Clioquinol Arrests Cell Cycle at G1 Phase and Triggers Intrinsic Apoptosis In Myeloma and Leukemia Cells by Inhibiting Histone Deacetylases

Abstract 1836

Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline, CQ) is an old and effective antifungal and amoebicidal drug but is restricted or discontinued in some countries because of its suspect causality in inducing subacute myelo-optic neuropathy, however it has been recently revitalized as an anti-cancer drug candidate at both in vitro and in vivo models. Our previous study suggested that CQ induced cell death and displayed anti-leukemia and anti-myeloma activity (Mao × et al, Leukemia, 2009), but its mechanisms are not well known. In this study, we demonstrated that clioquinol-induced cell death is via intrinsic rather than extrinsic apoptotic pathway, clioquinol-induced apoptosis depends on the activation of caspase-9 and -3. Because zinc is essential for the enzymatic activity of histone deacetylases (HDAC) which is currently emerging as a novel target for apoptotis and cancer treatment, and clioquinol is a strong chelator of zinc ion, we questioned whether clioquinol could interfere with HDAC activity by binding to zinc. Thus, we first examined the effects of clioquinol on the most often used hallmarks for HDAC inhibitions, including cyclin D, p21(CIP1), and p27(KIP1) (Nature Reviews Cancer, 2006). DNA microarray analysis indicated that clioquinol and its analogs significantly up-regulated the transcriptional level of p21 > 2–10 folds within 24 hr. The increased expression of p21(CIP1) protein was further confirmed by western blotting assay in various cell lines and primary patient samples. Clioquinol induced p21(CIP1) expression at concentrations as low as 5 μM witin 24 hr. At the same concentration, tumor suppressor and cell cycle regulator p27 (KIP1) was also increased. In addition, clioquinol also down-regulated expression of D-cyclins, including cyclin D2 and D3 in tested cell lines at a concentration consistent with that induced cell apoptosis. All these changes by clioquinol resulted in cell cycle arrest at G0/G1 phase in both leukemia and myeloma cell lines. Because these genes (cyclin D, p21 and p27) are most associated with histone acetylases, we next checked whether clioquinol is effective in inhibiting HDAC activity. Both leukemia and myeloma cell lines and primary patient samples were applied for the analysis of acetylation status of histone 3 (Ac-H3). Using trichostatin A (TSA) as a positive control, we found that clioquinol accumulated Ac-H3 in all tested cell lines and primary patient samples in a concentration- and time-dependent manner, which strongly suggested that clioquinol interfere with HDAC activity. Subsequently, we analyzed the interaction of clioquinol and HDAC. Just like TSA, clioquinol was docked into the active pocket of HDAC, where clioquinol competitively binds to zinc at the active center, which was confirmed by zinc addition. Zinc addition partially abolished the effects of clioquinol on HDAC activity. Thus, our study indicated that clioquinol-induced cell death in both leukemia and myeloma was via an apoptotic pathway by interfering with HDAC activity.