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 w ere 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 80 μM ZEB MDA-MB-231 breast cancer cells decreased by 59.7%. After 20 μM CAPE, viability in MCF-7 cells decreased by 31% in 120 μM CAPE and MDA-MB-231 cells decreased by 41%. The viability with 40 μM CAPE decreased by 19% in MDA-MB-231 cells. It was found that 20 μM CAPE concentration was associated with TP53 methylation in MCF-7 cell lines. The 80 μM ZEB concentration was found to be closely related to the unmethylated status of the TP53 gene. These results obtained with 50 μM ZEB + 5 μM CAPE application were found to be related to the methylated-unmetylated status of the TP53 gene in half (50%). For the caspase-9 gene of MDA-MB-231 cells, 80 μM ZEB concentration was found to be associated with unmetylated status. The effective use of drugs with low concentrations of the drug dose provides a more appropriate approach in terms of treatment.
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 [
Zebularine (ZEB), a novel DNMT inhibitor, is a sitidine analogue [
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 [
Apoptosis (programmed cell death) is a critical obstacle to tumorigenesis. Proteins such as calcium, ceramide, Bcl-2 family, P53, caspases, cytochrome-c and mitochondria serve in the regulation of apoptosis. Caspases are a cysteine protease family and function as central regulators of cell death. TP53 is mutated in most cancers and mutated in 30% - 50% of diagnosed breast tumors.
Chemotherapeutic agents may affect many effector and regulatory elements of the process of apoptosis. Several studies have shown that chemopreventive agents induce apoptosis in various cancer cells by affecting multiple proteins involved in programmed cell death. In accordance with the need for new compounds with both effective antitumor effect and high cancer cell selectivity and low normal cell toxicity, recently some compounds have increased the need for natural compounds to inhibit cancer cell growth [
In the first stage of this study, the DNA metabolite transferase inhibitor Zebularine is a new generation drug with propolis extract Caffeic Acid Phenethyl Ester (CAPE)’s MCF-7 breast cancer cell lines first and the combined use of drugs as a result of IC50 values The aim of this study was to determine the effects of ZEB, CAPE and ZEB + CAPE on cell viability, cell proliferation and to determine the relationship between epigenetic and caspases known to be associated with apoptotic pathway. Although different applications of ZEB and CAPE drugs in different breast cancer cell lines exist in the literature, the combined use of these two drugs is not observed. Therefore, the original value of this study is preserved.
MDA-MB-231 (ER (-), Mutant P53) and MCF-7 (ER (+), WTP53) human breast cancer cell lines 10% heat inactivated fetal bovine serum (FBS), 2 mM L gulutamine, 100 μg/ml were cultured in RPMI 1640 medium, containing penicilin-streptomycin, at 37˚C and 5% CO2 incubator. (Memmert CO2 Incubator, INCO153med, Germany).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test is performed on incubated breast cancer cell lines to determine the time-dependent effects of ZEB and CAPE in different concentrations. Cells are treated with 0 - 200 μM ZEB and 0 - 40 μM CAPE for 24 hours. At 570 nm wavelength spectrophotometric reading, the effect of drugs on relative cell viability was determined by controlling and proportioning to the drug applied cells. (Multiskan™ FC Microplate Photometer, ThermoFisher Scientific, Multiskan FC, China.)
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 IC50 values obtained for the drugs. Counting of cells was performed on Neubauer hemocytometer.
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).
