Radiosensitization of calreticulin‐overexpressing human glioma cell line by the polyphenolic acetate 7, 8‐diacetoxy‐4‐methylcoumarin

Abstract Background Calreticulin (CRT), an endoplasmic reticulum–resident protein generally overexpressed in cancer cells, is associated with radiation resistance. CRT shows higher transacetylase activity, as shown by us earlier, in the presence of the polyphenolic acetates (like 7, 8‐diacetoxy‐4‐methylcoumarin, DAMC) and modifies the activity of a number of proteins, thereby influencing cell signaling. Aim To investigate the relationship between CRT expression and radiation response in a human glioma cell line and to evaluate the radiomodifying effects of DAMC. Methods and results Studies were carried out in an established human glioma cell line (BMG‐1) and its isogenic clone overexpressing CRT (CROE, CRT‐overexpressing cells) by analyzing clonogenic survival, cell proliferation, micronuclei analysis, and protein levels by Western blotting as parameters of responses. CRT overexpression conferred resistance against radiation‐induced cell death in CROE cells (D37 = 7.35 Gy, D10 = 12.6 Gy and D0 = 7.25 Gy) as compared to BMG‐1 cells (D37 = 5.70 Gy, D10 = 9.2 Gy and D0 = 5.6 Gy). A lower level of radiation‐induced micronuclei formation observed in CROE cells suggested that reduced induction and/or enhanced DNA repair partly contributed to the enhanced radioresistance. Consistent with this suggestion, we noted that CRT‐mediated radioresistance was coupled with enhanced grp78 level and reduced P53 activation–mediated prodeath signaling, while no changes were noted in acetylation of histone H4. DAMC‐enhanced radiation–induced delayed (secondary) apoptosis, which was higher in CROE cells. Conclusion CRT overexpression confers resistance against radiation‐induced death of human glioma cells, which can be overcome by the polyphenolic acetate DAMC.


| INTRODUCTION
Glioma is the most common type of human cancer that is refractory to a variety of therapies with a 5-year survival rate of less than 10% post treatment. 1,2 Various forms of radiotherapy with temozolamide (an alkylating agent and DNA methyl transferase inhibitor) is the current standard of care in the management of malignant gliomas following surgery that modestly enhances patient survival. 3 Despite significant technological advances, success of radiation therapy is limited due to the radioresistance of glioma cells linked to alterations in multiple signaling pathways related to DNA damage and repair, apoptosis, cancer stem cells proliferation, and self-renewal. 4 Consequently, radiosensitizers targeting these pathways have shown promising clinical outcome, enhancing the specificity and efficacy. 5 However, acquired drug resistance and undue normal tissue cytotoxicity pose limitations to the widespread use of these agents preventing clinical benefits. 6 Therefore, there has been a considerable amount of effort in identifying and/or designing novel drugs or approaches which are effective in the management of radiation therapy.
Calreticulin (CRT), a calcium-binding, endoplasmic reticulumresident protein, has emerged as an important molecule of interest due to its multifunctional role in cells including maintenance of intracellular calcium homeostasis, molecular chaperoning function, cell adhesion, migration, apoptosis, etc. 7,8 Studies with CRT-knockout mice have revealed its indispensable role in tissue growth and development. 9 Role of CRT has been implicated in a variety of disease conditions like heart disease, immune dysfunction, wound healing, tissue repair, cancer, etc. 10 A strong correlation exists between CRT expression and tumorigenesis progression, and metastasis with overexpression in many types of solid tumors. 11,12 Gene expression profiling interactive analysis (GEPIA) shows high CRT expression in gliomas (low grade as well as glioblastoma multiform, GBM) and head and neck carcinomas ( Figure 1). Recent studies have shown that CRT expression is a powerful prognostic biomarker reflecting enhanced antitumor immune response in patients with non-small-cell lung carcinoma (NSCLC) and acute myeloid leukemia. 13,14 In response to stress like ionizing radiation (IR), CRT translocates to the surface of dying cancer cells and acts as "eat-me" signal for antigen-presenting cells (APCs). The APCs phagocytose the dying cells that leads to antigen presentation and tumor-specific cytotoxic T lymphocytes response. 15 CRT has been shown to regulate P53-mediated UV-induced apoptosis by affecting the rate of P53 degradation and nuclear localization in mouse embryonic fibroblast cells. 16,17 Although few studies have highlighted the role of CRT in determining the cellular responses to IR, further studies demonstrating a relationship between CRT expression and IR-induced glioma cell death are merited.
Protein acetylation regulates a variety of cell functions including chromatin remodeling and gene expression through transactivation, protein-protein interactions, and stabilization of the target proteins. [18][19][20] Canonical protein acetylation system involves lysine acetyltransferases (KATs) and deacetylases (KDACs) and acetylating/deacetylating lysine residues on histones and nonhistone proteins. KDACs are upregulated in many cancers such as leukemia, glioblastoma, melanoma, carcinoma, etc. 21,22 Consequently, KDAC inhibitors that induce apoptosis, cellular differentiation, and cell cycle arrest of cancer cells have emerged as promising anticancer therapeutics that target the altered epigenetic regulation in tumors. 23 However, acute side effects associated with HDACi like gastrointestinal toxicity, cardiac arrhythmia, thrombocytopenia, anemia, fatigue, nausea, vomiting, etc. limit their use as radiosensitizers in clinics. 24 Therefore, novel drugs or approaches which simulate effects similar to KDAC inhibitors, but are associated with lesser toxicity, are required to improve the efficacy of radiotherapy.
Our earlier investigations have established the transacetylase function of CRT wherein it efficiently transfers acetyl group from polyphenolic acetates (PAs) to target proteins while acetyl co-enzyme was a weak donor. [25][26][27] In vitro studies have established that among a variety of acetylated polyphenols with a varying number of acetyl group side chains, 7, 8-diacetoxy-4-methyl coumarin (DAMC) is an efficient acetyl group donor, acetylating the target proteins (enzymes) like cyto-P-450, NADPH-cyto c reductase, glutathione S transferase, and nitric oxide synthase modulating their activities and associated physiological effects. 28 Acetoxy drug: CRT transacetylase (CRTase) F I G U R E 1 Human glioma and head and neck cancers express a higher level of calreticulin gene compared to normal tissues. Expression of calreticulin gene in human glioma and head and neck cancers analyzed using the online tool-Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/). Each dot represents expression of samples. Y-axis represents log2 (TPM + 1) as the expression level, where TPM is transcripts per million; p < .01 protein acetylation system extends the realm of protein acetylation beyond the KAT/KDAC system. Our earlier studies have shown that DAMC sensitizes the human glioma cell line (BMG-1) by impairing the removal of radiation-induced DNA double-stranded breaks thereby enhancing mitotic and interphase death. 29 The radiosensitization correlated with hyperacetylation of histone H3 lysine (9/14) by DAMC is similar to radiosensitizing effects of classical HDAC inhibitors. 30 Although these circumstantial evidences suggest a role for CRT: DAMC in determining cellular responses to IR, a causal relationship between CRT:DAMC-mediated protein acetylation and radiosensitivity has not been established. Therefore, the present studies were undertaken in human glioma cell line (BMG-1) and its isogenic cells

