The aim of this study is to investigate the anti-proliferative effects of Zebularine on caspase-3 and 9 genes by methylation and protein expression on SKBR3 cells. The SKBR3 cells were treated with Zebularine at different concentrations (0 - 140 μM) for 24 - 96 hours and the effects on the cell viability were shown. The effects on the cell migration and cell transformation were also demonstrated at the specified dose (IC 50 = 40 μM). At the same time, the HRM method and western-blot analysis were used to understand the effects in the apoptotic mechanism. According to the obtained results, it was observed that Zebularine significantly decreased the cell proliferation, cell migration and the cell growth (p < 0.001). As a result of the methylation analyzes performed on SKBR-3 cells at the applied dose, it was observed that Zebularine caused a decrease in both caspase-3 and 9 methylation rates at the 72nd hour. However, both reductions in the methylation levels didn’t cause a significant change (p > 0.05). At the same time, it was detected by the western-blot analyses that there was a time dependent increase in caspase-3 genes while a decrease was detected in the caspase-9 gene in progress of time. The obtained results showed that the methylation changes occurring in the caspase genes weren’t related to the protein levels. According to these results, supportive results a re obtained showing that the Zebularine c an be used in chemotherapy and that this study is unique since it is the first study in the literature intending to investigate the effects of Zebularine on the SKBR3 cells.
Breast cancer is the most commonly observed type of cancer in women and the second most commonly cause of death by cancer [
Apoptosis is a defense mechanism that prevents the cells from forming tumors; moreover, it has a great importance in tumor regression in the response to chemotherapy [
The regulation of apoptosis is controlled by the caspases divided into two including initiator caspases (caspases 2 8, 9 and 10) and effector caspases (caspases 3, 6 and 7) [
Caspase-3 which is the focus point of our study is the most studied among the effector caspases. It plays an important role both in the extrinsic pathway initiated by caspase-8 and in the intrinsic pathway involving caspase-9 [
Based on these results, it was intended to investigate the effects of a new DNA methylation inhibitor and a cytidine analog called Zebularine on apoptotic mechanism [
The SKBR-3 which is a breast cancer cell line was obtained from Prof. Dr. Oğuz Öztürk who is working for the Research Institute for Experimental Medicine. The SKBR-3 cells were produced within an incubator (New Brunswick-Galaxy 170 R) including 5% CO2 at 37˚C in DMEM (Lonza) medium containing 10% fetal bovine serum (FBS) (Biological Industries) and 1% penicillin/streptomycin (Biological Industries). The Zebularine was purchased from the Cayman chemicals and was dissolved in DMSO. In the proliferation experiments, the cells were treated with different concentrations of Zebularine (0 - 140 µM). The cells were treated with 40 µM of Zebularine during the Clonogenic Survival Assay, Wound Healing Assay, Colony Formation Assay, HRM and Western Blot Analysis and all the experiments were repeated 3 times.
The cell proliferation was performed by using MTT Assay. MTT reagent (Glentham Life Sciences) was added to the cells exposed to Zebularine. 4 hours later, DMSO (Merck Millipore) was added to obtain formazan crystals and then the results were monitored in a microplate reader (Thermo scientific?Multi scan Go) at 570 and 650 nm.
The principle of this experiment is based on the interaction between the cells with damaged membrane and the trypan blue. First of all, SKBR-3 cells were cultured in 6-well petri dishes in a way that there was 1 × 105 cell in each well and the cells were allowed to adhere to the surface. Zebularine (40 µM) was applied to the cells at IC50 value determined at the end of the MTT test and the cells were incubated for different time periods. The treated cells were collected in tubes after being removed with the help of typsin-EDTA and centrifuged at 2000 rmp for 5 min. 50 µM medium and 50 µM trypan dye were added to the pellet. The cells were loaded to hemocytometer and count was performed under inverted line microscope (Nicon Eclipse TS100).
The wound healing test was performed in order to examine the effects of Zebularine on cell migration and on the cell-cell interaction. SKBR-3 cells were cultured in 6-well petri dishes in a way that there was 25 × 104 cell in each well and the cells were allowed to adhere. The adherent cells were scraped in a straight line from the center of each well by the tip of a 200 µM pipette. Wash was performed with 1× PBS in order to remove the cell remains. After washing 2 ml of fresh both medicated (Zebularine 40 µM) and non-medicated (control group) media was added. The movements of the cells were monitored at the 24th, 48th, 72nd and 96th hours and also their images were recorded.
