Cytotoxic and Radio-sensitizing Effects of ...

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Cytotoxic and Radio-sensitizing Effects of Polyphenolic Acetates in a Human Glioma Cell Line (BMG-1) Amit Verma1, Kavya Venkateswaran1, Abdullah Farooque1, Anant Narayan Bhatt1, Namita Kalra1, Anu Arya4, Ashish Dhawan4, Mohan B. Arya2, Hanumantharao G. Raj3, Ashok K. Prasad4, Virinder S. Parmar4, Bilikere S. Dwarakanath1,* 1

Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Majumdar Marg, Timarpur, Delhi-110054; 2SSVPG College, Hapur, U.P. India; 3V. P. Chest Institute, University of Delhi, Delhi-110054, India; 4Department of Chemistry, University of Delhi, Delhi110007, India Abstract: Background and Aims: Polyphenolic acetates (PAs) have antioxidant/ pro-oxidant properties and can also acetylate proteins (enzymes) by a novel acetoxy drug: calreticulin transacetylase acetylation system. While PAs have been investigated as pharmacological agents for the treatment of various diseases, their potential as anti-cancer agents or their efficacy as an adjuvant in anti-cancer therapeutics remains to be explored. In the present study we investigated the cytotoxic and radio-sensitizing effects of 7, 8- diacetoxy-4-methyl coumarin (DAMC) and 7- acetoxy-4-methyl coumarin (7-AMC) in a human glioma cell line (BMG-1). Methods: Cytotoxic and radio-sensitizing effects were investigated by studying reactive oxygen species (ROS) levels, metabolic viability, clonogenic survival, growth inhibition, cell cycle perturbation, DNA repair and cytogenetic damage, besides analyzing the histone (H3) acetylation. Results: Exposure of cells to DAMC and 7-AMC for 24 h showed a dose dependent increase in toxicity as indicated by reduced metabolic viability, clonogenic survival and cell proliferation, with DAMC being more toxic than 7-AMC. The degree of radiosensitization by DAMC was also higher as compared to 7-AMC as reflected by a decrease in the clonogenicity, enhanced radiation-induced cell cycle perturbation and apoptosis. DAMC impaired the removal of radiation-induced DNA double stranded breaks (measured by
H2AX immuno-fluorescence) and enhanced the cytogenetic damage (micronuclei formation), leading to an increase in mitotic death. While DAMC alone induced pan nuclear
H2AX fluorescence, both pan nuclear and spatially restricted foci was observed with the combined treatment (DAMC + Radiation) suggesting a complex nature of DNA damage and repair. DAMC- induced cytotoxic and radio-sensitizing effects were independent of its pro-oxidant activity, whereas histone H3 lysine (9/14) hyperacetylation appeared to be a potential target, regulating cellular responses to ionizing radiation (IR). Conclusions: The polyphenolic acetate 7, 8- diacetoxy-4-methyl coumarin (DAMC) demonstrates both anticancer effects and radiosensitizing potential under in vitro conditions.

Keywords: Calreticulin - histone deacetylase inhibitors - polyphenolic acetates – ionizing radiation – micronuclei –
H2AX INTRODUCTION Radiotherapy is the most common treatment regimen used for local tumor control where more than one half of all tumors are treated with radiation either alone or in combination with chemotherapeutic drugs and/or surgery. However, cancer cell resistance and normal tissue toxicity limit the therapeutic efficacy leading to relapse of tumors [1]. To overcome this limitation, radiosensitizers have been used in treating refractory tumors like glioma, improving the therapeutic outcome, preventing normal tissue toxicity and prolonging the median survival rate [2]. Unfortunately, lack of differential sensitization between tumor and normal tissues as well as systemic toxicity at effective doses has limited the efficacy of currently available sensitizers as effective adjuvants in the radiotherapy of tumors. Therefore, there has been considerable interest in developing new mechanism based radio-sensitizers that can enhance therapeutic gain. Polyphenols are naturally occurring heterocyclic compounds, which have been extensively investigated because of their immense biological and pharmacological importance [3]. Polyphenols like genistein, curcumin, resveratrol, flavopiridol, green tea polyphenols, etc. are considered as redox agents due to their ability to act as *Address correspondence to this author at the Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Majumdar Marg, Timarpur. Delhi-110 054, India; Fax (+91) 11-2391 9509; E-mails: [email protected]; [email protected] 1381-6128/14 $58.00+.00

