Investigation of Methylation Profiles of TP53, Caspase 9, Caspase 8, Caspase 3 Genes Treated with DNA Methyl Transferase Inhibitor (DNMTi) Zebularine (ZEB) and Caffeic Acid Phenethyl Ester (CAPE) on MCF-7 and MDA-MB-231 Breast Cancer Cell Lines

Purpose: MCF-7 (ER+, WTP53) and MDA-MB-231 (ER Met, Mutant P53) Caffeic Acid Phenethyl Ester (CAPE) and DNA Methyl Transferase Inhibitor (DNMTi) in breast cancer cell lines of Zebularine (ZEB) single and combined application of TP53, caspase-9, caspase 8 and caspase-3 genes as a result of the use of single and combined drug methylation profiles are aimed to be evaluated by specific PCR method. Material-Metods: In the MCF-7 and MDA-MB-231 breast cancer cell lines, MTT test and survival analysis were performed as a result of single and combined application of CAPE and Zebularine and Methylation Specific PCR was performed to examine the methylation of caspase-3, caspase-8, caspase-9 and TP53 genes. Results: According to the results of 24-hour drug administration, the IC50 for the MCF-7 cell line was determined as 200 μM, for CAPE 40 μM and for the combined values of 50 μM ZEB + 5 μM CAPE. The effects of caspase-3, caspase-8, caspase-9 and TP53 genes on the methylation level of ZEB, CAPE and ZEB + CAPE drug combination were determined by using bisulfite modified DNAs in MCF-7 and MDA-MB-231 cell lines. Discussion: In the MCF-7 cell line, the 120 μM ZEB viability rate was 51%, and the viability of How to cite this paper: Eroglu, O., Celik, EG.., Kaya, H., Celen, M., Karabicici, M. and Karacoban, E. (2019) Investigation of Methylation Profiles of TP53, Caspase 9, Caspase 8, Caspase 3 Genes Treated with DNA Methyl Transferase Inhibitor (DNMTi) Zebularine (ZEB) and Caffeic Acid Phenethyl Ester (CAPE) on MCF-7 and MDA-MB-231 Breast Cancer Cell Lines. Journal of Cancer Therapy, 10, 69-85. https://doi.org/10.4236/jct.2019.101006 Received: December 24, 2018 Accepted: January 20, 2019 Published: January 23, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
Breast cancer is a common type of cancer and, despite improvements in its treatment, it is still considered to be the main cause of death in women [1].
Studies have shown that breast cancer is a heterogeneous tumor that responds differently to treatments. Breast cancer is caused by the accumulation of genetic and epigenetic events. Most of the epigenetic modifications are reversible events that target the gene sequence after transcription and the inhibition of these mechanisms may be advantageous in the treatment of breast cancer [2]. DNA methylation is an important epigenetic modification that is most studied and involved in the regulation of many cell processes beyond the DNA sequence level [3]. Cancer cells often exhibit abnormally high DNA methylation at gene-specific CpG-rich promoter sites [4]. DNA methylation is controlled by DNA methyl transferase (DNMT), an enzyme that catalyses the transfer of a methyl group from S-adenosyl-1-methionine to the 5-position of cytosines in the CpG nucleotide. DNMT overexpression was detected in various malignancies, including lung, prostate, and colorectal tumors [3] [4]. Since DNA methylation is a reversible biochemical process, and DNMT inhibitors are not specific to cancer type, DNMT may be a viable target in cancer treatment, since it can be used in the treatment of various cancers [5]. By inhibiting DNMTs, genes that may be silenced by DNA methylation during the carcinogenic process can be reactivated and the non-carcinogenic state of the cell can be regenerated [6].
Because of this feature, DNA methylation changes are believed to be an alternative pathway for cancer and have the potential to be diagnostic markers that can be used in the clinical context [7]. Zebularine (ZEB), a novel DNMT inhibitor, is a sitidine analogue [8] [9] O. Eroglu  containing the 2-(1H)-pyrimidinone ring initially developed as a cytidine deaminase inhibitor to prevent deamination of nucleoside analogs. Zebularine is a versatile starting material for the synthesis of complex nucleosides and is a mechanism based on mechanism DNA cytosine methyl transferase inhibitor [10]. The mechanism of action of ZEB as a DNMTi is its incorporation into DNA after its phosphorylation to the diphosphate level and its conversion to a deoxynucleotide. Unlike other DNMTi, zebularine is less toxic for breast cancer cell lines and there are antimitogenic and angiostatic effects of ZEB. It has been shown that ZEB has antitumor activity by stimulating apoptosis and has an effect of inhibiting cell proliferation [2]. Caffeic acid phenethyl ester (CAPE) is polyphenol, a component of honeybee propolis. CAPE has a number of important biological activities including anti-bacterial, antiviral, antifungal, antioxidant, anti-inflammatory and anticancer properties [11]. CAPE has been supported by studies on cell cycle progression, cell proliferation, cell cycle arrest and apoptosis in many cancer models (colon, lung, pancreatic).
CAPE inhibits the activity of cancer cells using nuclear transcription factor NF-kB. Studies have shown that NF-kB may be one of the most important factors in oncogenesis and cancer progression. In addition, CAPE is capable of exhibiting different toxicity to tumor models without affecting normal cells [12].

