Synergistic cytotoxicity of Solanum aculeastrum with 4-hydroxytamoxifen in the MCF-7/TAMR-1 breast adenocarcinoma cell line

Abstract

Introduction: Breast cancer, the leading cause of death in woman worldwide, is characterised by its receptor status. Most patients (70%) are diagnosed with oestrogen receptor (ER)-positive breast cancer. Tamoxifen, a selective oestrogen receptor modulator (SERM), is the preferred chemotherapeutic treatment, however, breast cancer cells may become resistant to its active metabolite (4-hydroxytamoxifen [4-OH-TAM]). Chemoresistance may be due to alterations to ER, reducing chemoresponsiveness. Herbal medicines that are used for the treatment of cancer, such as Solanum aculeastrum (soda apple), may provide a combinational effect to reduce breast cancer cell growth and viability. The study assessed the synergistic cytotoxicity of S. aculeastrum with 4-OH-TAM in tamoxifen-resistant MCF-7 breast adenocarcinoma cells. Methods: Tentative phytochemical identification was conducted using ultra-performance liquid chromatography. Inherent cytotoxicity of methanol (ME) and hot water (HWE) extracts alone and in combination with 4-OH-TAM was assessed at 24 h, 48 h and 72 h via sulforhodamine B staining and resazurin conversion assays. Combinational effects were determined using a checkerboard assay and CompuSyn. The effect of the most synergistic combination on cell viability (fluorescence microscopy [Hoechst, acridine orange and propidium iodide]), redox status (reactive oxygen species [ROS] and reduced glutathione [GSH] levels via dichlorofluorescein and monochlorobimaine fluorescence. respectively) and ER-expression (Western blotting) was assessed after 24 h exposure. Results: A chromatographic fingerprint was produced. The methanol (ME) and hot water extract (HWE) displayed phytochemicals in each extract with solamargine, solasodine, solasonine, and solaculine. Dose-dependent cytotoxicity was observed after 24 h for 4-OH-TAM, ME, and HWE. The most synergistic combination was 10.05 µg/mL HWE and 5.83 µg/mL 4-OH-TAM, with a combinational index of 0.78289. The combination displayed significantly (p<0.001) reduced cell density (76.65%) The combinational treatment induced cellular blebbing and rounding, suggesting apoptosis in comparison to individual treatments. The combination abolished ROS levels after 24 h treatment, with a 30% decrease in GSH levels. A non-significant decrease in relative ER-α expression was observed after combinational treatment. Discussion: The combination showed signs of apoptosis with similar findings been reported for steroidal alkaloids identified in S. aculeastrum. The generation of ROS contributes to apoptosis of breast cancer cells, alongside a reduction of GSH availability. Although ROS was not increased after 24 h, the GSH levels reduced, suggesting destabilisation of the redox status of cells. This destabilisation appears to have caused reductive stress rather than oxidative stress, with reduced GSH levels that may hinder detoxification of 4-OH-TAM. The reduced expression of ER-α reduces the proliferative capabilities of cells as less oestrogen will be able to bind and promote growth of breast cancer cells. Solanum constituents, like solasodine have been shown to dysregulate ER, which may be like what has been observed in the present study. Conclusion: Combining 4-OH-TAM alongside the HWE increased the cytotoxicity in reference towards the MCF-7/TAMR-1 breast adenocarcinoma cell line via possible apoptotic changes, as well as destabilisation of redox status and dysregulated ER-α expression. Reduced GSH levels may hinder detoxification of 4-OH-TAM and contribute to reductive stress, impacting proliferation and cellular functions. Bioactivity is ascribed to steroidal alkaloids as seen in the extracts. Combinational treatment using S. aculeastrum needs to be further assessed to determine the potential for further development as an anticancer combination.