First of all, the 6-well petri dishes were plated with DMEM containing 20% FBS and 0.6% agarose in 1:1 ratio and they were allowed to freeze. A mixture of DMEM (containing or not containing 40 µM of Zebularine) including 10% FBS and 0.3% agarose in 1:1 ratio were added to the top of the petri dishes in a way that it included 5 × 103 cell. They were allowed for incubation for 20 days. Every two days the upper medium was replaced with fresh medium. After 20 days the wells were treated with 0.005% crystal violet and were kept for 20 minutes. The colonies formed after the staining was examined under microscope.
Genomic DNA was isolated from the magnified SKBR-3 cells at the 24th, 48th, and 72nd hours after the application of Zebularine. At the same time, the isolation was also performed on the cells which were not treated with Zebularine for control purposes. The cells were lysed with prepared lysis buffer (10 mM Tris-HCI (pH 8), 0.1 M EDTA (pH 8), 0.5% SDS) and the DNA isolation was carried out by the conventional phenol /chloroform method. The DNA amounts were measured by using BioSpec-nano (Shimadzu Corporation, Japan). The obtained DNA was subjected to bisulfite modification by using the EZ DNA Methylation Lightning Kit protocol (Zymo Research, Orange, CA) and after the modification the DNA samples were analyzed.
The MS-HRM Analysis was carried out by using the AriaMx Real Time PCR System device (Agilent Technologies, Santa Clara, CA). The used primers were given in
Genes | Primer Sequence | |
---|---|---|
Caspase-3 | Unmetile Forward | 5’-TGAGTTTTAGGGTGGGATTAAAGT-3’ |
Unmetile Revers | 5’-CACTACAACCCATCCCCTAA-3’ | |
Metile Forward | 5’-TTTAGGGCGGGATTAAAGC-3’ | |
Metile Revers | 5’-CTACGACCCGTCCCCTAA-3’ | |
Caspase-9 | Unmetile Forward | 5’-GTGGGGAGTGAAGATTGATTT-3’ |
Unmetile Revers | 5’-CCACTTCATCCATAACAAATAACC-3’ | |
Metile Forward | 5’-GGGAGC GAAGATTGATTC-3 | |
Metile Revers | 5’-CTTCGTCCATAACGAATAACC-3 |
the samples. The methylated and non-methylated DNAs were mixed in the ratio of 25%, 50%, and 75% in order to form dilutions and the methylated/non-methylated standard DNAs were used in each assay. The reaction mixture 2× Brillant HRM Ultra FastLoci Master Mix (Agilent Technologies) was prepared from 10 ng bisulfite transformed DNA sample in a way that the final volume included 20 µM. The reaction conditions were applied as given in
The protein samples were obtained from both the Zebularine-treated and not treated SKBR-3 cells. The samples were standardized by using the Bradford method. After the measurements, the proteins were mixed with laemmli solution (Bioland Scientific) in 1:1 ratio and were denatured for 5 minutes at 95˚C. The protein samples were loaded into 12% polyacrylamide gel and were conducted in 1× TAE solution at 90 V for 6 hours. The iBlot 2 Dry Blotting System (Thermo Fisher Scientific, US) was used for the transfer process and the transfer process was carried out according to the instruction of the device. After the transfer process, the membrane was blocked for an hour in the blocking solution (TBS-T (Tris-buffered saline, 0.1% Tween 20) (prepared with 5% milk powder). After the blocking, the membranes were treated with primer antibodies (anti-GAPDH (1:1000), anti-casp-9 (1:1000) and anti-casp-3 (1:1000) for 24 hours at 4˚C. After the washing process, the membranes were treated with secondary antibodies (1:5000 dilution) for 24 hours at 4˚C. The membranes which were passed through ECL solution after a repeated washing procedure were visualized by using the G-box gel imaging system (Syngene, USA).
