Combination of EGCG and BH3 Mimetic Inhibitor Enhances Apoptosis of MCF-7 and MDA-MB-231 Cancer Cells.

INTRODUCTION
Breast cancer is the most common and second leading cause of cancer death in women.[12] Based on the subtypes, breast cancer treatment strategies include surgical resection, radiotherapy, targeted antibodies, chemotherapy, and small-molecule inhibitors.[3] Drug resistance is one of the main obstacles to breast cancer treatment. Due to its heterogeneous nature, the drug-resistance mechanisms of breast cancer are not fully elucidated. Therefore, the development of effective therapeutic strategies to overcome chemoresistance is needed.[4]
B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra-large (Bcl-xL), and B-cell lymphoma 2-like protein 2 (Bcl-w) are the antiapoptotic members of the Bcl-2 family proteins in mammals, which contain one to four Bcl-2 Homology (BH) domains including BH1, BH2, BH3, and BH4.[5] The over-expression of Bcl-2 antiapoptotic proteins is often observed in different types of tumor cells, which is linked to the cell survival and treatment resistance.[67] Moreover, previous studies have shown that the expression levels of Bcl-2 antiapoptotic proteins such as Bcl-2 proteins are strongly related to the pathological grade and overall survival in breast cancer patients.[89] Therefore, the targeting of Bcl-2 prosurvival proteins could be a potential method for breast cancer therapy.
ABT-737 is a synthetic small-molecule BH3 domain mimetic that strongly binds to the hydrophobic groove of Bcl-2, Bcl-xL, and Bcl-w, thereby leading to the release of Bcl-2-associated X protein (Bak) and Bcl-2 homologous antagonist/killer (Bax) proapoptotic proteins and subsequently induction of apoptosis. However, ABT-737 binds weakly to some other prosurvival Bcl-2 members, such as myeloid cell leukemia 1 (Mcl-1) and Bfl-1/A1.[81011]ABT-737 demonstrated preclinical activity as a single agent or in combination with other chemotherapeutic drugs against types of malignancies such as cervical cancer, ovarian cancer, breast cancer, and oral cancer.[12131415] Other studies showed that cells with persistent or high levels of Mcl-1 are resistant to ABT-737.[81216] Therefore, combination treatment with ABT-737 and Mcl-1 inhibiting agents has been proposed as a potential strategy to overcome the resistance to ABT-737.[15171819]
Green tea is one of the most widely consumed beverages in the world, accounts for about 70% tea consumption. It contains a variety of compounds including polyphenols, caffeine, amino acids, catechins, chlorophyll, and vitamins.[20] Catechins are a type of polyphenol found in green tea, which are responsible for many of its potential health benefits.[212223] Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea that has anti-inflammatory, antiaging, and anticancer properties.[212223] Studies have shown that EGCG reduces the cell growth and triggers apoptosis in cancer cells. Moreover, EGCG significantly enhances the anticancer properties of chemotherapeutic agents.[212223] However, the combination effect of EGCG and ABT-737 remains to be fully explained. In this study, we hypothesized that EGCG can sensitize breast cancer cells to ABT-737 by inhibition of Mcl-1 expression. Thus, we investigated the effect of EGCG on Mcl-1 expression and sensitivity of the breast cancer cells to ABT-737 in vitro. We also investigated the impact of the compounds on the movement of cells migration. Matrix metalloproteinases (MMPs) are a group of enzymes that are involved in the breakdown of proteins and are known to have a significant role in the advancement of cancer, particularly in processes such as tumor invasion, neoangiogenesis, and metastasis. The MMPs are responsible for breaking down the extracellular matrix, which enables cancer cells to migrate and invade surrounding tissues. Previous research has shown that elevated levels of MMPs are associated with poor overall survival rates in various types of solid tumors, including breast, lung, colon, gastric, pancreatic, and prostate cancer.[24]

MATERIALS AND METHODS

Cell culture
The MDA-MB-231 and MCF-7 breast cancer cell lines (Pasteur Institute, Tehran, Iran) were cultured in RPMI-1640 medium (Gibco, Invitrogen, Life Technologies, Germany). supplemented with 15% fetal bovine serum (Gibco), 100 mg/ml streptomycin, 100 U/ml penicillin (Gibco), 2 mM glutamine, and 1% sodium pyruvate at 37˚C and 5% CO2.

