Using a high throughput screening (HTS) approach, we have identified and validated several small molecule Mcl-1 inhibitors (SMIs). Here we describe a novel selective Mcl-1 SMI inhibitor, 2 (UMI-77), developed by structure-based chemical modifications of the lead compound 1 (UMI-59). We have characterized the binding of UMI-77 to Mcl-1 by using complementary biochemical, biophysical and computational methods, and determined its antitumor activity against panel of pancreatic cancer (PC) cells and in vivo xenograft model. UMI-77 binds to the BH3 binding groove of Mcl-1 with Ki of 490 nM, showing selectivity over other members of anti-apoptotic Bcl-2 members. UMI-77 inhibits cell growth and induces apoptosis in PC cells in a time and dose-dependent manner, accompanied by cytochrome c release and caspase-3 activation. Co-immunoprecipitation experiments revealed that UMI-77 blocks the heterodimerization of Mcl-1/Bax and Mcl-1/Bak in cells, thus antagonizing the Mcl-1 function. The Bax/Bak-dependent induction of apoptosis was further confirmed by using murine embryonic fibroblasts that are Bax and Bak deficient. In an in vivo BxPC-3 xenograft model, UMI-77 effectively inhibited tumor growth. Western blot analysis in tumor remnants revealed enhancement of pro-apoptotic markers and significant decrease of survivin. Collectively, these promising findings demonstrate the therapeutic potential of Mcl-1 inhibitors against PC and warrant further preclinical investigations.

This study examined the effect of 3, 9-dihydroxy-2-prenylcoumestan (pso), a furanocoumarin, on PC-3 and C4-2B castration-resistant prostate cancer (CRPC) cell lines. Pso caused significant G0/G1 cell cycle arrest and inhibition of cell growth. Molecular analysis of cyclin (D1, D2, D3, and E), cyclin-dependent kinase (cdk) (cdks 2, 4, and 6), and cdk inhibitor (p21 and p27) expression suggested transcriptional regulation of the cdk inhibitors and more significant downregulation of cdk4 than of cyclins or other cdks. Overexpression of cdk4, or silencing of p21 or p27, overcame pso-induced G0/G1 arrest, suggesting that G0/G1 cell cycle arrest is a potential mechanism of growth inhibition in CRPC cells.

LH/human chorionic gonadotropin receptor (LHR) undergoes down-regulation during preovulatory LH surge or in response to exposure to a supraphysiological concentration of its ligands through a posttranscriptional mechanism involving an RNA binding protein designated as LHR mRNA binding protein (LRBP). miR-122, a short noncoding RNA, has been shown to mediate the up-regulation of LRBP. In the present study, we show that inhibition of miR-122 using a locked nucleic acid (LNA)-conjugated antagomir suppressed human chorionic gonadotropin (hCG)-induced up-regulation of LRBP as well as its association with LHR mRNA, as analyzed by RNA EMSA. Most importantly, inhibition of miR-122 resulted in the abolishment of hCG-mediated LHR mRNA down-regulation. We also show that the transcription factor, sterol regulatory element binding protein (SREBP) (SREBP-1a and SREBP-2 isoforms), is an intermediate in miR-122-mediated LHR mRNA regulation. HCG-stimulated increase in the activation of both SREBP-1a and SREBP-2 was inhibited by pretreatment with the miR-122 antagomir. The inhibition of cAMP/protein kinase A (PKA) and ERK pathways, upstream activators of miR-122, abolished SREBP activation after hCG treatment. SREBP-mediated regulation of LRBP expression is mediated by recruitment of LRBP promoter element to SREBP-1a, because chromatin immunoprecipitation assay revealed that association of LRBP promoter to SREBP was increased by hCG treatment. Pretreatment with miR-122 antagomir suppressed this response. Inhibition of SREBP activation by pretreating the rats with a chemical compound, fatostatin abrogated hCG-induced up-regulation of LRBP mRNA and protein. Fatostatin also inhibited LHR-LRBP mRNA-protein complex formation and LHR down-regulation. These results conclusively show that miR-122 plays a regulatory role in LH/hCG-induced LHR mRNA down-regulation by increasing LRBP expression through the activation of SREBP pathway.