Verapamil potentiates anti-glioblastoma efficacy of temozolomide by modulating apoptotic signaling

Accepted Manuscript Verapamil potentiates anti-glioblastoma efficacy of temozolomide by modulating apoptotic signaling

Farina Hanif, Kahkashan Perveen, Saima M. Malhi, Huma Jawed, Shabana U. Simjee PII: DOI: Reference:

S0887-2333(18)30343-6 doi:10.1016/j.tiv.2018.07.001 TIV 4321

To appear in:

Toxicology in Vitro

Received date: Revised date: Accepted date:

14 February 2018 5 July 2018 6 July 2018

Please cite this article as: Farina Hanif, Kahkashan Perveen, Saima M. Malhi, Huma Jawed, Shabana U. Simjee , Verapamil potentiates anti-glioblastoma efficacy of temozolomide by modulating apoptotic signaling. Tiv (2018), doi:10.1016/ j.tiv.2018.07.001

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ACCEPTED MANUSCRIPT Verapamil potentiates anti-glioblastoma efficacy of temozolomide by modulating apoptotic signaling Farina Hanif1,2 [email protected]; Kahkashan Perveen2,4 [email protected]; Saima M. Malhi1,3 [email protected]; Huma Jawed3,5 [email protected] and Shabana U. Simjee2,3

[email protected] 1

Institute of Biomedical Sciences, Dow University of Health Sciences, Ojha Campus, SUPARCO Road, Karachi, Pakistan 2

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Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan 3

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H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan Department of Biochemistry, Baqai Medical University, Karachi, Pakistan

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Mohammad Ali Jinnah University, Karachi, Pakistan

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Corresponding author.

ACCEPTED MANUSCRIPT Abstract Glioblastoma Multiforme (GBM) is the most malignant and invasive tumor of the CNS. Although temozolomide (TMZ) has improved the survival, long-lasting responses have not been reported. Therefore, there is a need to develop improved treatments, one of which might be newly identified drugs which can be used in combination therapy with low doses of standard drugs. Verapamil (VP) a known antihypertensive drug has been shown to

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enhance the activity of bis-chloroethylnitrosourea (BCNU), a drug used to treat GBM. Since, TMZ has replaced BCNU as the standard GBM chemotherapy; therefore, we aimed to study in vitro interaction of VP and TMZ against

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GBM. Anti-proliferative and apoptotic activities were studied using MTT, TUNEL assay and DAPI staining. Synergy was assessed using combination index method. Apoptotic markers were evaluated by RT-PCR, and

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immunocytochemistry. Both VP and TMZ significantly inhibited the growth of U87 cells in dose dependent manner. The combine effect of TMZ with VP was synergistic with a CDI value of <1. Combination of TMZ and VP

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increased the ratio of Bax to Bcl-2 expression and thus shifted the equilibrium of cells towards apoptosis. Our findings suggest that the synergistic growth inhibition that was observed in combination treatment group may in part

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relate to increase in apoptosis. The combine administration of VP and TMZ may be therapeutically exploited for the

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management of GBM.

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Keywords: Glioblastoma, Temozolmide, Verapamil, Apoptosis

ACCEPTED MANUSCRIPT Introduction Glioblastoma multiforme (GBM) is the most common, invasive and undifferentiated type of tumor and has been designated Grade IV by world health organization (WHO). Almost 60% of all brain tumors in adults are GBM. In spite of several international efforts, GBM treatment is still the most challenging task in clinical oncology (Mrugala, 2013). Advances in surgical approaches, radiotherapy, and adjuvant chemotherapy have shown gradual

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improvements in survival and quality of life of the GBM patients but the prognosis is still depressing. However, a

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much more significant pace needs to be made to see positive outcomes, analogous to those seen in certain other

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cancers that can now be treated successfully (Hanif et al., 2017; Mrugala, 2013; Ohka et al., 2012). The current standard of care for high-grade gliomas patient is surgery along with radiation and concomitant adjuvant

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temozolomide (TMZ) therapy (Mrugala, 2013). Although temozolomide has improved the survival, the prognosis is still extremely poor with patients usually having a median survival of approximately 14-15 months after diagnosis