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to analyze the effect of EGCG on the sensitivity of breast cancer cells to ABT-737. The experiment was divided into five groups: ABT-737, EGCG, ABT-737 and EGCG, blank control and solvent control. Briefly, cells were cultured at concentrations of 3 × 103 cells per well in 96-well culture plates. On the following day, the cells were treated with different concentrations of ABT-737 and EGCG (0-64 µM) alone and in combination. Cells treated with 1% dimethyl sulfoxide were served as the solvent control group. After 48 hours treatment, the cytotoxicity of the compounds was evaluated using the MTT cell proliferation kit (Roche Diagnostics GmbH, Mannheim, Germany). The absorbance (A) at 570 nm was determined by using a microplate reader (Awareness Technology, Palm City, FL, USA). The survival rate (SR) was calculated from the equation as follows: SR (%) = (ATreatment/AControl) ×100%. The IC50 values were calculated using GraphPad Prism 6.01 software (GraphPad Software Inc., San Diego, CA, USA).

Analysis of combined drug effects
The combination indices (CIs) theorem of the Chou-Talalay method[25] was used to determine the synergy, additivity, or antagonism of the ABT-737 and EGCG combinations. The MTT assay was used to determine the cell survival fractions, which were then converted to fraction affected (Fa) values ranging from 0 to 1. A Fa value of 0 represents 100% cell survival, while a Fa value of 1 indicates 0% cell survival. These values were further analyzed using CompuSyn software (ComboSyn Inc., Paramus, NJ, USA). CI <1, CI = 1, or CI >1 indicates synergistic, additive, or antagonistic effect, respectively.

Cell growth assay
The effect of ABT-737 and EGCG on tumor cell growth was determined by the trypan blue staining. Breast cancer cell lines (5 × 104 cells/well) were exposed to ABT-737 and EGCG, alone and in combination, in 6-well cell culture plates for duration of 24-120 h as described above. At various time intervals, the cells were harvested and subsequently the cell suspensions stained with 0.4% trypan blue (Merck KGaA, Darmstadt, Germany) and incubated for 2 min. Afterward, the count of viable cells was determined using a hemocytometer and an inverted microscope (Nikon Instrument Inc., Melville, NY, USA). By dividing the number of viable cells in the test group by the number of viable cells in blank control group and multiplying the results by 100, the quantification of cell viability was determined. The cell viability percentage in the control group at each time point was regarded as 100%.

Reverse transcription quantitative polymerase chain reaction
Following 48 h of treatment, total cellular RNA was isolated from the cells using the RNA extraction kit (Parstous, Tehran, Iran) following the manufacturer’s protocols. Complementary DNA (cDNA) was synthesized from 1 μg of purified RNA using an Easy cDNA Synthesis Kit (Parstous) and oligo-dT primer, according to the manufacturer’s instructions. LightCycler 96 System (Roche Diagnostics GmbH, Mannheim, Germany) and SYBR Green qPCR MasterMix (Yekta Tajhiz Azma, Tehran, Iran) were employed for qPCR analysis to qualify the relative gene expression. The qPCR was performed in a 20 µl reaction system containing 10 µl of SYBR Green qPCR MasterMix, 1 µl of each of the primers, 1 µl of cDNA template, and 7 µl of nuclease-free distilled water. The sequences of PCR primers were as follows: forward, 5’-TAGTTAAACAAAGAGGCTGGGA-3’, reverse, 5’-CCTTCTAGGTCCTCTACATGG-3’, for Mcl-1, forward, 5’-ATACCATCGAGACCATGCG-3’, reverse, 5’-CCAATGATCCTGTATGTGATCTG-3’, for MMP-2, and forward, 5’-GACATCCGCAAAGACCTGTA-3’, and reverse, 5’-GGAGCAATGATCTTGATCTTCA-3’, for β-actin. The design of qPCR primers was carried out using the NCBI Primer-BLAST tool. The qPCR reactions were performed under the following conditions: 95°C for 5 min and then 35 cycles at 95°C for 10 s, 58°C for 15 s, and 72°C for 15 s. Relative mRNA expression was measured with the 2- (∆∆Ct) method[26] using β-actin as the reference gene.

