Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline, CQ) is an anti-fungal and anti-parasitic drug and is also a strong chelator of divalent metal ions such as zinc, and copper. Recent studies suggested by formation a metal complex, CQ becomes an inhibitor of proteasomes and displays anti-cancer activity in several types of solid cancers including prostate cancers and breast cancers. Although addition of copper and other divalent mental ions increases the activity of CQ in terms of proteasomal inhibition and cell death, our recent studies found that CQ and its analog 5-amino-8-hydroquinoline display potent anti-leukemia and anti-myeloma activity without addition of such metal ions. Because CQ is a potent chelator of zinc that is indispensable for many biological enzymes, such as histone deacetylases (HDACs). HDACs are a class of zinc-dependent enzymes regulating gene expression, cell survival and cell death. We questioned that whether CQ induces apoptosis by inhibiting HDAC activity via interfering with zinc in the active sites of these enzymes. To answer this question, we first analyzed the effects of clioquinol on transcription of HDAC-regulated genes including p21, p27 and p53. After 24 hr treatment, expression of these genes was significantly increased by CQ in a concentration-dependent manner. Consistent with these findings, CQ also induced cell cycle arrest and cell apoptosis, a sign of HDAC inhibition. We then examined HDAC activity by evaluating the expression level of acetylated histone 3 (Ac-H3). As expected, Ac-H3 was increased by CQ in all examined cell lines and bone marrow cells from primary leukemia and myeloma patients. CQ also induced accumulation of acetylated p53 and acetylated HSP90. In the mechanistic study, we further surveyed the effects of CQ on a panel of HDACs, including HDAC-1, −2, −3, −5 and −8, and found that most enzymes but HDAC2 were decreased by CQ at concentrations of 20 mM or higher in both myeloma and leukemia cells. Since CQ increased Ac-H3 at a concentration as low as 5 mM, we wondered whether CQ binds to HDACs thus directly interfering with their activity. To this end, we next screened the effects of CQ on all 11 zinc-dependent HDACs, including Class I (HDAC1, 2, 3, 8), Class 2A (HDAC4, -5, -7, -9) and Class 2B (HDAC6, -10) and Class IV (HDAC11) and measured the values of IC50 to each enzyme. The results showed that CQ had no effects on activities of Class 2B (HDAC6-, -10) and Class IV (HDAC11). Compared with trichostatin, the classic HDAC inhibitor, CQ displayed similar inhibition to Class 2A HDACs, but the IC50 values to Class I HDACs were 1000 fold higher than trichostatin. Thus, CQ probably mainly targeted Class 2A HDACs. To demonstrate this hypothesis, we analyzed the interaction between CQ and HDAC by computer modeling. The result indicated that CQ was well docked into the active pocket of the enzyme, where the oxygen and nitrogen atoms in CQ formed stable coordinate bonds with the zinc ion, and the hydroxyl group from CQ formed an effective hydrogen bond with Asp267. Moreover, CQ formed extensive van der Waals interactions with hydrophobic residues Trp141, Phe152 and Try306. To further verify this prediction, we co-treated cells with CQ and zinc chloride, and found that CQ-induced accumulation of Ac-H3 was attenuated by zinc. Thus, the proteasomal inhibitor CQ can induce apoptosis in leukemia and myeloma by inhibiting the HDAC activity, especially Class 2A enzymes. This study proposed a new line of mechanism for understanding CQ in the treatment of leukemia and myeloma.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.