anti-oxidants and pro-oxidants and are implicated in a variety of diseases like Alzheimer’s, heart malfunctioning and cancer [4,5]. They have been widely used as chemo-preventive agents and are also investigated for their chemo- or radio-sensitization potential, by altering the damage response pathways, regulating dynamic interplay between various processes like induction and repair of DNA damage, activation of cell cycle arrest and apoptosis [6]. Gossypol, a plant polyphenol represses the repair, directly or indirectly by inositol phosphate metabolism [7]. The polyphenols are also implicated in chemo-sensitization of refractory tumors like hepato-cellular carcinomas by altering the prosurvival signaling [8]. The principal limitation to the in vivo use of polyphenols is their low bioavailability; in addition polyphenols like free curcumin are highly hydrophobic and difficult to be administered systemically. Several efforts are now underway for the development of analogues, pro-drugs or delivery systems (liposomes, nanoparticles and others) to improve the pharmacokinetic characteristics of the polyphenols and enhance their disposition to the target tissues and tumors [9]. Semi-synthetic acetyl derivatives of polyphenols have been investigated extensively for their pro-oxidant/ anti-oxidant functions and demonstrated as an acetyl group donor in protein acetylation by a novel acetoxy drug: calreticulin transacetylase (CRTAase) acetylation system [10,11, 12, 13, 14, 15, 16]. Earlier in vitro observations suggest that among a variety of acetylated polyphenols with varying number of acetyl group side chains, like 7, 8-

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diacetoxy- 4- methyl coumarin (DAMC) and 7-acetoxy-4-methyl coumarin (7-AMC), DAMC is an efficient acetyl group donor, facilitating the acetylation of target proteins (enzymes) like cyto-P450, NADPH- cyto c reductase, glutathione S transferase and nitric oxide synthase modulating their activity and associated physiological effects [17, 18]. Acetylation and deacetylation of histones and non-histone proteins by a coordinated action of histone/lysine acetyl transferases (HATs/ KATs) and histone/lysine deacetylases (HDACs/ KDACs) have been widely studied due to their role in chromatin remodeling and gene expression. They are also known to participate in maintaining the native state of a protein, leading to equilibrium and cellular homeostasis [19- 21]. However, loss of balance in the protein acetylation/ deacetylation results in indefinite signals for excessive proliferation leading to cancer. Restoration of acetylation dependent processes by HDAC inhibitors (HDACi) that creates hyperacetylation condition has been implicated in cancer therapy. Many HDAC inhibitors like suberoylanilide hydroxamic acid (SAHA, Vorinostat) and Romidepsin (Depsipeptide, FK228) are at various stages of preclinical and clinical trials [22, 23]. Identification of a unique protein acetylation system involving polyphenolic acetates and CRTAase extends the realm of protein acetylation beyond HATs/ HDACs system, which can simulate the effects similar to HDAC inhibitors (i.e. induction of hyper-acetylation condition), leading to the death of cancer cells when used alone or in combination with radio-or chemo-therapy. Our recent studies in human glioma (BMG-1; low CRT) and head and neck carcinoma (KB; high CRT) cell lines suggests that intracellular CRT level influences DAMC induced cell death linked to alterations in protein acetylation status, which was confirmed by both over expression of CRT in BMG-1 cells (CROE) and siRNA knock down of CRT in KB cells, which resulted in proportional changes in sensitivities [24]. In the present study we investigated the cytotoxic and radiosensitizing effects of DAMC and 7-AMC (varying in their number of acetyl group side chains); in a human glioma cell line (BMG-1). Results show that the cytotoxic as well as radio-sensitizing effects of DAMC are higher than those of 7-AMC as revealed by inhibition in clonogenic survival, cell proliferation and enhanced mitotic as well as inter-phase (apoptotic) death. Further, DAMC compromised the kinetics of removal of radiation-induced DNA double stranded breaks (measured by
H2AX immuno-fluorescence) resulting in enhanced cytogenetic damage in the form of micronuclei. The cytotoxic and radio-sensitizing effects of DAMC were independent of its pro-oxidant action and were related to histone H3 hyperacetylation (lysine 9/ 14). The study discusses the possible role of PAs as new anti-cancer agents and adjuvant, based on their cytotoxic effects as well as the ability to enhance radiation-induced death of tumor cells. MATERIAL AND METHODS Material Dulbecco’s Minimum Essential Medium (DMEM), Hank’s Basal salt solution (HBSS), Phosphate buffered saline (PBS), Penicillin G 50000 unit/l, Streptomycin 38850 unit/l and Nystatin 9078 unit/l, 3-4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dimethyl sulfoxide (DMSO), Ethylene-di-amine-tetra acetate (EDTA), HEPES, Hochest-33258 (H33258), propidium iodide (PI), RNAase, 2’,7’ Dichlorofluorescin diacetate (DCFHDA), Annexin V-FITC kit and Foetal bovine serum were procured from Sigma chemicals Co. (St Louis, USA). Primary antibody phospho detect H2AX (phospho serine 139) was procured from Calbiochem (USA), acetylated histone H3 antibody (lysine9/ 14), secondary antibody (goat anti-rabbit, HRP conjugate) from Santa Cruz biotechnology Inc. USA and secondary antibody (goat antirabbit, FITC conjugate) from BD Pharmingen (USA) ; Antifade Reagent from Molecular Probe. 7, 8- Diacetoxy -4-methyl coumarin (DAMC) and 7-acetoxy -4-methyl coumarin (7-AMC) were synthesized by Department of Chemistry, University of Delhi.
Ray irradiation (source: 60Cobalt) was carried by using Bhabhatron-II,