Wound healing
Breast cancer cell migration was measured using the wound healing assay. Briefly, cells were seeded into 6-well plates with complete culture medium and grown to 80%–90% confluence. Then, we inflicted a uniform scratch in each plate using a micropipette tip, washed twice with PBS to remove the floated cells. Following 48 h of incubation, the wound closure was photographed using bright-field microscopy. The gap distance was measured using ImageJ 1.62 software (National Institutes’ of Health, Bethesda, Maryland, USA).

Clonogenic assay
The breast cancer cells (MCF-7 and MDA-MB-231) were seeded in 6-well culture plates (5 × 103 cells per well) and incubated for 24 h in complete growth medium. Then, cells were treated with IC50 doses of ABT-737 and EGCG, alone and in combination, for 48 h. The growth medium was replaced with fresh medium every 4 days. After 2 weeks, the colonies were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. The number of cell colonies was quantified in each well.

Cell apoptosis analysis by Hoechst 33342
The MDA-MB-231 and MCF-7 BC cells were seeded at 5 × 104 cells/well in 12-well flat-bottomed plates for 24 h, and then treated with ABT-737 and EGCG, alone and in combination. After 48 h of treatment, the cells were washed with PBS, fixed with 3.7% formaldehyde, and stained with Hoechst 33342 (Beyotime, Jiangsu, China) for 15 min. Apoptotic morphological changes were observed under a fluorescent microscope.

Apoptosis ELISA
The assessment of cell death was conducted using a cell death detection ELISA plus kit (Roche Diagnostics GmbH), which enables the detection of mono- and oligonucleosomes released into the cytosol of apoptotic cells. The tumor cells were grown in 6-well culture plate at a density of 1 × 105 cells/well in 6-well culture plate. Following this, the cells were exposed to IC50 doses of ABT-737, EGCG, and their combination, as mentioned before. After 48 h of incubation, the cells were collected and lysed, and then the ELISA was carried out according to the manufacturer’s instructions. In summary, 20 µl of the supernatants and 80 µl of a mixture containing anti-DNA-peroxidase and anti-histone-biotin were transferred into each well of a streptavidin-coated plate. Subsequently, the plate was incubated for 2 h at 25°C. Next, the wells were carefully washed with incubation buffer, and 100 µl of ABTS solution was added. To terminate the reactions, the ABT stop solution was added and the absorbances of the samples were assessed with an ELISA plate reader at 405 nm (with a reference wavelength at 540 nm). The results displayed the difference in absorbance between the treatment and control groups, expressed as a fold change.

Caspase-3 activity assay
The in vitro assessment of caspase-3 activity was carried out using a colorimetric caspase assay Kit (Abnova, Taipei, Taiwan). Briefly, the treated cells were collected and suspended in 50 µl cold cell lysis buffer. Following a 10 min incubation, the cells were centrifuged for 1 min at 10,000 rpm. Next, 50 µl of a 2 × reaction buffer containing 10 mM DTT and DEVD-pNA substrate were added to each sample and incubated at 37°C for 2 h. The spectrophotometric analysis was conducted to measure the absorbance using a microplate reader at a specific wavelength of 405 nm.

Statistical analysis
Quantitative data are expressed as mean ± standard deviation. Statistical comparisons were made using analysis of variance followed by Student’s t-test. The value of P < 0.05 was considered to indicate a statistically significant difference. Prism statistical software version 5.0 (GraphPad, San Diego, CA, USA) was used for all statistical analyses.

RESULTS

Effect of epigallocatechin gallate enhanced the sensitivity of breast cancer cells to ABT-737
The effects of EGCG and ABT-737, alone and in combination, on breast cancer cell survival were explored using the MTT assay. The results demonstrated that monotherapy with each of these compounds significantly inhibited the cell survival rate in a dose-dependent manner (relative to the blank control) [Figure 1]. The IC50 values of EGCG and ABT-737 for 48 h treatment were 61.7 and 2.7 µM in MDA-MB-231 cells, and 46 and 4.1 µM in MCF-7 cells, respectively [Table 1]. Furthermore, the EGCG in combination with ABT-737 further reduced the cell survival rate and the IC50 value, relative to the single treatment (P < 0.05). These data suggest that EGCG can inhibit the cell survival rate and also increase the sensitivity of the breast cancer cells to ABT-737.