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(Hanif et al., 2017; Mrugala, 2013). The principal mechanism responsible for the cytotoxicity of TMZ is to methylate DNA at the N7 and O6 position on guanine which leads to the failure of DNA miss match repair system to

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find a complementary base for methylated guanine thus resulting in long lived nicks in DNA and consequently

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blocks the cell cycle at the G2-M boundary that triggers apoptosis (Scott et al., 2011). However it has been reported that high levels of methyl guanine methyl transferase (MGMT) activity in tumor cells is associated with poor

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temozolomide response. It has also been reported to produce cytotoxic effect by pro-autophagic (Kanzawa et al., 2004) and/or apoptotic pathway (Hanif et al., 2014). Although TMZ has slightly increased the survival of patients, it

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is also responsible for inducing many side effects (Dario and Tomei, 2006; Friedman et al., 2000; Sengupta et al.,

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2012; Singhal et al., 2007).

Verapamil (VP) is an anti-hypertensive calcium channel blocker used in the treatment of hypertension, arrhythmias, and certain vascular diseases of the brain (Conlin and Williams, 1998). VP has been found to overcome Pglycoprotein mediated multidrug resistance and to increase sensitivity to cytotoxic anticancer drugs. It was also reported that meningiomas that were treated with VP were smaller in size and less vascular, with lower cell proliferation and greater apoptosis. It’s cytotoxic and apoptotic effect has also been observed on human colon and breast cancer cells (Zhang et al., 2009). It has been reported to enhance the activity of anticancer drug BCNU (Bowles et al., 1990). These studies have indicated that VP could be a potential anticancer agent or synergist in cancer treatment.

ACCEPTED MANUSCRIPT Since, TMZ has replaced BCNU as the standard initial chemotherapy in the treatment of GBM (Azzalin et al., 2017; Vinjamuri et al., 2009) therefore; we aimed to study the in vitro interaction of VP and TMZ in inhibiting GBM. According to best of our knowledge no data has been published on the combined effect of VP and TMZ.

Material and Methods:

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Drugs/Compound Preparation and Cell Culturing:

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The stock concentrations of TMZ and VP (Sigma Chemical Company, MO, USA) were prepared in sterile 100%

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dimethyl sulfoxide (DMSO) and stored at -20C. Six different working concentrations of TMZ (0.025 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.5 mM) and VP (0.01mM, 0.02 mM, 0.05 mM 0.1 mM, 0.2 mM and 0.4 mM)

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were prepared in culture media and used to treat the cells. In addition to the above mentioned concentrations cells were also treated with various combinations of TMZ and VP (0.1 mM + 0.01 mM, 0.1 mM + 0.025 mM, 0.1 mM +

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0.05mM, 0.2 mM + 0.01 mM, 0.2 mM + 0.025 mM, 0.2 mM + 0.05 mM) to see their combined effect. Cells treated

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with only DMSO were used as controls. Final Concentration of DMSO used was 0.1%. Human glioblastoma cell line U87 (ATCC number: HTB-14TM) was cultured in DMEM (Sigma Chemicals)

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supplemented with 1% penicillin and streptomycin, 1%, amphotericin B, 1% sodium pyruvate, 1% L-glutamine and

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10% fetal bovine serum (FBS) in a humidified atmosphere at 37C containing 5% CO2.

(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) Assay:

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The effect of TMZ and VP on GBM tumor cell growth was evaluated by the MTT assay (Mosmann, 1983). Cells were treated with both the drugs as a single agent and in above mentioned combinations. The cells (3x10 3 cells/100

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µl) were plated in 96-wells plate and incubated at 37C containing 5% CO2 for 24 hrs. Following incubation, media was aspirated and cells were treated with 100 µl of different test concentrations of VP and TMZ and their combinations in media having 1% FBS. Cells treated with 0.1% DMSO in media (1% FBS) were used as untreated controls. The plates were re-incubated for 24 hrs and on the following day MTT dye (Promega, USA) was added to each well and after 3 hrs incubation, 100 µl of DMSO (100%) was added to each well to solubilize formazan and the absorbance was recorded at 490 nm (Wilson and Anne, 2000). All assays were performed in triplicate and the growth inhibition (%) was calculated (Patel et al., 2009). IC50 was calculated using the formula mentioned by Mathieu et al. (2008).