Verma et al.

Teletherapy machine from Panacea, Medical Technologies Pvt. Ltd (Bangalore, India). Source of Human Tumor Cell Lines Human cerebral glioma cell line (BMG-1; diploid, wild type p53; passage no: 18) was established in Bangalore, India and was used in the study [25]. The cells were maintained as monolayer at 37ºC in 25 cm2 tissue culture flasks (Tarsons, India). The cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum, HEPES and antibiotics. Cells were passaged routinely in exponential growth phase twice a week using 0.05% trypsin solution (with 0.02% EDTA, 5.5mM glucose and 0.002% phenol red) in phosphate-buffered saline (PBS). All experiments were carried out in exponentially growing cells. Drug Treatment DAMC and 7-AMC were dissolved in DMSO and diluted in Hanks Basal salt solution (HBSS) before filter sterilizing and added to the cells in the required concentration after serial dilution in growth media for 24 h. Irradiation A 60Cobalt Teletherapy unit (Bhabatron II, Panacea Medical Technoligies, Bengaluru, India) was used for irradiation at a dose rate of 2 Gy/min (at 80 cm Source to Sample Distance and 35x35 cm field size). All irradiations were carried out at room temperature. Cells were pretreated with DAMC or 7-AMC (100
M) for 22h before irradiation and kept for an additional 2h in presence of the drug before plating to study growth/clonogenicity or processing for various assays. Macrocolony Assay Cells ranging from 150 – 4000 (depending on the treatment) were plated 24 h before treating with varying concentrations of DAMC or 7-AMC in DMEM at 37º C and irradiated according to the protocol discussed above. After the treatment, cells were replaced with fresh growth media and incubated in CO2 incubator for 7- 8 days with BMG-1 cells. Colonies (more than 50 cells) were fixed with methanol and stained with 1% crystal violet and were counted to calculate the plating efficiency (PE) and the surviving fraction (SF). PE = (No. of colonies counted/ No of cells plated) x 100 SF = PET /PEC Where PET is the plating efficiency of the treated group and PEC is the value of the control Cell Proliferation Following different treatments, cells were harvested at every 24 h time interval. Both floating and attached cells were counted and fixed in 70 % chilled ethanol for the analysis of cell cycle distribution. Cell proliferation was calculated by computing the increase in cell number and an index of proliferation P was calculated as: P = Nt/N0 Where, Nt= number of cells at time “t”. N0= number of cells at the time of treatment. Micronuclei Formation Cells were washed twice in phosphate buffered saline (PBS, pH 7.2) and fixed in Carnoy's fixative (Methanol: Acetic acid; 3: 1) at 4ºC for 2-4 hours. Fixed cells were spread on clean pre-chilled microscopic slides. Following overnight air drying, slides were stained with 10
g/ml hoechst-33258 in phosphate buffer [Na2HPO4.2H2O, Tween-20 0.5%, and (citric acid 0.1 M)] in the ratio 9: 1, final pH 7.4 for 30 minutes in dark at room temperature as described earlier [25]. After washing the excess stain with distilled water followed