The combination effect of epigallocatechin gallate and ABT-737 on breast cancer cells was synergistic
To explore whether the effect of EGCG with ABT-737 on cell survival is responsible for their synergistic interaction, the CI analysis was performed using CompuSyn software. The CI–Fa plots demonstrated that the interaction between EGCG (0-64 µM) and ABT-737 (0-64 µM) exhibited a synergistic effect (CI < 1) at various concentrations. The detailed results of CI analysis are presented in Figure 1.

Effect of epigallocatechin gallate increased the growth inhibitory effect of ABT-737
We then investigated whether EGCG could enhance the growth inhibitory effect of ABT-737 in breast cancer cells. The MDA-MB-237 and MCF-7 cells were treated with EGCG and ABT-737, alone and in combination and then the cell viability was measured by trypan blue dye exclusion assay during a period of 120 h. The cell growth curve showed that compared with the blank control group, the percent of viable cells in EGCG, ABT-737 and combinatorial group markedly decreased time-dependently. In MDA-MB-231 cells, the cell viability in EGCG, ABT-737 and combinatorial group dropped to 65%, 66% and 60% respectively, after 24 h of treatment, and then to a further 26%, 22% and 20%, respectively, at the end of the fifth day [P < 0.05; Figure 1]. Similar results were observed in the MCF-7 cells. The results showed that when two compounds were used together, the IC50 value was significantly reduced compared to when they were used separately. This suggests that combination of these compounds has a synergetic effect on cell growth inhibition.

Effect of epigallocatechin gallate lowered the expression levels of Mcl-1 and MMP-2
We used the quantitative real-time reverse transcription polymerase chain reaction method to investigate the effect of EGCG and ABT-737 on mRNA expression. The results showed that after 48 h of treatment of with EGCG, the expression levels of Mcl-1 and MMP-2 significantly decreased compared to the blank control group. Moreover, ABT-737 did not cause a significant change in the expression level of MMP-2 mRNA, while the expression of Mcl-1 mRNA increased [compared to the blank control group, Figure 2]. In the combination group, a marked decrease in the expression of MMP-2 was observed compared to the ABT-737 and blank control groups [Figure 2, P < 0.05]. The effect of combination treatment on MMP-2 expression was significantly less than EGCG-treated group. In addition, there was a significant difference in Mcl-1 expression between the combination group with the single treatment and blank control groups [Figure 2, P < 0.05]. These data suggest that EGCG has the ability to counteract the impact of ABT-737 on the expression of Mcl-1. No significant change was observed between the solvent control and blank control groups (P > 0.05). The results were consistent across both MDA-MB-231 and MCF-7 cell lines.

Effect of epigallocatechin gallate enhanced the inhibitory effect of ABT-737 on breast cancer cell migration
The wound-healing assay was employed to investigate the effects of EGCG and ABT-737, both individually and in combination, on breast cancer cell migration. The results demonstrated that both EGCG and ABT-737 alone were effective in inhibiting cell migration. However, when these two treatments were combined, the same inhibitory effect was achieved at a lower dose [Figure 3]. This suggests that EGCG has the ability to enhance the inhibitory effects of ABT-737 on breast cancer cell migration. Quantitative analysis of wound healing was depicted in Figure 3.

Effect of epigallocatechin gallate enhanced the inhibitory effect of ABT-737 on breast cancer cell colony formation
By employing the colony formation assay, the impact of EGCG and ABT-737 on the growth of MDA-MB-231 and MCF-7 cells was investigated in terms of their antitumor properties. The results exhibited a substantial decline in the colony formation ability of both cell lines upon exposure to EGCG and ABT-737 [Figure 3]. Moreover, when the two compounds were administered together, a significant decrease in colony formation was observed in comparison to the control cells (P < 0.05).