ACCEPTED MANUSCRIPT Morphological Features of the U87 Cells Following Drug Treatment Cells were grown in 25 cm3 flasks and treated with IC50 concentration of drugs and DMSO (vehicle control) for 24 hrs. The images were captured at 10X and 20 X magnifications viewing under Nikon inverted microscope.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Assay for Apoptosis:

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U87 glioblastoma cells were grown in two chambered slides (Lab-Tek®) and treated with IC50 doses of drugs and

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DMSO for 24 hrs at 37⁰C and 5% CO2. After incubation, DeadEndTM TUNEL assay system kit (Promega

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Corporation, USA) was used to detect apoptosis in cell as per manufacturer protocol. Cells with dark brown nuclei were considered apoptotic (Yu et al., 2001).

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4, 6-diamidino-2-phenylindole (DAPI) Staining:

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It has been documented that DAPI staining can identify chromatin condensation which is one of the hallmarks of apoptosis. (Kobayashi et al. 2017), therefore we have used DAPI staining to show chromatin condensation in treated

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cell. Briefly, U87 glioblastoma cells were plated in two chambered slides (Lab-Tek®) and incubated at 37C and 5% CO2 for 24 hrs with IC50 doses of drugs and DMSO. Following incubation, cells were fixed with 4%

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paraformaldehyde, washed with PBS (5X) and blocked with blocking solution (2% bovine serum albumin (BSA), 2% normal goat serum and 0.2% Tween20 all prepared in PBS) at 37°C for 1 hr. Then cells were washed again and

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counterstained with DAPI in 1:500 dilutions in PBS. The stained slides were then mounted with PBS/glycerol (1:1)

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and viewed under a fluorescent microscope (NIKON). Synergy analysis of combine drug effects:

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To determine the effect of drug combination on growth inhibition of U87 cells, the coefficient of drug interaction (CDI) was calculated using formula, CDI = AB / (A X B). Where, AB, and A or B represent the ratio of the combination groups to control group (DMSO) and the ratio of single agent (drug) to control group (DMSO) respectively. The combined effect said to be synergistic, additive or antagonistic if the CDI value is less than, equal to or greater than 1 respectively (Qiu et al., 2012).

ACCEPTED MANUSCRIPT Semi-quantitative RT-PCR: Cells were cultured in 75cm3 flasks and treated for 24 hrs with VP (0.025 mM) and TMZ (0.1 mM) alone, and in combination (VP 0.025 mM + TMZ 0.1 mM). Cells treated with DMSO served as vehicle control. Following incubation, cells were trypsinized, centrifuged at 1000 rpm for 8 minutes and pellets were used to isolate total RNA using SV Total RNA Isolation System kit (Promega, USA). The RNA samples were reverse-transcribed into cDNA

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using RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Maryland, USA). The transcribed cDNA (1µl) was then amplified using PCR Master Mix (Fermentas, USA) and oligonucleotide primers corresponding to transcripts

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of the respective genes. The PCR products were resolved on ethidium bromide stained 1% agarose gel and intensity of bands was quantified using Quantichrome software. Density of each mRNA band was normalized to its respective

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housekeeping gene i.e. β-actin. The primer sequences used in this study were β-Actin (sense, 5’GTCCTGTGGCATCCACGAAAC-3’) (antisense, 5’ - GCTCCAACCGACTGCTGTCA-3’) Bax (sense, 5’(antisense,

5’-GGCCCCAGTTGAAGTTGC-3’),

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AAGAAGCTGAGCGAGTGTC-3’)

(antisense,

(sense,

5’-

5’-GGCAGGCATGTTGACTTCAC-3’),

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ACTTCGCCGAGATGTCCAGC-3’)

Bcl-2

Immunocytochemical Analysis:

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CAAGCTTGTCGGCATACTGTTTCAG-3’), (Ning et al., 2010; Rzeski et al., 2006; Winter et al., 2001).