Survival/Trypan Blue Coloring Test
The aim of this study is to determine the time-dependent effect of the drugs administered with survival test at a certain concentration. For this purpose, the MDA-MB-231 and MCF-7 breast cancer cell lines are seeded in 6-wells and the cells are treated with drugs at 24, 48, 72 and 96 hours according to the IC 50 values obtained for the drugs. Counting of cells was performed on Neubauer hemocytometer.

DNA Isolation
Total DNA isolation from breast cancer cells was performed to determine the effect of applied drugs on methylation and total DNA concentrations isolated were measured in nanodrop device. (Nanodrop, Shimadzu Corporation, BioSpec-nano, Japan).

Bisulfite Modification and Methylation Specific PCR (MSP)
It is the process of converting unmetylated cytosine residues to uracil without any change in methylated cytosines. Zymo EZ DNA Methylated-GoldTM kit (D5005, Zymo Research Corp., Orange, CA) was used for the bisulfite conversion of genomic DNA. The bisulfite transformed DNA was used as a template and MSP method was applied to determine the methylation status of the genes that obtained significant results, especially caspase 3, caspase 8 and caspase 9 (Table 1). (Thermal Cycler Block, ThermoFisher Scientific, 5020, Finland.) PCR reaction 95˚C 5 min initial denaturation, 40 cycles; 94 -30 s, 50˚C -30 s, 72˚C 30 s and finish 72˚C 7 min and the PCR results were examined on the non-denaturing gel.

The Effect of Combined Application of ZEB, CAPE and ZEB + CAPE on Cell Viability in MCF-7 and MDA-MB-231 Breast Cancer Cell Lines
In

The Effect of CAPE, ZEB, CAPE + ZEB Combination on Cell Survival in MCF-7 and MDA-MB-231 Breast Cancer Cell Line
In order to demonstrate the effect of CAPE (20 -

Investigation of the Methylation Profile of Caspase-9, Caspase-8, Caspase-3 and TP53 Genes by MSP Method in MCF-7 and MDA-MB-231 Breast Cancer Cell Lines with CAPE, ZEB, CAPE + ZEB Combination
The methylation profile evaluation of the respective genes on 2 different cell lines used in the study is shown in Figure 6 (p < 0.5).
As a result, it was concluded that the combined treatment in MCF-7 breast cancer cell lines was highly effective. However, it has been observed that the same doses of drugs in different genes may cause different methylation conditions. For example, the CAPE 80 μM dosage is closely related to the unmethylated condition in caspase-8 and caspase-9 in TP53 and caspase-3. On the other hand, in combination applications, the effect is constant and half is associated with methylation-unmethylation. This result is important in terms of the efficiency of the treatment.
In general, drugs administered on MDA-MB-231 cell lines did not have a methylation effect on caspase-8, but stimulation was carried out via the intrinsic pathway, which is the internal signaling pathway, or via a different signaling pathway. In addition, it is seen that the combined drug application is successful.

