Effect of sulforaphane on BCL2 and BCL2L1 expression at the gene and protein levels in breast cancer cells

Background and Objectives Breast cancer remains the most commonly diagnosed cancer among women globally. The complicated tumor microenvironment, aggressive behavior, heterogeneous nature, elevated proliferation rate, and treatment resistance are among the most characterized hallmarks of breast cancer. Many of these processes relate to the capacity of cancer cells to avoid apoptosis. Overexpressing anti-apoptotic genes is one strategy by which cancer cells prevent apoptosis. Consequently, anti-apoptotic gene expression reduction can promote the apoptosis mechanism and enhance the cells' sensitivity to cancer therapy. Chemical therapies are effective in treating cancer, but their adverse effects and resistance frequently result in treatment failure. Consequently, plant-based substances have recently received a lot of attention for their ability to inhibit cancer cell survival and increase sensitivity to treatment. One of these phytochemicals is sulforaphane (SFN), which can be found in cruciferous vegetables like broccoli and cauliflower. SFN possesses various attributes, including anti-inflammatory, cardioprotective, antioxidative, cytoprotective, and antimicrobial properties; however, its anticancer property is the most significant. Several studies demonstrated that SFN might induce apoptosis and inhibit the expression of cancer-specific genes. Thus, this study aimed to investigate the in vitro effects of SFN on breast cancer cells by evaluating BCL2 and BCL2L1 expression at both the gene and protein levels. Material and Method In this study, MCF-7 and MDA-MB-231 breast cancer cell lines were used. Cells were seeded in plates and incubated overnight. The next day, cells were treated with 25 µM and 50 µM concentrations of SFN or with DMSO as a control (0 µM). After 48 hours of incubation time, the expression of BCL2 and BCL2L1 genes was determined using reverse transcription-quantitative PCR (RT-qPCR). The total RNA was extracted from cells using the RNeasy Mini Kit. One microgram of total RNA was converted into cDNA using the High-Capacity RNA-to-cDNA Kit. RT-qPCR analysis was performed on a QuantStudio3 Real-Time PCR System. Relative gene expression was normalized to β-actin. BCL-2 and BCL-XL protein levels were assessed using Western blot analysis, involving separation by gel electrophoresis and transfer onto a membrane utilizing the semi-wet transfer unit Mini Blot module. Target proteins were detected using specific primary and secondary antibodies. The visualization of chemiluminescent imaging was done utilizing the Azure 280 system. The target protein expression level was normalized using the GAPDH protein as an internal control. Statistical analysis was performed using IBM SPSS Statistics 30 (version 30.0.0.0), and statistical significance was defined as a p-value less than 0.05. Results In MCF-7 cells, after the treatment with 25 µM and 50 µM concentrations of SFN, the BCL2 gene expression was reduced to 0.44 and 0.33, and BCL2L1 gene expression was 0.83 and 0.73, respectively. Additionally, BCL-2 protein levels decreased to 0.56 and 0.33 after the same SFN concentrations. The level of the BCL-XL protein did not alter significantly. In MDA-MB-231 cells, the BCL2 gene expression reduced to 0.88 and 0.82, and BCL2L1 gene expression was 0.73 and 0.32 after the treatment with 25 µM and 50 µM concentrations of SFN, respectively. Also, the concentrations of SFN used statistically significantly reduced protein levels. BCL-2 protein levels decreased to 0.85 and 0.78, and BCL-XL was 0.84 and 0.63. Conclusions and Recommendations Our study results revealed that SFN reduce the gene expression and protein levels of BCL2 and BCL2L1 in breast cancer cells. This suggests that SFN may be a potential anticancer agent whose effects are associated with the regulation of BCL-2 family proteins in the treatment of breast cancer.