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Cells were plated in two chambered slides (Lab-Tek®) and incubated at 37C and 5% CO2 for 24 hrs with effective individual doses involved in synergistic activity of TMZ and VP and their respective combination. After incubation,

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cells were fixed with 4% paraformaldehyde, washed with PBS (5X) and blocked with blocking solution (2% bovine

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serum albumin (BSA), 2% normal goat serum and 0.2% Tween20 all prepared in PBS) at 37°C for 1 hr. Next, cells were re-washed with PBS (5X) and incubated with mouse anti-Bax (2d2) monoclonal antibody and mouse anti-Bcl2 monoclonal antibody (Santa Cruz Biotechnology) separately in 1:100 dilutions at 4°C overnight. On subsequent day cells were washed with PBS three times and then incubate for 1 hr at room temperature with secondary antibodies; Alexa Flour®546 anti-mouse IgG (for Bax) and Alexa Flour

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anti-mouse IgG (for Bcl-2) at 1:200

dilutions in PBS. Then cells were washed again and counterstained with 4, 6-diamidino-2-phenylindole (DAPI) in 1:500 dilutions in PBS. The stained slides were then mounted with PBS/glycerol (1:1) and viewed under a fluorescent microscope (NIKON). The captured images were analyzed using ImageJ software (NIH, USA). Briefly, 5 fields were selected blind based and the intensity of fluorescence was quantified in cells and subsequently the

ACCEPTED MANUSCRIPT background intensity was subtracted from it to represent Bax or Bcl-2. Cells were counted and % intensity of expression was calculated. Data from three different experiments were combined and expressed as means ± standard error of the mean (SEM).

Statistical Analysis:

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The results obtained from various experiments were analyzed with SPSS-19 software. Data were expressed as mean

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Bonferoni post hoc test. P < 0.05 was considered to be statistically significant.

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± SEM of separate experiments (n ≥ 3) and compared by one-way analysis of variance (ANOVA) followed by

Results

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TMZ and VP Inhibited growth of U87 Cells

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Data analysis of MTT assay for TMZ demonstrated significant inhibition of the U87 cells growth in dose dependent manner compared to vehicle control group (ranging from P < 0.001 to P < 0.05). The IC50 dose of TMZ was

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calculated as 0.45 mM. The Bonferoni’s multiple comparison test revealed a significant difference in growth inhibition among TMZ 0.025 mM : 0.05 mM (P < 0.01), TMZ 0.05 mM : 0.1 mM (P < 0.01), TMZ 0.1 mM : 0.2

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mM (P < 0.001) and, TMZ 0.4 mM and 0.5 mM (P < 0.05) treated groups (Fig. 1a).

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Verapamil treatment demonstrated a significant inhibition of the U87 cells growth (P < 0.001) in dose dependent manner compared to vehicle control group in MTT assay. The IC50 dose of VP was calculated as 0.134 mM. A trend

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of dose dependent growth inhibition was observed from the 0.025 mM dose of VP, however, the Bonferoni’s multiple comparison tests revealed that the significant difference exist between VP 0.025: 0.05 mM and VP 0.2 mM:

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0.4 mM (P < 0.001) treated groups (Fig. 1b).

Effect of TMZ and VP on Cellular Morphology, DNA Fragmentation and Chromatin Condensation The U87 cells were treated for 24 hrs with DMSO, and IC50 doses of TMZ (0.45 mM) and VP (0.134 mM). The cells were then visualized by phase-contrast microscopy under 20X magnification to evaluate their effects on cellular morphology as well as on DNA fragmentation and chromatin condensation both are hallmarks of apoptosis.

Dramatic morphological changes were observed in the cells treated with TMZ and VP for 24 hrs (Fig. 1c). The cells were prominently rounded and detached from the surface. Phase contrast photomicrographs after TUNEL assay

ACCEPTED MANUSCRIPT clearly showed an increase in apoptotic cells in the treated groups (Fig. 1c). The DAPI staining showed a prominent chromatin condensation in both TMZ and VP exposed cells, while no condensation was observed in vehicle control groups (Fig. 1c).

