The Mechanism Of Action Of Clioquinol In Human Cells

Clioquinol(CQ), a quinoline drug, is commonly used as antifungal, antibacterial or seborrhea medicine. Because there are several side-effects after CQ treatment, such as retinopathy, myopathy, cardiopathy, peripheral neuropathy, and others, it is prohibited in some areas. However, some recent researches pronounced that patients with neurodegenerative disease or tumor may benefit from CQ treatment, therefore, CQ becomes attractive again. To be safely applied to clinical treatment, it is essential to explore the molecular mechanisms including the side-effects of CQ. In this study, we investigated some cellular and molecular mechanisms of CQ in human cells.The cytotoxicity assay revealed that CQ does have an inhibition effect on the growth of SH-SY5Y, a neuroblastoma cell line, HeLa, a cervical cell line, SMMU7721, a hepatoma cell line and QSG7701, a normal hepatocyte line. Among them, the cytotoxicity of CQ was minimal in QSG7701cell line when treated at a low dose for a long time. By flow cytometry, we found CQ can induce autophagy in SH-SY5Y and SMMU7721. The result was confirmed by acridine orange staining with increased acidic vacuoles,3-MA (autophagy specific inhibitor) treatment with inhibited autophagy and western blotting for autophagy marker protein LC3with increased LC3Ⅱ expression. Meanwhile, CQ can induce apoptosis in SMMU7721, but not in SH-SY5Y, which was proved by Hoechst33258staining microscope analysis. The cell cycle analysis showed that CQ significantly induced s phase arrest of SH-SY5Y and SMMU7721. Amprolium, one of the thiamine transporters competitive inhibitors, exhibited the similar effect on cell cycle arrest of SH-SY5Y and SMMU7721. We assumed that CQ affects the two cellular programmed death pathways, and is also associated with the absorption, transportation, and metabolism of thiamine.To prove the hypothesis, we evaluated thiamine and thiamine pyrophosphate content in the live cells using HPLC and fluorescence detection. The result showed that CQ decreased the content of thiamine and increased that of thiamine pyrophosphate. Meanwhile, the activity of transketolase which is a thiamine pyrophosphate dependent enzyme was assayed and became lower by CQ treatment. By RT-PCR and Western-blotting, we found that there was no effect of CQ on the transketolase mRNA while CQ downregulated the expression of transketolase.In summary, we conclude that the effect of CQ treatment on cell growth was implemented through several signaling pathways, and varies according to the type of the cell. We confirmed that clioquinol treatment affect the absorption and metabolism of thiamine besides the apoptosis and autophagy of the cell.