Discussion
Although the monotherapy approach is still a very common treatment for many different types of cancer, this method is generally considered less effective than a combined therapy approach. Combined therapy can prevent toxic effects on normal cells and can also produce cytotoxic effects on cancer cells. Zebularine is a DNMT inhibitor that exhibits anti-tumor effect against various types of cancer. CAPE significantly affects the viability of breast cancer cells and increases the effect of standard anti-cancer drugs, suggesting the potential role of bioactive compounds in chemotherapy. In this study, it was aimed to investigate the relationship between apoptosis induced by caspases and epigenetic regulation of the anticancer and antiproliferative effects of CAPE, ZEB and CAPE + ZEB combined applications in MCF-7 and MDA-MB-231 cell lines and the combined application of CAPE, ZEB single and CAPE + ZEB in MCF-7 and Journal of Cancer Therapy MDA-MB-231 cell lines. The combined use of CAPE and ZEB drugs was applied for the first time in this study.
In this study, it was determined that single and combined application of ZEB and CAPE in two breast cancer cell lines reduced cell proliferation and induced apoptotic pathway (Figures 2-4 [17]. CAPE has been confirmed to be cytotoxic for cells similar to that in this study. The nuclear factor of CAPE showed that it inhibited NFKB1. They also studied CAPE to confirm that death-inducing receptors were clustered. They demonstrated that CAPE induces apoptosis and that Fas death-inducing receptors are harvested by a Fas-L independent mechanism in MCF-7 cells [18]. The data obtained in combination with CAPE and DNMT inhibitor ZEB, which is known to be effective in apoptotic pathway in accordance with the literature, has been found to be effective in reducing the cytotoxicity caused by drugs. CAPE + ZEB combined drug therapy is thought to be promising for new treatments. This difference between the two cell lines makes MDA-MB-231 cells more susceptible to cytotoxicity of the drug because ER is negative. According to the data obtained in the literature, it was revealed that high dose concentrations of ZEB caused cytotoxic effects in the cell. Recent studies have shown that the incidence of P53 mutations is different between the various types of cancer (~% 96 for serous ovarian cancer, <10% for thyroid cancer). The incidence of P53 mutation for primary breast carcinomas is 18% -25% [23]. He stated that CAPE plays an important role in apoptosis and cell cycle. It has also been found in several studies that CAPE can reduce the expression of the MDR-1 gene and thus increase the susceptibility of cancer cells to chemotherapy, as the high expression and activity of the MDR1 gene causes the cancer cells to become resistant to treatment with many agents [24] [25].
The application of CAPE and ZEB to breast cancer cell lines induces p53 tumor suppressive activity and thus leads to apoptosis sensitivity of the cancer cell. In our study, 20 μM CAPE concentration was found to be associated with methylation of TP53 in MCF-7 cell lines (Figure 6(a)). The low dose ZEB concentration (80 µM) was found to increase the expression of the TP53 gene according to the high dose ZEB concentration (120 µM) (Figure 6(a)). These results obtained with 50 µM ZEB + 5 µM CAPE application were found to be related to the methylated and unmethylated status of the TP53 gene in half ( Figure 6(a)). MDA-MB-231 breast cancer cell line with increasing the CAPE dose of the TP53 gene has been found to change from methylated to unmethylated state (p < 0.05) (Figure 6(b)). In a study conducted by You et al.
(2014), it has been shown that TP53 activity, which is a tumor suppressor gene, has been increased in ZEB administered lung cancer cell line A549 and HPF cells [25]. In accordance with the literature, the data showed that the TP53 gene, µM CAPE on the caspase 9 gene was found to be more effective than the Zebularine, as opposed to the CAPE effect and a more efficient unmetilized condition, which was thought to be associated with a more active apoptotic stimulation and the efficacy of the combined application on the cells was higher (Figure 6 showed cytotoxic effect on cells and showed that this cytotoxic effect varies with both the applied CAPE concentration and the duration of the cells exposure [30]. When the data were analyzed, it was found that incubation of MCF-7 and MDA MB 231 cells with ZEB, CAPE and their combination triggered activation of caspase-9 and significantly increased caspase-3 activity (Figure 6(a) and Figure 6(b)). These results showed that combined therapy may be more effective in stimulating the apoptotic pathway in humans with breast cancer than in the ad-Journal of Cancer Therapy ministration of drugs alone.
In MCF-7 cells, 80 µM CAPE concentration was found to be more effective than 20 µM CAPE concentration in activation of caspase-8 gene (Figure 6(a)). It can be said that the increase in dose concentration has a parallel effect with the activation of caspase-8 and is involved in the activation of the methylated gene by unmetrification. Combined administration of 50 µM ZEB + 5 µM CAPE has been found to be associated with halved methylated-unmethylated status of caspase-8 gene (Figure 6(a)). MDA-MB-231 breast cancer cells cannot be stimulated via caspase-8 ie extrinsic route. Because the MDA-MB-231 cells on a 120 µM CAPE and 80 µM ZEB concentrations alone when applied with a 50% methylated-unmetylated profile (p < 0.01), 20 µM ZEB + 30 µM CAPE combined application was found to be associated with the methylated situation ( Figure 6(b)). It can be said that apoptotic stimulation is via the intrinsic (caspase-9) pathway.
The 80 µM CAPE application showed a 50% methylated-unmetylated profile of the caspase-8 gene in the cells. It was found that the caspase-3 gene was associated with methylene status in cells with 80 μM CAPE (p < 0.01) ( Figure   6(a)). Although the caspase-9 gene associated with the intrinsic pathway of the drugs is related to the unmethylated state, the caspase-3 gene is associated with the methylated condition, and other mediators may be effective in stimulating the caspase-9 to induce apoptosis.
Tolba et al. (2013) found that CAPE combined with Paclitaxel and Docetaxel in prostate cancer was effective in reducing the IC50 volumes of these drugs, and showed that combined application with CAPE was 1.3 times more effective in the effectiveness of caspase-3 compared to drug administration alone [31]. Our study, which is supported by literature research, is to suggest that interactions of ZEB + CAPE combined drug therapy are more effective in cell proliferation and apopototic mechanism compared to ZEB and CAPE alone application to MDA-MB231 breast cancer cell line cells (Figure 3(b)). The explanation of the association of CAPE with apoptosis and the similarity of caspase gene induction processes in different cancer cell lines has been supported by literature studies.
In general, it has been observed that the drugs administered in MDA-MB-231 cell lines have no effect on the methylene status via caspase-8, according to the results obtained, it was thought that the stimulation was carried out via the intrinsic pathway, which is the internal signaling path, or through a different signaling pathway affected. It can also be said that the combined drug application is successful. Effective combination with low concentrations of dose ratios in the combined drug provides a more appropriate approach in terms of treatment.
Based on our findings, it was observed that CAPE, ZEB and CAPE + ZEB