Combine Effect of TMZ and VP Treatment on U87 cells Growth Inhibition

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To determine whether the growth inhibitory effects of TMZ on U87 cells could be enhanced by VP, cells were

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treated in various combinations of the drugs for 24 hrs and MTT assay was performed. When statistically analyzed it was found that growth inhibition increased significantly when TMZ 0.1 mM combined with 0.025 mM of VP (P <

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0.001) as compared to their individual treatment (Fig. 1d). Coefficient of drug interaction (CDI) was also calculated to determine whether the combined effect of TMZ and VP was synergistic or additive. The combined effect of TMZ

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0.1 mM with 0.025 mM of VP was found to be synergistic with a CDI value of 0.915. Lowest doses showing

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synergistic effect were used for further studies.

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Modulation of Bcl-2 and Bax mRNA Expression after Treatment with TMZ, VP and their Combination i.e., TMZ + VP

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Figure 2 shows the expression of Bcl-2 mRNA in U87 cells. The intensity of gene expression was quantified using Spot Density tool of Gel-Doc system for comparison purpose. The housekeeping gene β-actin was amplified as an

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internal control and the integrated density value (IDV) of each band was normalized with their respective β-actin IDV. The gel pictures clearly showing that Bcl-2 mRNA expression has become faint in VP treatment group. No

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observable difference in the intensity of band was noticed when we compared vehicle control (VC) group with the TMZ alone treatment group and VP alone with TMZ + VP. When analyzed statistically it was found that low doses

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of VP (Fig. 2a) (P < 0.01) and its combination with TMZ i.e., TMZ + VP has significantly down-regulated the expression of Bcl-2 (P < 0.001) as compared to vehicle control. Next we analyzed the data employing Bonferoni’s multiple comparison tests and found significant difference in Bcl-2 mRNA expression when TMZ alone compared with TMZ + VP combination treatment group (P < 0.001). However no statistically significant decrease in expression was noticed with TMZ alone compared with vehicle control and among VP and TMZ + VP.

In case of Bax, the gel image (Fig. 2b) showed a faint band in vehicle control group whereas bright band was detected in TMZ treatment group; indicating increase in intensity of Bax mRNA expression. While in the case of VP

ACCEPTED MANUSCRIPT treatment, the intensity of band was comparable to that of vehicle control (Fig. 2b). When data was analyzed, significant increase in Bax mRNA expression was observed in TMZ (P < 0.001), and TMZ + VP (P < 0.001) treatment group as compared to control. No significant difference in Bax mRNA expression was observed among VP and vehicle control group. When the expression was compared among the individual drug treatment group, a

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significant increase was observed in TMZ + VP treatment group as compared to VP.

Immunocytochemical Analysis of Bcl-2 and Bax Protein Expression in U87 Cells after Treatment with TMZ,

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VP and their Combination i.e., TMZ + VP

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The effect of TMZ and VP as individual drug and their combination on Bcl-2 and Bax protein expression was determined immunocytochemically. Figure 3 shows a marked decrease in the level of Bcl-2 immunofluorescence in

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VP treatment. While in TMZ treated group, the Bcl-2 immunofluorescence was comparable to that of vehicle control group. The intensity of immunofluorescence was measured and data was quantified using ImageJ software.

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A significant decrease in the Bcl-2 protein expression after 24 hrs treatment with VP (P < 0.05) and TMZ + VP (P <

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0.01) was observed. The Bonferoni’s multiple comparison tests revealed that the expression was further downregulated in combination treatment group, however, the decrease was not statistically significant among VP and

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TMZ + VP group.

In contrast to Bcl-2, negligible Bax immunofluorescence was detected in vehicle control whereas increased level of

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immunofluorescence was detected in all treatment groups as compared to control (Fig. 4). When the data was analyzed using One-way ANOVA test, significant increase in Bax protein expression in all treatment group

observed.