All the analyses were carried out by using Microsoft Excel and SPSS (SPSS INC., Chicago, IL, USA) programs. The Two-tailed Student’s T test and the One-way
Segment | Cycle | Temperature | Duration |
---|---|---|---|
Hot Start | 1 | 95˚C | 3 minutes |
Amplification | 40 | 95˚C | 5 seconds |
60˚C | 20 seconds | ||
High Resolution Melt | 1 | 95˚C | 30 seconds |
65˚C | 30 seconds | ||
95˚C | 30 seconds |
ANOVA analyses were used according to the number of the variable. The statistical significance levels were determined as *p < 0.05, **p < 0.01 and ***p < 0.001.
In this study, SKBR3 breast cancer cell line with ER (-) and PR (-) phenotype was used. Although there are many studies conducted on SKBR3 and the chemotherapy agent, the effects of Zebularine have not been fully explained yet. MTT Assay and Survival Assay were applied in order to investigate the effects of Zebularine on the proliferation of SKBR3 cells. During the MTT Assay applications various doses of Zebularine (0 - 140 µM) were applied to the cells for 24 hours. As a result, the SKBR3 cells were susceptible to Zebularine and a decreased was observed in the proliferation within the increased doses (
Wound Healing Assay was carried out in order to examine in more detail how Zebularine affects the migration of the cells. While the wound was almost completely closed in the cells which were not treated with Zebularine, the wound healing was prevented in the cells to which Zebularine was applied (
Anchorage?Independent Colony Formation Assay was performed in order to evaluate the effects of Zebularine on the independent growth of the cells on the surface. The SKBR3 cells were suspended in soft agar and they were allowed to grow for 20 days in the presence and absence of Zebularine. The obtained data showed that the colony diameter of the cells treated with Zebularine was smaller than the diameter of the untreated cells (
cancer cells that could grow independently for the surface were prevented from the growing by the Zebularine.
MS-HRM studies were carried out to evaluate the effects of Zebularine on the methylation changes in the SKBR3 breast cancer cell line. The MS-HRM analyzes were performed after the bisulfite medication process was carried out with the DNAs obtained from the cell culture.
MS-HRM used the melting profiles obtained from the DNA amplicons derived from bisulfite modified samples. The obtained melting curve provides a combination of the melting curves of the methylated and non-methylated samples. The PCR product obtained from the non-methylated sample had lower melting temperature compared to the PCR product obtained from the methylated sample. The PCR product obtained from the non-methylated sample starts to melt at a relatively lower temperature and then there is a significant decrease in fluorescence [
In this study, the methylation conditions of caspase 3 and 9 which were the important components of the apoptotic mechanism before and after the application of Zebularine, were analyzed. According to the obtained findings significant change was not found in the methylation levels of the caspases although the methylation levels of caspase-3 and 9 was observed to be decreased at the 72nd hour (
At the same time, the protein levels of the caspase-3 and caspase-9 involved in the apoptotic mechanism were evaluated through the western blot analysis conducted on the protein samples obtained from the SKBR-3 cells. There was a time-dependent increase at the inactive caspase-3 level after treating the SKBR-3 cells with Zebularine (
Gene Name | Methylation Rate | ||||||
---|---|---|---|---|---|---|---|
0% - 25% | 25% - 35% | 35% - 45% | 45% - 55% | 55% - 75% | 75% - 100% | ||
Untreated | Casp3 | X | |||||
Casp9 | X | ||||||
Treated | Casp3 | X | |||||
Casp9 | X |
evaluated through the western blot analyses. The results showed that the levels of inactive caspase-9 were decreased time-dependently in the SKBR-3 cells treated with Zebularine (
In cancer, covalent changes such as the addition of methyl group to genomic DNA more often occur compared to genetic changes [
One of the reasons why Zebularine is chosen among the other DNMTi inhibitors is the fact that Zebularine can be effective at lower concentration as well. Moreover, this medication has low toxicity. In the conducted study, it was observed that Zebularine which was a DNA methyl transferase inhibitor had anti-tumor effects on SKBR3 breast cancer cell line. It was detected that the treatment of Zebularine induced the apoptosis mechanism in the SKBR cells and prevented the tumor formation. With the help of MS-HRM studies it was observed that changes occurred in the methylation status of the caspase genes that were effective on apoptosis mechanism.