| Source of human tumor cell lines
Human cerebral glioma cell line (BMG-1; diploid, wild-type P53) was established in the Department of Biophysics, National Institute of Mental Health and Neuro Sciences, Bangalore, India, and was used in the study. 31 Cells were periodically checked for ploidy, growth rate, and clonogenic potential (plating efficiency) and revived after 40 passages. Calreticulin-overexpressing BMG-1 cells (CROE) were prepared as described earlier. 32 The cells were maintained as monolayers at 37 C in 25 cm 2 tissue culture flasks (Tarsons, India). The cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum, HEPES, sodium bicarbonate, and antibiotics. Cells were passaged routinely in exponential growth phase twice a week using 0.05% trypsin solution (with 0.02% EDTA, 5.5 mM glucose, and 0.002% phenol red) in phosphate-buffered saline (PBS). All experiments were carried out in exponentially growing cells.   α/β ratio was calculated by the following equation:

| Irradiation
where D is a radiation dose (Gy). From this regression curves, D 37 , D 10 , and D 0 values were obtained by GraphPad Prism software. Using  House, Inc., USA) software was used for the cell cycle analysis.

| Micronuclei expression
where N m is the number of cells with micronuclei and N t is the total number of cells analyzed.
Since proliferation influences radiation-induced micronuclei expression as they are expressed in postirradiation daughter cells and radiation causes perturbations in cell cycle progression, we normalized the values of cells with micronuclei linked to proliferation as follows:

| Apoptosis
Flow cytometric measurement of cellular DNA was performed with ethanol fixed cells as described earlier. 32 Presence of sub G 1 (hypodiploid) population is indicative of apoptotic cell death and was calculated using Modfit software (Becton and Dickinson), which was subsequently reconfirmed from regional statistics in bivariate plots of DNA versus forward scatter or side scatter.

| Intracellular calcium (Ca 2+ ) levels
The intracellular levels of calcium in CROE cells and parental BMG-1 cells were measured by fura-2 that binds to the cytosolic calcium. The cells were grown on cover slips and, 48 h later, were washed with PBS and stained with Fura-2 (3 μM) for 15 min. The samples were washed, and antifade mounting solution was applied to prevent bleaching. Cells were visualized using fluorescence microscope with UV excitation (Zeiss, Germany).

| Statistical analysis
All the experiments were carried out in triplicates. Data are expressed as mean values with SD. The statistical significance between groups was calculated using two-tailed Student's "t"-test. Interestingly, an increase in the glucose uptake was also noted in CROE cells as compared to the parental BMG-1 cells ( Figure 2D).

| Overexpression of CRT induces resistance against radiation-induced cell death
To study the effects of CRT on cellular response to radiation, we investigated various cytotoxic parameters and the potential contributing factors.  Figure 3C), thereby suggesting that decrease in the mitotic death is a significant contributing factor for the enhanced radioresistance in CROE cells.

| Protein levels
To investigate the potential contributing factors for CRT-mediated radioresistance, we analyzed the level of few proteins implicated in the  Figure 3D). 34 We also observed increased grp78 (a molecular chaperone) levels in CROE cells compared to BMG-1, suggesting a higher level of unfolded protein response implicated in rescuing cells from radiation-induced cell death. 35 The level of acetylated H4 (lysine 16) was 2-fold higher in BMG-1 cells, while no significant change was noted in CROE cells. However, the endogenous level of CROE cells was higher than that of BMG-1 cells.

| Radiosensitization by DAMC is higher in CRT-overexpressing CROE cells
Our earlier studies have shown that DAMC has both cytotoxic and radiosensitizing effects in BMG-1 cells and the radiosensitizing effects are additive in nature. 29 The radiosensitization by DAMC was profoundly higher in CROE cells as compared to BMG-1 cells ( Figure 4A), and the shoulder on the dose-response curve was abolished in CROE cells