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including TMZ, (P < 0.001), VP (P < 0.05) and TMZ + VP (P < 0.001) as compared to vehicle control was Furthermore,

posthoc

Bonferoni’s

multiple

comparison

tests

revealed

that

the

level

of

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immunofluorescence is significantly higher in combination treatment group TMZ + VP as compare to VP alone. However no significant difference was observed between TMZ alone and TMZ + VP treatment group. Analysis of Bax/Bcl-2 ratio after Treatment with TMZ and VP alone and their Combination A balance between the expression of pro-apoptotic protein (Bax) and anti-apoptotic protein (Bcl-2) plays an important role in the initiation of apoptosis. We therefore studied the ratio of Bax to Bcl-2 at translational level. Figure 5 shows that all the treatment groups including TMZ (P < 0.01), VP (P < 0.01) and combination therapy TMZ + VP (P < 0.001) induces significant increase in the Bax/Bcl-2 ratio and the highest increase was observed in

ACCEPTED MANUSCRIPT combination treatment group. Level of significance was found to be (P < 0.05), and (P < 0.001) when individual treatment group of TMZ and VP were compared with TMZ + VP treatment group respectively.

Discussion: Poor prognosis of glioblastoma with survival rate of 14-15 months after diagnosis makes it a crucial public health

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issue. In spite of several efforts, GBM treatment is still the most challenging task in neuro-oncology. Over the last

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decade, a range of different treatments were investigated with very limited success. Main challenges in therapy of

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GBM are related with the location of the disease and its complex and heterogeneous biology. Therefore, more significant pace needs to be made to see positive outcomes. New drug development is highly resource-intensive and

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expensive. Another potentially fruitful area is the repurposing of drugs, in which a drug originally licensed for one disease is found to work well for another either alone or in combination with available standard drugs. However,

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combining multiple cytotoxic drugs for cancer therapy may sometimes leads to risk of toxicity which is associated with drug-drug interaction (DDI) and might compromise the quality of patients’ life. Before prescribing multiple

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drugs, patients at high risk of DDI should be screened first, for example patients with many comorbidities. If

2014).

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possible physicians should substitute perilous drugs to safer alternatives (Riechelmann and Girardi 2016; Feng et al. The present study is providing very first time the data of combine action of verapamil a known

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antihypertensive drug reported to be used in certain vascular diseases of the brain (Kunert‐Radek et al., 1989) and

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temozolomide (standard drug for GBM) on human glioblastoma cell line U87.

We confirmed that both TMZ and VP inhibit the growth of U87 glioblastoma cells in dose dependent manner and

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these findings are in accord with the study of Schmidt et al. (1988) and Carmo et al. (2011) which showed that TMZ and VP inhibit the proliferation of glioma cells respectively (Carmo et al., 2011; Schmidt et al., 1988). To determine whether apoptosis is involved in the TMZ and VP mediated growth inhibition of U87 glioblastoma cells, the effect of IC50 value of these drugs was observed on morphology. The cells showed apoptotic morphology in both treatment group as described by Jiang et al. (2004). Presence of apoptosis was confirmed by DNA fragmentation and chromatin condensation which are hallmarks of apoptosis. In both the treatment groups DNA fragmentation was evident in treatment groups as dark brown stained nuclei which resulted from the labeling of fragmented DNA at free 3’ OH with

biotinylated nucleotide using recombinant enzyme rTdT. DNA fragmentation along with

ACCEPTED MANUSCRIPT characteristic chromatin condensation showed that cells are undergoing apoptosis. Thus the growth inhibitory activity of TMZ and VP was at least in part found to be related to apoptosis. Our results are supported by the studies demonstrating the ability of, TMZ (Günther et al., 2003) and VP (Cao et al., 2005) to induce chromatin condensation, fragmentation and thus apoptosis.

