Synergistic Effects of the Combination of Quercetin, Naringin, and Rutin on Cervical Cancer: Roles of Apoptosis and Autophagy

Abstract

Cervical cancer is, to date, the second most common cause of cancer in female patients worldwide. Most of the present-day research activities have concentrated on studies of antitumor medicinal plants and bioactive compounds obtained from natural flavonoids such as quercetin (Q), naringin (N), and rutin (R). The present study reported an in vitro method for assessing the combined anticancer activity of quercetin, naringin, and rutin in combination in HeLa cervical cancer cells. The anticancer potential of the compounds was investigated via experiments involving apoptosis, detection of ROS levels, rhodamine 123 staining, AO/EtBr staining, scratch tests, real-time methods, and Western blot techniques in protein-mediated autophagy pathways. On the basis of the results from the MTT assay and combination index, reasonable combinations of RQ and RQN were selected. Experiments revealed significant upregulation of Beclin1 protein levels and corresponding downregulation of P62, indicating enhanced autophagic flux in HeLa cells treated with flavonoid combinations. Assays for apoptosis revealed that the total number of apoptotic cells increased in a time-dependent manner, and the percentages of apoptotic cells enriched with the RQN combination were 16.07%, 65.02%, and 8.91% at 24, 48, and 72 h, respectively. The combination of RQ resulted in the following percentages of apoptotic cells at the same time points: 31.04%, 32.36%, and 11.46%. Real-time PCR and Western blotting of autophagy-related marker proteins revealed that the flavonoid combinations drastically modulated the autophagy pathway in HeLa cells by increasing the expression levels of Beclin1 (protein and expression levels) and LC3 (expression), whereas the expression level of P62 (protein and expression levels) notably decreased. Thus, these data suggest that flavonoid combinations may induce cervical cancer cell death through an autophagy-mediated mechanism and apoptosis. These flavonoid combinations represent potential therapeutic strategies for treating cervical cancer.