Abstract
BackgroundDisulfiram (DSF) is a drug used for treatment of alcoholism that has also displayed promising anti-cancer activity. It unfolds its effects by inhibiting the enzyme activity of aldehyde dehydrogenase (ALDH) isoforms. MethodsMTT assay, spheroid formation, clonogenicity assay, qRT-PCR, and ALDH enzyme activity analysis were performed using ovarian cancer cell lines IGROV1, SKOV3 and SKOV3IP1. Cell cycle analyses and measurement of intracellular reactive oxygen species (ROS) were carried out by flow cytometry. ALDH+ and ALDH− cells were isolated by FACS sorting. ResultsALDH activity was inhibited in ovarian cancer stem cells (the proportion of ALDH+ cells was reduced from 21.7% to 0.391%, 8.4% to 0%, 6.88% to 0.05% in cell lines IGROV1, SKOV3, and SKOV3IP1, respectively). DSF with or without the cofactor copper (Cu2+) exhibited cytotoxicity dose- and time-dependent and enhanced cisplatin-induced apoptosis. DSF + Cu2+ increased intracellular ROS levels triggering apoptosis of ovarian cancer stem cells (CSC). Significantly more colony and spheroid formation was observed in ALDH+ compared with ALDH- cells (P < 0.01). Moreover, ALDH+ cells were more resistant to cisplatin treatment compared with ALDH-cells (P < 0.05) and also exhibited a lower basal level of ROS. However, no significant difference in ROS accumulation nor in cellular viability was observed in ALDH + cells in comparison to ALDH- cells after pre-treatment with DSF (0.08 μM). ConclusionOur findings provide evidence that DSF might be employed as a novel adjuvant chemotherapeutic agent in combination with cisplatin for treatment of ovarian cancer.
Highlights
Epithelial ovarian cancer (EOC) is the most lethal gynaecological cancer [1]
DSF showed a linear increase of cytotoxicity with increasing concentration of the drug in SKOV3 cells, and biphasic cytotoxicity in IGROV1 and SKOV3IP1 cell lines, with the relative viability of cells increasing slightly at DSF 10 μM (Fig. 1A)
The results showed that in the IGROV1 cell line early apoptosis increased from 0.893% to 6.42%, late apoptosis increased from 4.15% to 19.8%, and necrosis increased from 2.11% to 2.74% in control cells and in cells treated with cisplatin alone, respectively
Summary
Epithelial ovarian cancer (EOC) is the most lethal gynaecological cancer [1]. many patients initially benefit from surgery and chemotherapy [2,3], recurrent disease develops in more than 80% of patients with advanced stage and 25% of early stage ovarian cancer [4]. There are several suggested mechanisms for the chemo-resistance of CSCs. First, traditional chemotherapeutic agents typically induce apoptosis by damaging DNA and inhibiting cell cycle distribution. Disulfiram (DSF) is a drug used for treatment of alcoholism that has displayed promising anticancer activity. It unfolds its effects by inhibiting the enzyme activity of aldehyde dehydrogenase (ALDH) isoforms. Methods: MTT assay, spheroid formation, clonogenicity assay, qRT-PCR, and ALDH enzyme activity analysis were performed using ovarian cancer cell lines IGROV1, SKOV3 and SKOV3IP1. DSF + Cu2+ increased intracellular ROS levels triggering apoptosis of ovarian cancer stem cells (CSC). ALDH+ cells were more resistant to cisplatin treatment compared with ALDH-cells (P < 0.05) and exhibited a lower basal level of ROS. Conclusion: Our findings provide evidence that DSF might be employed as a novel adjuvant chemotherapeutic agent in combination with cisplatin for treatment of ovarian cancer
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