Discovery of Vanoxerine Dihydrochloride as a CDK2/4/6 Triple-Inhibitor for the Treatment of Human Hepatocellular Carcinoma

Abstract

Background Cyclin-dependent kinases 2/4/6 (CDK2/4/6) play critical roles in cell cycle progression, and their deregulations are hallmarks of hepatocellular carcinoma (HCC).Methods Here we combined computational and experimental approaches to discover a CDK2/4/6 triple-inhibitor from FDA approved small-molecule drugs for the treatment of HCC.Results Based on molecular docking results, vanoxerine dihydrochloride was found to exhibit strongest cytotoxic effect on human HCC QGY7703 and Huh7 cells (IC50: 3.79 µM for QGY7703and 4.04 µM for Huh7 cells). Vanoxerine dihydrochloride treatment caused G1arrest, induced apoptosis, and reduced the expressions of CDK2/4/6, cyclin D/E, retinoblastoma protein (Rb), as well as the phosphorylation of CDK2/4/6 and Rb in QGY7703 and Huh7 cells. In addition, combined vanoxerine dihydrochloride and 5-Fu produced synergistic cytotoxicity in Huh7 cells. Finally, in vivo studies in preclinical animal model of BALB/C mice subcutaneously xenografted with Huh7 cells, we showed that injection of vanoxerine dihydrochloride (40 mg/kg, i.p.) produced significant antitumor activity (p < 0.05), comparable to that achieved by 5-Fu (10 mg/kg, i.p.), with the combination treatment resulted in strongest effect.Conclusions The present study is the first to identify a CDK2/4/6 triple inhibitor vanoxerine dihydrochloride, and demonstrate that it represents a novel therapeutic strategy for HCC treatment alone or in combination with 5-Fu.

Over-expression of long non-coding RNA insulin-like growth factor 2-antisense suppressed hepatocellular carcinoma cell proliferation and metastasis by regulating the microRNA-520h/cyclin-dependent kinase inhibitor 1A signaling pathway

“…Association between different genes was analyzed with Spearman's correlation coefficient. p < 0.05 indicates statistical significance [14].…”

“…Briefly, cells to be tested were plated at an initial density of 1 × 10 4 cells/well per 100 μL of cell culture medium in 96well plates; 10 μL of CCK-8 reagent was added into 100 μL of cell culture medium. The optical density (OD) values were measured at 450 nm using a microplate reader [12,14].…”

“…The protein concentration was measured using the BCA method following the manufacturer's protocol. Then, equal amount proteins (15 μg/sample) were loaded and separated on 10% SDS-PAGE, transferred to PVDF membrane, blocked with 5% fat free milk, blotted with primary antibody (antibody against CDKN1A at 1:2000, antibody against Ki-67 at 1:200 or antibody against GAPDH at 1:1000) at 4°C overnight and secondary antibody at room temperature for 1 h, respectively, developed with SuperSignal Chemiluminescent HRP Substrate, and photographed with software Image Lab version in ChemiDoc MP Imaging System (Bio-Rad, USA) [11,12,14].…”

“…The minimum (W) and maximum (L) length of tumors were measured with a vernier caliper every week to calculate the tumor volume with the formula of ½LW [2]. The mice were sacrificed by cervical dislocation after 5 weeks; the tumor tissues were isolated for immunohistochemical assay [14].…”

“…The total and positive cell numbers were counted in five randomly selected fields of each section under a microscope at 400× magnification to quantitate the staining intensity. The positive cell percentages from five random fields were used for statistics analysis [14].…”