Radio-sensitizing Effects of Polyphenolic Acetates

by PBS, the slides were mounted in PBS-glycerol (1: 1) and observed under fluorescence microscope (Olympus BX60, Japan) using UV excitation filter. A total of 1000 cells were scored per group. The frequency of cells with micronuclei, called the M-fraction (MF) was calculated as: MF (%) = Nm/Nt x 100, Where, Nm is the number of cells with micronuclei Nt is the total number of cells analyzed. Apoptosis Flow cytometric measurement of cellular DNA was performed with ethanol fixed cells as described above. The presence of sub G0-G1 (hypodiploid) population is indicative of apoptotic cell death. The percentage of apoptotic cells and/ or sub G0-G1 was calculated using Modfit software (Becton and Dickinson) and reconfirmed from regional statistics in bivariate plots of DNA vs forward scatter or Side scatter. Surface exposure of phosphatidylserine in apoptotic cells was measured by Annexin V-FITC/PI apoptosis detection kit I. FITC fluorescence was analyzed by Cell-Quest software (Becton Dickinson, CA, USA). Percentage of cells undergoing apoptosis was also determined microscopically from the slides used for micronuclei detection (cytogenetic damage). At least, 1000 cells were counted and percent apoptotic cells were determined as follows: Apo (%) = Na/Nt x 100, Where, Na is the number of cells with apoptosis Nt is the total number of cells analyzed. Metabolic Viability Following treatment, cells were plated in 96 well plates (3000 cells/200μl/well); the metabolic viability was measured by MTT assay. After different time intervals (24-72h) of seeding the cells, stock MTT solution (20μl) was added and cells were incubated at 370 C in the dark for two hours. The medium was removed and formazan crystals formed by the cells was dissolved using 150 μl of DMSO. The absorbance was read at 570 nm using 630 nm as reference wavelength on an ELISA reader (biotech instruments). Determination of Intracellular Redox Levels DAMC induced changes in the intracellular ROS levels of irradiated BMG-1 cells were measured by DCF fluorescence as described earlier [26]. The cells after treatment were washed twice in PBS and held in PBS with Ca2+, Mg2+, H2DCF-DA (10
g/ml) and 5 mM glucose and subsequently incubated for half an hour at 37°C. After incubation cells were washed with PBS and analyzed by flow cytometry. Immuno-histochemistry The appearance of H2AX loci were examined by fluorescence microscopy using anti- H2AX antibody. The cells were plated with density of 0.2X106 on glass cover slips and incubated overnight at 370C in 5% CO2. Next day the cells were treated with 100
M DAMC alone and in combination with radiation for 24 h. The cells following treatment were fixed and permeabilized in acetone: methanol (1: 1) at -20oC for 15min. After fixation and permeabilization the cells were washed with chilled TTBS (20mM Tris, 150mM NaCl, 0.2% Tween 20; pH 7.4) and blocked with 5% goat serum in TBST for 1h at room temperature. The cells were than incubated with primary H2AX antibody in dilution buffer (1% Bovine serum albumin in TTBS) for 1h at room temperature followed by washing. FITC labeled secondary antibody (1: 1200) was then added and the cells were incubated for 1h at room temperature. The cells were washed and mounted with anti-fade reagent containing DAPI. The cells were examined under the fluorescence microscope and images were captured using 40X objective.

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Immuno-flowcytometry For measuring H2AX by immuno-flowcytometry the cells were plated and treated with DAMC alone and in combination with radiation for 24 h. The cells following treatment were trypsinized/ fixed and permeabilized in acetone: methanol (1: 1) at -20oC for 15min. After fixation and permeabilization the cells were washed with chilled TTBS (20mM Tris, 150mM NaCl, 0.2% Tween 20; pH 7.4). Blocking and H2AX immuno-staining was done same as described above. The cells were washed thrice and resuspended in phosphate buffered saline (PBS) and analyze. Green (FITC) fluorescence was recorded for at least 10,000 events by flow cytometer. SDS-PAGE and Western Blot Preparation of cell lysates and immunoblot analysis from untreated, DAMC and DAMC+IR (5Gy) treated cells were harvested after 24 h and lysed in ice cold RIPA lysis buffer (Tris–HCl: 50mM, pH 7.4, NP-40: 1% , Na-deoxycholate: 0.25%, NaCl: 150 mM, EDTA: 1 mM, PMSF: 1 mM, aprotinin 1 lg/ml, leupeptin 1 lg/ml and pepstatin: 1 lg/ml, Na3VO4: 1 mM, NaF: 1 mM). The protein content in the lysate was measured by BCA protein assay. Protein (50–60
g) was resolved on 15% SDS–PAGE and electroblotted onto PVDF membrane (Amersham). The membrane was then incubated in 4% skimmed milk for 1 h followed by primary antibody incubation (anti-acetylated histone H3 (lysine9/ 14) for 2h. After washing, corresponding secondary antibody (horseradish peroxidase conjugate) was added and incubated for 1 h. After washing, the blots were developed using ECL chemiluminescence detection reagent (Pierce). Probes were stripped from the membranes using the stripping buffer (25mM Glycine, 1% SDS, pH 2 adjusted with HCl) for 1 hour, washed twice in TTBS for 10 minutes each and re-probed with
-Actin (cytoplasm) as loading control. RESULTS Cytotoxicity To investigate the cytotoxic effects, we analyzed metabolic viability, cell growth, clonogenic cell survival and apoptosis as parameters of response after incubating cells with DAMC and 7AMC for 24 h. Both DAMC and 7-AMC induced a concentration dependent decrease in the metabolic viability (Fig. 1a), with DAMC showing a higher effect as compared to 7-AMC indicating that DAMC is more potent than 7-AMC. The cell proliferation and clonogenic survival data corroborated the findings on metabolic viability (Fig. 1b, c). DAMC also induced a higher degree of apoptosis (Annexin V/ PI +ve cells and Sub G1 population) as compared to 7-AMC (d). These observations indicate that the biological activity (cytotoxic effects) of DAMC is higher than that of 7-AMC and also suggest that PAs with varying acetyl group side chains demonstrate differential cytotoxic effects in these (BMG-1) cells. Earlier in vitro studies have also shown that DAMC is more cytotoxic to various cancer cells compared to other polyphenols [27, 28] and also a preferred acetyl group donor over 7-AMC for CRT dependent acetylation of proteins [29]. Radio-sensitizing Effects The cytotoxic effects of PAs have been well established in a variety of tumors [15, 24, 17]. However, their effects in combination with radiation have not been investigated so far. Therefore, we investigated the potential of PAs to function as a radio-sensitizing agent in a human glioma cell line (BMG-1) established earlier [22]. Cells were treated with DAMC or 7-AMC (100
M ) for 22h followed by exposure to IR (5Gy) and kept for additional 2h in the presence of these PAs before growing them further to observe modifications in radiation-induced loss of clonogenic survival. Results clearly showed that DAMC enhanced the radiation-induced cell death (loss of clonogenic survival), which was additive in nature, while 7-AMC did not significantly alter the radiation-induced

