Metformin Potentiates the Anticancer Effect of Everolimus on Cervical Cancer In Vitro and In Vivo.

Ya-Hui Chen,Shun-Fa Yang,Yi-Hsuan Hsiao,Mei-Ling Chen,Tze-Ho Chen,Jyun-Xue Wu

doi:10.3390/cancers13184612

Abstract

Simple SummaryRecent studies have shown that metformin combined with clinical chemotherapeutic drugs could cause decreased cell toxicity and attenuate tumor resistance in various types of cancer. The aim of the present study was to elucidate whether combined treatment with metformin and everolimus has a synergistic anticancer effect in human cervical cancer in vitro and in vivo. The results showed that this combined treatment synergistically inhibited the growth of human cervical cancer cell lines and xenografts in nude mice, and induced caspase-dependent apoptosis, promoting sub-G1- and G0/G1-phase arrest and enhancing mtROS production. Combined treatment also synergistically inactivated PI3K/AKT signaling and activated MAPKs signaling in cervical cancer. Our data suggested that metformin potentiates the anticancer effect of everolimus on cervical cancer, and combined treatment provides a novel therapeutic strategy for patients with cervical cancer.Cervical cancer is globally the fourth most common cancer in women. Metformin is a widely used drug for the treatment of type II diabetes and has been shown to possess important anticancer properties in cervical cancer. Everolimus is an mTOR inhibitor and is widely used to treat NETs, RCC, TSC, and breast cancers. The present study investigated the anticancer effects of metformin and everolimus in cervical cancer, when used alone or in combination. CaSki and C33A human cervical cancer cells were treated with different concentrations of everolimus alone or in combination with metformin. Cell viability was assessed using a CCK-8 assay. Cell apoptosis, cell-cycle, and mtROS analyses were conducted using flow cytometry. Target protein levels were analyzed by Western blotting. Related mechanisms were confirmed using appropriate inhibitors (z-VAD-fmk and BIRB796). The in vitro results were further confirmed in a xenograft tumor study. Both metformin and everolimus, when used alone, were moderately effective in inhibiting cell proliferation and inducing cell apoptosis of CaSki and C33A cells. When used in combination, these two drugs synergistically inhibited the growth of human cervical cancer cells and xenografts in nude mice, promoted sub-G1- and G0/G1-phase cell-cycle arrest, and enhanced mtROS production. The protein expressions of PI3K (p110α) and p-AKT were significantly downregulated, while P27, P21, p-p38, p-ERK, and p-JNK were upregulated following combined treatment. These results revealed that metformin potentiates the anticancer effect of everolimus on cervical cancer, and combination treatment with metformin and everolimus provides a novel therapeutic strategy for patients with cervical cancer.

Highlights

Cervical cancer is globally a major gynecologic carcinoma among women; in 2018, approximately 570,000 women were diagnosed and more than 310,000 women died from the disease [1]
20 mM metformin with 20 μM everolimus for C33A cells. These results suggested that metformin combined with everolimus could have a synergistic effect against the cell proliferation of cervical cancer cells
G0/G1-phase was significantly higher (77.3 ± 0.8%) in C33A cells, as compared with cells treated with metformin or everolimus alone (Figure 3, p < 0.05). These results suggested that combined treatment with metformin and everolimus was more effective than treatment with either alone in promoting human cervical cancer cell apoptosis and causing cell-cycle arrest in the sub-G1 and G0/G1 phases

Summary

Introduction

Cervical cancer is globally a major gynecologic carcinoma among women; in 2018, approximately 570,000 women were diagnosed and more than 310,000 women died from the disease [1]. In studies regarding international trends in the incidence of cervical cancer based on pathological type, squamous cell carcinoma represents threequarters of cases; adenosquamous cell carcinoma and adenocarcinoma account for 10–15%; and other types or those of unspecified histology account for the remaining 10–15% of cases [4,5]. Histology has been shown to be a statistically significant variable in cause-specific mortality [6]. Relative to non-microinvasive squamous cell carcinoma, the cause-specific mortality hazard ratios for microinvasive squamous cell carcinoma and adenosquamous carcinoma were reported to be 0.28

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