Respectively, MTT assay, survival assay, anchored-dependent colony formation assay and wound healing assay were carried out in order to evaluate the effects of Zebularine on the cell death, growth and migration in the SKBR3 cells depending on time and dose. Previously conducted studies revealed that Zebularine had anti-tumor effects on cancers such as head and neck cancer, cholangiocarcinoma, colorectal cancer and leukemia [
On the other hand, the invasion of the cancer cells into the surrounding tissues is the first stage of the tumor metastasis and it requires the metastasis to adhere to the extracellular matrix and the chemotactic migration of the cancer cells which are directed by the protruding movement of the cell membrane [
These results also showed that Zebularine prevented the cell metastasis by suppressing the cell migration and cell to cell interactions in the SKBR3 breast cancer cell.
In addition to this, with the help of the soft-agar colony formation test it was investigated whether the Zebularine grew independently from the surface. Through cell transformation the cells can undergo a malignant transformation to form a tumor. Due to this transformation, growth may happen independently from the surface. As a result of the research, it was found that the cell transformation of the SKBR-3 cells treated with Zebularine was largely suppressed and prevented from growing (p < 0.001). Consequently, the tumor formation of the cells was prevented by a malignant transformation.
Another important feature of Zebularine is that unlike the other DNMT inhibitors it tends to prefer the cancerous cells rather than the normal fibroblasts. Therefore, compared to normal fibroblasts, it seemed to suppress the cell growth at the tumor cell line more. At the same time, it primarily blocks DNMT1 and can stimulate the cancer-associated antigen genes in cancer cells. Since it stimulates the cancer-associated antigen genes, when it was combined with immunotherapy, a group of cancer and apoptosis-associated genes were found which supported the possibility of the anti-tumor potential [
The apoptosis mechanism is one of the most important and effective mechanisms in the fight against cancer. It is regulated in two ways including intrinsic and extrinsic ways. There are many components which are involved in the activation and inhibition of apoptosis. Caspase is one of the most important components which are involved in stimulating apoptosis. They are studied in three groups including initiators, effectors and inflammatory caspases. Caspases are present in the cell as zymogen in inactive form. The initiator caspases cause dimer formation and becomes activated by passing through a series of cutting processes after being stimulated. The activated initiator caspases are effective in inducing the effector caspases. They allow the effector caspases to undergo the proteolytic cleavage and to become activated by stimulating them [
Caspase-3 is one of the major caspases which is effective in apoptosis and also affected by caspase-9. The caspase-3 is included within the class of effector caspases and it can be stimulated by both apoptotic pathways. Caspase-3 is the best defined effector caspase. The conducted studies also revealed that it had distinct and overlapping roles with caspase-7 and caspase-6. It is regulated by post-translational modifications just as in caspase-3 and caspase-9 [
Epigenetic mechanisms are one of the mechanisms which are effective in the development of cancer. DNA methylation is the most studied one among them and many studied were carried out in the recent years to understand its effect in cancer. In a research conducted by Eroglu et al. (2018) on breast cancer tissues by using MS-HRM method, the levels of GSTP1 and CDH1 genes were investigated and this is one of the examples to be shown in this regard [
Consequently, as a result of the conducted studies it has been shown for the first time in the literature that Zebularine suppressed significantly the cancer cell growth, cell migration and cell transformation in the SKBR-3 breast cancer cells. In addition, the effects of Zebularine on the methylation of caspases in SKBR3 cells have been investigated for the first time in the literature and it has been detected that Zebularine did not cause a significant change in the methylation of caspases at the applied doses and times. This shows that the caspase stimulation that is responsible for the apoptosis mechanism is not caused by methylation. According to these results, supportive results were obtained about the fact that Zebularine can be used in chemotherapy. This research is unique in the literature since it is the first study to investigate the effects of Zebularine on SKBR3 cells. However, in order to understand the effect mechanism of Zebularine better, the caspase-dependent apoptosis mechanism should be clarified in the future studies.
The authors declare no conflicts of interest.
Eroglu, O. and Celen, M. (2019) Investigation of the Effects of Zebularine on Caspase-3 and Caspase-9 Involved in Anticancer and Apoptotic Mechanisms in SKBR3 Breast Cancer Cell Line. Journal of Cancer Therapy, 10, 229-244. https://doi.org/10.4236/jct.2019.103019