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Keeping in mind the effect of IC50 dose of TMZ and VP on U87 cells we decided to explore the therapeutic benefits

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of VP in combination with standard drug TMZ that may not only reduce the dose of the drugs to produce therapeutic effect but will also help to reduce the side effects of the standard drug. In the combination study, it was observed

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that VP synergistically increases the growth inhibition of U87 cells when given in combination with TMZ. Observing the effect of IC50 doses of these drugs, we hypothesized that the synergistic activity observed might be a

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consequence of changes in the expression of the apoptotic markers. Deregulated apoptosis is a common occurrence in oncogenesis and contribute to drug resistance (Fisher, 1994). It is also found to be deregulated in gliomas

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(Steinbach and Weller, 2004) and therefore it is interesting to target underlying molecular markers involved in the

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apoptotic process. Therefore we also investigated the effect of the doses that are used in combination study of these drugs on the expression of Bax and Bcl-2 at both transcriptional and translational level. Pro-apoptotic (Bax) and

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anti-apoptotic (Bcl-2) proteins are subgroup of Bcl-2 family proteins that promotes and inhibit apoptosis respectively (Cohen, 1997). Over expression of Bcl-2 protein is observed in various tumor cells which then increase

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the tumor cell growth and resistance by decreasing Bax-Bcl-2 ratio. Bax in itself does not cause cell death but by countering Bcl-2 activity it favors an entry into the apoptotic program. Thus imbalance in the intracellular

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environment towards an increased Bax/Bcl-2 ratio will favor apoptosis (Oltval et al., 1993).

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The present study revealed significant down-regulation of Bcl-2 in VP alone and combination treatment group i.e. VP + TMZ (at both transcriptional and translational level) as compared to vehicle control group. However, no significant difference in down-regulation of Bcl-2 was observed when these two groups are compared to each other. Nevertheless, the difference was significant between TMZ alone and VP + TMZ treatment group. Hence we can say that the down-regulation of Bcl-2 which is observed in combination treatment group may be due to individual effect of VP. On the other hand Bax expression was significantly pronounced in TMZ alone and combination treatment group (VP + TMZ) at both mRNA and protein level as compared to vehicle control group. However no significant increase in Bax expression was observed when these two groups were compared. The difference was significant

ACCEPTED MANUSCRIPT between VP alone and combination treatment group (VP + TMZ). Previous studies have also shown that TMZ can induce Bax expression and has no effect on Bcl-2 expression (Gupta et al., 2006). There are only few studies which have shown the effect of VP on Bax and Bcl-2 expression. Our results are in accord with the study of Meister et al. (2010) who showed the down regulation of Bcl-2 mRNA and up-regulation of Bax mRNA in melanoma cells following exposure to verapamil (Meister et al., 2010). It has been shown that apoptotic activity of VP can be

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abrogated by inducing the over expression of Bcl-2 (Karwatsky et al., 2003). Verapamil also reduce the Bcl-2 and

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increase Bax expression in rat eggs in calcium deficient medium (Chaube et al., 2009). However another study

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shows that VP did not modulate the expression of Bax and Bcl-2 in different tumor cell lines (El-Moselhy et al., 2012). In the present study because of the significant inhibition of Bcl-2 by VP treatment only and significant

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increase in Bax expression by TMZ as compared to VP, the Bax/ Bcl-2 was highest in combination treatment group i.e. VP + TMZ. It is reasonable here to assume that increased Bax/ Bcl-2 ratio of expression may have shifted the

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equilibrium of the cells towards apoptosis and hence could be responsible for the synergistic activity in combination

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treatment group.

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Conclusion:

The present study demonstrated that VP has the ability to potentiate the in vitro efficacy of TMZ when given in

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combination with TMZ. Further VP may increase the efficacy of TMZ by increasing Bax/Bcl-2 ratio. Still there is a need to explore the interaction of VP and TMZ at molecular level targeting major signaling pathways associated

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with the progression of glioblastoma. In future, this approach might help in halting the progression of the tumor by targeting selective molecular markers and for better management of GBM.

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Conflict of Interest

The authors declare they have no competing interests as defined by Toxicology In Vitro.

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Combination of temozolomide and verapamil synergistically inhibited the growth of U87 glioblastoma cells Growth inhibitory activity of Temozolomide and verapamil was related to increased apoptosis Combination of TMZ and VP increased apoptosis by increasing ratio of Bax to Bcl-2 expression Combine administration of VP and TMZ may be therapeutically exploited for the management of GBM.

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