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 We used the combination of computational and experimental approaches to discover a CDK2/4/6 triple-inhibitor from FDA approved small-molecule drugs for the treatment of HCC. Results We identified vanoxerine dihydrochloride as a new CDK2/4/6 inhibitor, and a strong cytotoxicdrugin human HCC QGY7703 and Huh7 cells (IC50: 3.79 μM for QGY7703and 4.04 μM for Huh7 cells). In QGY7703 and Huh7 cells, vanoxerine dihydrochloride treatment caused G1-arrest, 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. Drug combination study indicated that vanoxerine dihydrochloride and 5-Fu produced synergistic cytotoxicity in vitro in Huh7 cells. Finally, in vivo study in BALB/C nude mice subcutaneously xenografted with Huh7 cells, vanoxerine dihydrochloride (40 mg/kg, i.p.) injection for 21 days produced significant anti-tumor activity (p < 0.05), which was comparable to that achieved by 5-Fu (10 mg/kg, i.p.), with the combination treatment resulted in synergistic effect. Immunohistochemistry staining of the tumor tissues also revealed significantly reduced expressions of Rb and CDK2/4/6in vanoxerinedihydrochloride treatment group. Conclusions The present study isthe first report identifying a new CDK2/4/6 triple inhibitor vanoxerine dihydrochloride, and demonstrated that this drug represents a novel therapeutic strategy for HCC treatment.

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

“…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 96-well 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 ].…”

Transcriptome Analysis Reveals the Anti-cancerous Mechanism of Licochalcone A on Human Hepatoma Cell HepG2

“…CDK2 in cyclin, as an essential component of cell cycle mechanism, can form CDK2/cyclin E complex with cyclin E, and it is mainly active CDK2/cyclin A complex and phosphorylated E2F in the S phase of the cell cycle and remains in the nucleus in the form of CDK2 in G2 phase. A previous study revealed that phytochemicals could target the CDK2 to treat HCC (48). PDCD4, also known as a programmed cell death protein, participates in the apoptotic signaling pathway and acts as an inhibitor of tumorigenic transformation.…”