Effect of epigallocatechin gallate augmented the apoptotic effect of ABT-737 in breast cancer cells
To assess whether the cytotoxicity of compounds was related to the induction of apoptosis, the MDA-MB-231 and MCF-7 cells were treated with the IC50 doses of EGCG, ABT-737 and their combination for 48 h, as described before. Afterward, we executed Hoechst 33342 staining and an ELISA cell death assay. Nuclear morphological change is a prevalent attribute associated with cell death. So, apoptotic morphological changes in breast cancer cells were demonstrated using Hoechst 33342 staining. The presence of apoptotic cells with nuclear fragments was evident in cells treated with EGCG and ABT-737 [Figure 4]. Conversely, the control cells did not exhibit such characteristics. In addition, there was no significant difference observed in the number of apoptotic cells between the combination treatment group and the cells that treated with only one type of compound.
The findings from the ELISA cell death assay revealed a significant rise in apoptosis following a 48 h exposure to EGCG and ABT-737. The extent of apoptosis in MDA-MB-231 cells were determined to be 4.75 and 4.81 times higher in EGCG or ABT-737 treated cells, respectively, in comparison to the blank control group (P < 0.05). Intriguingly, when the two substances were combined, the level of apoptosis increased to 4.72 times at the specified time point (P < 0.05). Conversely, no noteworthy alterations in apoptosis were observed in the solvent control group when compared to the blank control group (P > 0.05). Similar results were observed in the MCF-7 cell line.
Our data indicated that the IC50 dose of the combination is lower than that of either compound alone. This suggests that the combined use of these two agents has a more pronounced impact on inducing apoptosis compared to the individual treatment with each agent. The observed increased in apoptosis effect with the combination aligns with the results obtained from the MTT assay.

Combination therapy enhanced the caspase-3 activity in breast cancer cells
A caspase-3 activity assay was performed to investigate the molecular mechanism of apoptosis induced by EGCG and ABT-737. The revealed a notable increase in caspase-3 activity in the cells that were exposed to EGCG or ABT-737. Furthermore, the percentage of caspase-3 activity in the breast cancer cells treated with the combination of EGCG and ABT-737 did not differ significantly from the caspase-3 activity observed in cells treated with EGCG or ABT-737 alone [P > 0.05, Figure 4].

