Isorhamnetin Reverses Doxorubicin Resistance in MCF-7/Doxo Cells by Suppressing P-glycoprotein-mediated Drug Efflux and Reactivating Apoptosis
Abdulmajeed M. Jali, Ali Hanbashi, Faroq Kamli, Khalid Zoghebi, Hamad Al Shahi, Awaji Y. Safhi, Fahad Y. SabeiBackground
Doxorubicin resistance is a major challenge in breast cancer treatment, leading to cancer recurrence and poor therapeutic outcomes. Therefore, new agents that can overcome chemoresistance are urgently needed.
Purpose
Chemotherapy is widely used as a frontline treatment for breast cancer (BC), but chemoresistance to the drug relapses cancer, leading to the treatment being unsuccessful. The objective of this study is to explore the ability of the plant flavonoid isorhamnetin to overcome chemoresistance and subsequently guide it to the apoptotic pathway.
Materials and Methods
To achieve the objective of this study, wild-type MCF-7 BC cells were exposed to increasing doses of doxorubicin for an extended duration to obtain a doxorubicin-resistant BC cell line (MCF-7/Doxo). Then, the cytotoxic potency of isorhamnetin was evaluated using a tetrazolium-based assay, and morphological alterations were observed under phase-contrast microscopy. Apoptosis and deoxyribonucleic acid (DNA) fragmentation were investigated by annexin/propidium iodide (PI) staining and a comet assay, respectively. Mitochondrial membrane potential was evaluated using the Muse Mito Potential Kit by flow cytometry. Anti-proliferative activity was further determined by Ki-67 expression analysis, cell-cycle distribution studies, and clonogenic survival assays. The ability of isorhamnetin to reverse chemoresistance was investigated by evaluating the gene expression levels of P-glycoprotein.
Results
Isorhamnetin exhibited dose-related cytotoxicity against MCF-7/Doxo cells, with a half maximal inhibitory concentration (IC 50 ) of 39.508 µg/mL. Annexin/PI staining resulted in a marked increase in the percentage of late apoptotic and necrotic cells. Comet assay showed increased tail and head length, confirming fragmented DNA upon exposure to isorhamnetin. Investigation of mitochondrial membrane potential using the Muse Mito Potential Assay Kit revealed a damaged and depolarized mitochondrial membrane in isorhamnetin-treated group. Ki-67 proliferation analysis indicated a high proliferative index in untreated MCF-7/Doxo cells, which was significantly reduced after isorhamnetin treatment. Cell-cycle analysis further demonstrated cell accumulation in S phase and G2/M phase in treated cells. In addition, gene expression studies revealed substantial downregulation of P-glycoprotein, suggesting that isorhamnetin may overcome chemoresistance by inhibiting drug efflux and promoting apoptosis in resistant BC cells.
Conclusion
Isorhamnetin effectively associates with the drug reversal mechanism in MCF-7/Doxo cells by suppressing P-glycoprotein-mediated drug efflux and inducing apoptosis through mitochondrial dysfunction, DNA fragmentation, and anti-proliferative activity.