM response. It is worth noting that the human astrocytes may also be considered as control; however, they might be composed of multiple subtypes and characterized by different genetics and therapy response [33].

To summarize, combining a PARP inhibitor, OLA, with TMZ or OXA or both may offer a promising treatment approach for GBM, regardless of MGMT promoter methylation status. Multidrug repositioning can help minimize side effects by lowering drug doses while significantly enhancing chemotherapeutic efficiency in GBM cell lines compared to single-drug treatment. Although our results are preliminary, the synergistic effect of the three compounds might be proposed as a modern chemotherapy strategy for GBM. Further studies on the synergistic action of repositioned drugs (OXA, OLA) in combination with TMZ may bring a breakthrough, which will ultimately enable more effective and safer pharmacotherapy for the patient. Therefore, in vitro and in vivo molecular studies on OXA and OLA in combination with TMZ in GBM different models (i.e., 2D and 3D) are necessary and will be performed in the next part of the ongoing project.

Limitations of the study

One limitation of the present study is the lack of experimental groups receiving specific drug combinations: 25 µM OXA + 100 µM TMZ on all cell lines; 25 µM OXA + 25 µM OLA on H4 cell line; 25 µM OLA + 100 µM TMZ on H4 cell line; 25 µM OXA + 75 µM OLA on U118 cell line; 7 µM OLA + 100 µM TMZ on U118 cell line; 25 µM OXA + 75 µM OLA on U87 cell line; 75 µM OLA + 100 µM TMZ on U87 cell line; and the lack of different drug combinations on U251 cell line. This limitation prevented the use of multivariate analysis of variance (ANOVA) to fully investigate potential drug interactions. Instead, we used one-way analysis of variance (ANOVA) to examine the effects of drug combinations at the same concentration on selected glioma cell lines. In future research, we would aim to employ a more complete factorial design to enhance the robustness and validity of drug–drug interaction analyses.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AD

Adenine–ribose

ART

ADP–ribosyltransferase

BER

Base excision repair

BRCA

Associated breast and ovarian cancers

CDI

Carbonyl diimidazole

CHK1

Checkpoint kinase 1

CNS

Central nervous system

CRC

Colorectal cancer

DCC

N,N’–dicyclohexylcarbodiimide

DCM

Dichloromethane

DIC

N,N’–diisopropylcarbodiimide

DMF

Dimethylformamide

DMSO

Dimethyl sulfoxide

DSBs

Double–strand breaks

GBM

Glioblastoma multiforme

GSCs

Glioma stem–like cells

HATU

Hexafluorophosphate azabenzotriazole tetramethyl uronium

HBTU

O–benzotriazole–N,N,N’,N’–tetramethyl–uronium–hexafluoro–phosphate

HCL

Hydrochloric acid

HD

Helical

hFib

Human fibroblast line

HPLC

High–performance liquid chromatography

HRD

Homologous recombination deficiency

IC₅₀

Half maximal inhibitory concentration

IDH

Isocitrate dehydrogenase

MGMT

O6methylguanine–DNA methyltransferase

MMR

Mismatch repair

MTS

3–(4,5–dimethylthiazol–2–yl)–5–(3–carboxymethoxyphenyl)–2–(4–sulfophenyl)–2 H–tetrazolium

NI

Nicotinamide

OLA

Olaparib

OXA

Oxaliplatin

O6

MeG–O6–methylguanine

PARP1

Poly(ADP–ribose) polymerase 1

PH

Phosphate

RT

Radiotherapy

SI

Supplementary information

TBTU

O–(benzotriazol–1–yl)–N, N,N’,N’–tetramethyluronium tetrafluoroborate

TEA

Triethylamine

TMZ

Temozolomide

Author contributions

Anna Zając-Grabiec*, conceptualization, data curation, funding acquisition, investigation, resources, methodology, roles/writing - original draft, writing - review & editing. Anna Czopek*, investigation, data curation, visualization, methodology, funding acquisition, roles/writing - original draft, writing - review & editing. Karolina Pazdan, investigation, methodology. Jakub Jończykb, software, data curation, visualization, methodology, funding acquisition, roles/writing - original draft. Filip Michałkiewicz, software, formal analysis, validation, visualization. Tomasz Skόra, investigation. Monika Krzyżowska, investigation, methodology. Beata Biesaga, supervision. Dominik Wiśniewski, investigation, methodology. Paula Ajersch, formal analysis, validation, visualization. Justyna Miszczyk, funding acquisition, supervision, writing - review & editing.

Funding

These studies were performed at Cyclotron Centre Bronowice IFJ PAN under the project Miniatura 8, No. 2024/08/X/NZ7/00625, funded by the National Science Centre, Poland, and partially funded from the state budget under the Ministry of Education and Science (Poland) program entitled Science for Society II, No. NdS-II/SP/0295/2023/01, total project amount 1 mln PLN. The synthesis and in silico study were financially funded by JU MC Funds (N42/DBS/000412 and N42/DBS/000415).

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Human Bioethical Committee of the Regional Medical Board in Kraków (Approval No. 163/KBL/OIL/2023, dated 06.07.2023). Written informed consent was obtained from the participant prior to the collection of the skin fragment.

Consent for publication

Written informed consent for publication was obtained from the participant.

Institutional review board

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Human Bioethical Committee of the Regional Medical Board in Kraków (No. 163/KBL/OIL/2023, 06.07.2023). Written informed consent was obtained from the participant involved in the study before the fragment of skin was taken.

Competing interests

The authors declare no competing interests.