DISCUSSION
The treatment of breast cancer involves a combination of local and systemic approaches, such as surgical tumor removal, targeted antibody therapy, chemotherapy, and small inhibitory molecules. However, the effectiveness of chemotherapy in treating breast cancer is often hindered by the complex and diverse nature of the disease, resulting in drug resistance.[134] This underscores the importance of developing new and improved treatment methods to effectively combat breast cancer. Over-expression of antiapoptotic proteins have been linked to enhanced cellular proliferation, resistance to pharmaceutical treatments, reduced cell apoptosis, and unfavorable outlook for individuals diagnosed with breast cancer.[567] Studies showed that cells with persistent or high levels of Mcl-1 are resistance to ABT-737.[81216] Therefore, combination treatment with ABT-737 and Mcl-1 inhibiting agents has been proposed as a potential strategy to overcome the resistance to ABT-737.[151819] Thus, we investigated the effect of EGCG on Mcl-1 expression and sensitivity of the breast cancer cells to ABT-737.
The results of our study revealed that treatment with either EGCG or ABT-737 individually led to a marked decline in cell viability and initiated apoptosis. Nevertheless, when EGCG and ABT-737 were administered together, there was a substantial decrease in the IC50 value and a synergistic reduction in the cell survival rate compared to using either compound alone. These findings indicate that combined treatment of these compounds results in a more significant effect on cell survival and apoptosis compared to using each compound individually. So far, numerous researches have been carried out to explore the link between the expression level of the Mcl-1 gene in cancer cells and their resistance to ABT-737. For example, in a study by Konopleva et al., the focus was on exploring the factors that impact the sensitivity and resistance of AML cells to ABT-737.[16] The results of the study demonstrated that AML cells with increased levels of antiapoptotic proteins like Bcl-xL, Mcl-1, or Bcl-2, and decreased levels of Bim, a proapoptotic protein, are more likely to display resistance to ABT-737. Moreover, the study identified the activation of survival pathways, such as the PI3K/Akt pathway, as a key contributing factor to the resistance of AML cells to ABT-737. Tahir et al. in a separate study examined how Bcl-2 family member proteins influenced the cellular response of various small-cell lung cancer cells to ABT-737.[27] Their findings revealed that small-cell lung cancer cell lines with increased expression of Bcl-2, Bcl-xL, Bim, and Noxa, and reduced levels of Mcl-1, exhibited increased sensitivity to ABT-737. Wang et al. demonstrated the efficacy of A-1210477, a targeted inhibitor of Mcl-1, in overcoming resistance to ABT-737 in AML cells that had up-regulated Mcl-1.[28] The researchers found that when A-1210477 was combined with ABT-737, a remarkable synergistic effect occurred. This synergistic effect resulted in the induction of apoptosis in AML cells. Woo et al. conducted a study to investigate the impact of YM155, a survivin inhibitor, and ABT-737 on cellular apoptosis in lung cancer, glioma, and renal cell carcinoma cells.[29] Their findings revealed that YM155 played a significant role in increasing the susceptibility of tumor cells to ABT-737 by suppressing the expression of Mcl-1. In this study, we showed that EGCG has the ability to reduce the levels of Mcl-1 mRNA, leading to an increase in apoptosis induced by ABT-737 in MDA-MB-231 and MCF-7 breast cancer cells. These findings are in agreement with the above studies and confirm that EGCG can enhance the susceptibility of breast cancer cells to ABT-737 through the inhibition of Mcl-1 expression.
We also assessed the impact of EGCG and ABT-737 on gene expression. The findings from qPCR analysis indicated that ABT-737 led to an increase in the expression of Mcl-1 mRNA while having no effect on the expression of MMP-2 mRNA. Additionally, it was observed that EGCG decreased the expression levels of both MMP-2 and Mcl-1 mRNA in cancerous cells. When the two compounds were used in combination, EGCG was found to counteract the up-regulation of Mcl-1 mRNA caused by ABT-737. These changes in gene expression were associated with the inhibition of cell proliferation, reduced colony formation, and decreased cell migration. Furthermore, sensitivity of the breast cancer cells to ABT-737 was increased after treatment with EGCG. Our study aligns with previous research that has explored the relationship between EGCG and its effects on gene expression and cancer cellular processes. A previous study explored the effect of EGCG treatment on growth and invasion in a breast carcinoma cell line resistant to tamoxifen. Results show that expression of EGFR and ERK decreased by EGCG treatment and in vitro cell growth and invasion decreased. Moreover, MMP-2 and MMP-9, which are implicated in cell invasion and metastasis, were significantly reduced. This study also demonstrated that EGCG could attenuate the tamoxifen-resistant phenotype of MCF-7 breast cancer cells.[30] Another study investigated the impact of EGCG on the activation of the HER-2 receptor in human HNSCC and breast carcinoma cells. The findings indicate that when these cells were treated with EGCG, it resulted in the suppression of cell growth and the inhibition of HER-2 phosphorylation in both types of cells. This inhibition was linked to the suppression of STAT3 activation and a decrease in the levels of cyclin D1 and Bcl-xL proteins within the cells. Additionally, EGCG notably increased the susceptibility of both cell lines to growth inhibition caused by Taxol.