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 (
Genes | Primers |
---|---|
Caspase 8 | M-F: 5’-TGTTGTTTGGGTAACGTATCGA-3’ M-R: 5’-CCCTACTTAACTTAACCCTACTCGAC-3’ U-F: 5’-TTGTTGTTTGGGTAATGTATTGA-3 U-R: 5’-CAACCCTACTTAACTTAACCCTACTCA-3 |
Caspase 3 | M-F: 5’-TTT AGG GCG GGA TTA AAG C-3’ M-R: 5’-CTACGACCCGTCCCCTAA-3’ U-F: 5’-TGAGTTTTAGGGTGGGATTAAAGT-3’ U-R: 5’-TTCGGTAGGCGGATTATTTG3’ |
Caspase 9 | M-F: 5’-GGGAGC GAAGATTGATTC-3 M-R: 5’-CTTCGTCCATAACGAATAACC-3 U-F: 5’-GTGGGGAGTGAAGATTGATTT-3’ U-R: 5’-CCACTTCATCCATAACAAATAACC-3 |
P53 | M-F: 5’-TTCGGTAGGCGGATTATTTG3’ M-R: 5’AAATATCCCCGAAACCCAAC-3’ U-F: 5’-TTGGTAGGTGGATTATTTGTTT-3’ U-R: 5’-CCAATCCAAAAAAACATATCAC-3’ |
In order to determine the appropriate dose of Zebularine MCF-7 and MDA-MB-231 breast cancer cells, changes in cell viability after 24 hours of drug application at increasing dose concentrations of 0 - 200 μM were examined by MTT cell viability test (
In order to determine the appropriate dose of CAPE on MCF-7 and MDA-MB-231 breast cancer cells, changes in cell viability were determined by MTT cell viability test as a result of drug administration at an increasing dose range of 0 - 40 μM for 24 hours. After 24 hours of administration with 20 μM CAPE, viability in MCF-7 cells decreased by 31% (
To determine the effective combination doses of ZEB and CAPE on MCF-7 and MDA-MB-231 breast cancer cells, over 1/4 of the doses determined by the MTT test using ZEB and CAPE alone and supported by the survival test combined drug range applied. In the MCF-7 breast cancer cells, ZEB: 50 μM + CAPE: 5 μM combined dose decreased by 50% after 24 hours of administration (
In order to demonstrate the effect of CAPE (20 - 40 µM) and ZEB on the proliferation of 200 µM MCF-7 and CAPE (120 µM) and ZEB (80 µM) MDA-MB-231 breast cancer cells, the cells were stained with trypan blue and then counted with hemocytometer. The counts showed that cell growth was decreased in time depending on time in both cell lines (
ZEB: 50 µM + CAPE: To demonstrate the effect of 5 µM administered drug combinations on the proliferation of MCF-7 breast cancer cells and the effects of ZEB 20 µM + CAPE 30 µM on the proliferation of MDA-MB-231 breast cancer cells, cells were stained with trypan blue and then counted with hemocytometer.
The counts showed that cell growth was decreased in time depending on time in both cell lines. When the data were examined, it was seen that combined therapy was effective in both cell lines without producing cytotoxic effect (
The methylation profile evaluation of the respective genes on 2 different cell lines used in the study is shown in
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.
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 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). MDA-MB-231 breast cancer cell line MCF-7 cell line compared to MDA-MB-231 cell line because of the mutant TP53 gene is more susceptible to cytotoxic agents. In addition, one of the reasons for MCF-7 cells being less susceptible to the effect of ZEB is the low rate of growth in the cells due to the zebularine.
Billam et al. (2010) in his study showed that high concentrations of ZEB may cause cytotoxicity in cells, while inhibiting cell proliferation. Billam et al. found that ZEB, which is a DNMT inhibitor, is less toxic in many cell lines compared to other DNTMs. Therefore, it is thought that ZEB can be used alone or in combination with DNMT inhibitors such as other 5-aza-dC for prolonged administration [
The combination of 50 μM ZEB + 5 μM CAPE combined dose of 50 μM in MCF-7 breast cancer cells after the 24-hour administration showed that the combined efficacy could be used effectively (
(Omene et al. (2012)), in vitro and in vivo in the use of CAPE and taxol drug at appropriate dose concentrations were observed to inhibit cell growth when applied together [
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% [
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 (
Hurt et al. (2006) in a study similar to normal plasma cells found in hypermethylated myeloma cells P53 re-expression in the study of the application of 200 μM ZEB in compared to the control group increased 2-fold increase in P53 activity and also increased the amount of protein observed [
Eroglu et al. (2018) have shown that hypermethylation of tumor-related genes in breast cancer has an important role in carcinogenesis and tumor formation. They showed that GSTP1 and CDH1 tumor suppressor genes were hypermethylated in breast cancer tissues at 82% and 95%, respectively [
The effects of single and combined administration of ZEB and CAPE on caspase-9/-3 and -8, which are important factors related to apoptosis in cancer cells, were investigated. It was observed that the MCF-7 and MDA-MB-231 administered CAPE significantly increased the activation of caspase-9 gene (p < 0.001) (
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 (
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 (
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) (
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 [
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 combined drugs inhibited MCF-7 and MDA-MB-231 cell proliferation and were more effective in apoptosis mechanism. It has been shown that the apoptosis-related genes were unmethylated and significantly increased apoptosis by caspases. These findings suggest that ZEB’s MCF-7 cells induce apoptosis along the intrinsic mitochondrial route, and that treatment with ZEB induces apoptosis in both MDA-MB-231 cells by intrinsic and extrinsic pathways. With the application of CAPE, caspase 3/7 in MCF-7 and MDA-MB-231 cells is shown to be effective for apoptosis activity and induce apoptosis events. Our findings are very important in combating the cytotoxic effect and combating cancer with combined therapy.
This work was financially supported by both Bilecik Şeyh Edebali University Research Projects (Project No. 2014-02-BİL.04.02) and Scientific and Technological Research Council of Turkey (TUBITAK Project No. 1919B011402145). We are grateful both to TUBITAK and Bilecik Şeyh Edebali University.
The authors declare no conflicts of interest regarding the publication of this paper.
Eroglu, O., Celik, E., 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.2018.101006