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Verma et al. treatment. Each point is the mean of three replicates; bars represent the SEM *P < 0.05. (B) Effects on cell proliferation measured after DAMC and 7AMC (24 h; 100
M) treatment in the cells *P < 0.05. (C) Effects on clonogenicity after exposure of cells to DAMC and 7-AMC (24 h; 100
M). Each point is the mean of three replicates; bars represent the SEM *P < 0.05. (D) Induction of apoptosis by DAMC and 7-AMC (24 h; 100
M) measured by flow cytometer showing the presence of sub G0-G1 (hypodiploid) population which is indicative of apoptotic cell death in BMG-1 cells *P < 0.02 and AnnexinV-FITC binding. The percentage apoptosis is measured immediately after the drug treatment (24h) and the values are the mean of three independent experiments; bars represent the SEM #P < 0.01.

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1D Fig. (1). Cytotoxic effects of PAs in a human glioma cell line (BMG-1). (A) Concentration dependent effects on metabolic viability evaluated by MTT assay after exposure of cells with DAMC and 7-AMC (5, 20 and 100
M) for 24 h. The percent metabolic viability is shown at 48h post

cell death under these conditions (Fig. 2a). Considering the higher biological activity of DAMC compared to 7-AMC, we carried out further investigations on the radio-sensitizing effects of PAs with DAMC at an absorbed dose of 5 Gy. Similar to the effects on clonogenic survival, DAMC enhanced also the radiation-induced growth inhibition, which was additive in nature (Fig. 2b). Measurements of cell cycle distribution carried out at various postirradiation times using flow cytometric analysis of DNA content indicated that DAMC induced inhibition in the cell proliferation was partly due to accumulation of cells in the S and G2+M phases of the cell cycle, while in combination with radiation a delay in the transition of cells to S-phase was also evident (Fig. 2c). Mitotic Death Kinetics of micronuclei induction showed a peak value of radiation-induced M-Fraction (32%) at 24h post irradiation, followed by a decrease at 48h due to an increase in the population of undamaged cells; the classical dilution effect [30]. Although the M-fraction values were lower in cells treated with DAMC alone or in combination with radiation as compared to radiation at 24h, it continued to increase as no dilution effect was observed (Fig. 3a). Since micronuclei are expressed only in post-mitotic daughter cells after various treatments, we analyzed the micronuclei induction by normalizing the M-fraction values with reference to cell proliferation from the plot generated with M-Fraction values vs proliferation index (increase in cell number relative to the value at the time of treatment). Data presented in (Fig. 3b) clearly shows that while irradiated cells could complete nearly 3 population doublings in 48 h (Nt/ No= 8), DAMC treated cells had less than 3 doublings (Nt/ No< 6) and the combined treatment resulted in less than 2 doublings (Nt/ No