[31] In their study, Hu et al. investigated the impact of EGCG, cisplatin, and oxaliplatin on the growth of DLD-1 and HT-29 human colorectal cancer cells.[32] The findings of their research demonstrated that the combination of EGCG with either cisplatin or oxaliplatin exhibited a synergistic effect in terms of suppressing cell proliferation and promoting apoptosis. Chen et al. conducted a study to investigate the effect of EGCG or sulforaphane (a major component of cruciferous vegetables) on both paclitaxel-sensitive (SKOV3-ip1) and -resistant (SKOV3TR-ip2) ovarian cancer cell lines alone or in combination.[33] The findings of this research indicate that sulforaphane inhibits cell viability of both ovarian cancer cell lines and that EGCG potentiates the inhibiting effect of sulforaphane on ovarian cancer cells. Their results also demonstrated that the combined treatment of EGCG and sulforaphane has the ability to trigger apoptosis through the suppression of hTERT and Bcl-2, while simultaneously enhancing the DNA damage response in paclitaxel-resistant ovarian cancer cell lines. Moreover, the employment of these compounds offers a solution to the problem of paclitaxel resistance in ovarian cancer cells. However, the results obtained from the previously mentioned reports are consistent with our own data, suggesting that EGCG possesses the capability to inhibit the proliferation and migration of cancerous cells. Moreover, it has the ability to augment the susceptibility of tumor cells towards chemotherapeutic agents such as ABT-737 by initiating apoptosis.
There are two main signaling pathways that control cellular apoptosis: the mitochondrial or intrinsic pathway and the extrinsic pathway.[34] The intrinsic pathway is activated when the cell is exposed to harmful stimuli, which leads to the release of cytochrome c and the activation of caspases-9. On the other hand, the extrinsic pathway is triggered by the binding of ligands to death receptors on the cell’s surface, resulting in the activation of caspase-8. Both pathways ultimately converge at caspase-3, which plays a central role in the apoptotic process. Once caspase-3 is activated, it initiates a proteolytic cascade, activating other caspases and initiating a series of events that ultimately lead to apoptosis.[34] The regulation of this intrinsic pathway is performed by the pro- and antiapoptotic members of the Bcl-2 family proteins.[35] In apoptotic conditions, the proapoptotic members such as Bak and Bax are activated. Activated Bak and Bax cause the mitochondrial outer membrane permeability (MOMP), release of cytochrome c into the cytoplasm, and subsequently activation of caspases. The antiapoptotic proteins such as Bcl-2 and Mcl-1, when not sequestered by proapoptotic members, inhibit apoptosis.[36] The synthetic compound ABT-737 acts as a mimic of BH3 and displays a remarkable ability to bind tightly to Bcl-2, Bcl-Xl, and Bcl-w proteins. However, its interaction with Mcl-1 is not as strong. Scientific investigations have revealed that an increase in Mcl-1 levels is associated with a reduction in the toxic effects of ABT-737 and the emergence of resistance. To overcome this challenge, researchers have proposed inhibiting the Mcl-1 protein as a potential strategy to heighten the responsiveness to ABT-737.[8101112]
EGCG has been demonstrated to cause changes in the expression of Bcl-2, Bcl-xL, P21, P53, and Cyclin D1 through NF-Kb, AKT, and PI3K-mTOR signaling pathways by which inhibits cell cycle and proliferation, and triggers apoptosis.[23] EGCG has been shown to induce apoptosis in various cancer cells through both intrinsic and extrinsic pathways. However, the exact mechanism of action is still unclear.[22] Our study provides evidence of the ability of EGCG to reduce the expression of Mcl-1. The observed alteration in gene expression was strongly associated with the initiation of cellular apoptosis. Moreover, the decrease in Mcl-1 levels was linked to an enhanced sensitivity to ABT-737. These findings suggest that EGCG possesses the capacity to not only induce cell death but also enhance the sensitivity of breast cells to chemotherapeutic agents such as ABT-737. This effect is achieved through the modulation of Mcl-1 expression, a crucial protein involved in promoting cell survival.
Our findings from this study demonstrated that the administration of either ABT-737 or EGCG resulted in a reduction in the rate of cell migration. Specifically, the inhibition of cell migration by EGCG was linked to the suppression of MMP-2 gene expression in breast cancer cells. These results are consistent with previous studies and provide further evidence that EGCG plays a role in inhibiting cell migration and metastasis by down-regulating the expression of MMP-2. This suggests that EGCG may have potential therapeutic implications in the management of cancer progression and metastasis.[23]

CONCLUSIONS
In summary, our findings highlight the promising therapeutic potential of combining EGCG and ABT-737 in breast cancer treatment. This combination treatment not only showed a significant reduction in the IC50 value but also exhibited synergistic effects in inhibiting colony formation, impeding cell growth, and promoting cell death. Moreover, EGCG demonstrated its ability to inhibit cell migration and induce apoptosis through the down-regulation of MMP-2 and Mcl-1, respectively. Interestingly, the expression of Mcl-1 was found to increase after treatment with ABT-737. Furthermore, EGCG was able to enhance the apoptotic effect of ABT-737 by suppressing Mcl-1. These findings provide valuable insights into potential therapeutic strategies for breast cancer treatment.

Conflicts of interest
There are no conflicts of interest.