Bromodomains and extra-terminal (BET) proteins inhibitors are promising cancer therapeutic agents. However, tumor cells often develop resistance to BET inhibitors, greatly limiting their therapeutic potential. To study the mechanism underlying the resistance of BET inhibitors in hepatocellular carcinoma (HCC) cells, we herein investigated the impact of BET inhibitor JQ1 on the gene expression of Bcl-2 family members by RNA sequencing analysis, and found that acute treatment with JQ1 triggered upregulation of Mcl-1 in HCCLM3 and BEL7402 cell lines. This JQl-triggered Mcl-1 upregulation was further confirmed by quantitative reverse transcription polymerase chain reaction and western blotting analysis, both at mRNA and protein levels. Inhibition of Mcl-1 by RNA interference dramatically enhanced JQ1-triggered caspase-3 activation, cleavage of poly (ADP-ribose) polymerase and apoptotic cell death induction in multiple HCC cell lines. Moreover, JQ1 in combination with cyclin-dependent kinase inhibitor flavopiridol at a subtoxic concentration that reduced expression of Mcl-1, triggered massive apoptotic cell death in HCCLM3 and BEL7402 cell lines. Together, these data suggest that Mcl-1 is a major contributor to BET inhibitor-resistance in HCC cells, and that combining drugs capable of down-regulating Mcl-1 may promote therapeutic potential in human HCC. (C) 2018 Elsevier Inc. All rights reserved.