The tumor originates from monoclonal cell and appears to contain a heterogeneous population of tumor cells. According to the cancer stem cell hypothesis, among these heterogeneous cell populations, only a few subpopulation of cancer cells, possess enhanced self-renewal capacity, differentiation potential, tumorigenicity and chemoresistance . Our previous studies have demonstrated that high aldehyde dehydrogenase (ALDH) activity may represent a functional marker for cervical CSCs and LGR5 can promote cervical cancer stem cell traits and chemoresistance [29, 30]. Here, we explored to find a drug targeting cervical CSCs for cancer therapies. DS has been shown to be effective against some cancer types in preclinical studies. It has also been observed that the patients who continuously used DS have a lower risk of death from cancer compared with those who stopped using DS at their diagnosis in an epidemiological study . However, little is known about the mechanisms and therapy of disulfiram in cervical cancer. In the present study, we show, for the first time, the cytotoxicity of disulfiram may be superior to cisplatin caused by targeting LGR5-positive cervical cancer stem-like cells in cervical cancer.
In this study, DS with exogenous Cu2+ supplementation exhibited dose-dependent and time-dependent cytotoxicity in cervical cancer cells, but disulfiram alone had no therapeutic effect in votro. Researches reported recently demonstrated that disulfiram/copper treatment eliminated the stem cell-like ALDH + cells pool in non-small cell lung cancer [34, 35]. We also found disulfiram/copper complex alone or in combination with DDP strongly inhibited ALDH activity in SiHa cells and DDP treated HeLa cells. Although there is no ALDH expression in untreated HeLa cells, disulfiram/copper complex induced more remarkable cell apoptosis and exhibited greater efficacy of tumor growth inhibition on HeLa cells compared with DDP in vitro and in vivo. Previous studies manifested that overexpression of LGR5 promotes cervical cancer cell stemness and chemoresistance. According to this study, disulfiram/copper complex exhibited equivalent effect whether LGR5-positive cervical cancer stem-like cells or LGR5-negtive cells both in vitro and in vivo systems. In other words, disulfiram/copper complex treatment was superior to cisplatin by its ability of eliminating the stem cell-like LGR5 + cells pool in HeLa cells.
It has also been reported that DS/Cu treatment causes accumulation of cells in G2 and inhibits DNA synthesis . In our study, the decrease in the proportion of cells in S phase following DS/Cu treatment was accompanied by an increase in the proportion of SiHa cells in G2 phase. Although the decrease in the proportion of cells in S phase following DS/Cu treatment without increase of G2 phase in HeLa cells, both SiHa and HeLa cells were corroborated the observation that DS/CuCl2/DDP caused arrest in G2. In addition, the expression of p53 and p27 was significantly increased in DS/CuCl2/DDP treated SiHa and HeLa cells (P < 0.05, Fig. 2). When DNA damage occurs in the G2 phase, the expression levels of p53 and p27 are increased, cells undergo G2/M phase cell cycle arrest, p53 and its downstream gene p27 promotes apoptosis [36–38]. However, in detail, the changes of these genes caused by DS/CuCl2 are asynchronous in HeLa and SiHa cells, which need further study. Bcl-2, an anti-apoptotic protein, has been reported to regulate the apoptotic pathways and protect against cell death [39, 40]. The multidrug resistance gene ABCG2 is also an important proliferation-promoting oncogene in cervical cancer . We also observed that DS/CuCl2 treatment resulted in a marked decrease of bcl-2 and ABCG2 protein expression in SiHa and HeLa cells, which was superior to that of cisplatin.
Previous studies developed a vaginal tablet of DS to be used in HeLa and Ca-Ski cells for the localised treatment of cervical cancer [28, 42]. This study showed animal experiments provided a basis for systemic application of DS. In addition, disulfiram/copper complex could inhibit the expression of stemness marker in cervical cancer cells. It is attractive to consider DS as an independent treatment or adjuvant treatment combined with cisplatin to establish a new chemotherapy protocol for targeting CCSCs. In the combination treatment, DS could suppress the cisplatin-resistant LGR5+ stem-like population to cisplatin treatment. At the same time, the cytotoxicity of cisplatin could be increased once combined with DS. However, more research is still needed to further explore the effect of DS on primary tumors from patients.