Cancer cells undergo genetic or epigenetic changes that result in uncontrolled proliferation and escape from apoptosis. Cyclin-dependent kinases (Cdks) are the master regulators of cell division. RO-3306, a selective small-molecule inhibitor of CDK1, has been shown to inhibit proliferation, induces a G2/M cell cycle arrest and apoptosis in cancer cell lines. The mechanism of action of RO-3306 involves binding to the ATP pocket of CDK1 and it also affects p53 downstream targets. The purpose of the present study was to investigate additional effects of RO-3306 through screening for changes in mRNA expression of genes known to be involved in human carcinogenesis in the human hepatocarcinoma cell line HepG2 using the RT² Profiler PCR Array. In the present study, RO-3306 reduced the viability, inhibited colony formation assay, induced a G2/M cell cycle phase arrest, as well as apoptosis in HepG2 cells. On the mRNA level RO-3306 increased the expression of the two cell cycle inhibitors p21 and p16 , as well as the expression of tumour necrosis factor (tnf) relative to control and it downregulated the expression of tissue inhibitor of metalloproteinase 3 (timp3). We confirmed the increased expression of p21 and p16 on the protein level by Western blot. Furthermore, RO-3306 induced the expression of p53 protein, while it did not alter the protein levels of CDK1 or its inhibitory phosphorylation on tryrosine 15. In conclusion, our study confirms the previous results that RO-3306 has multiple anticancer effects, and it provides further insight into the molecular events associated with these effects including the induction of the expression of cell cycle inhibitory and apoptotic genes.
Key words: Cyclin-dependent kinase 1, RO-3306, HepG2 cells, p53, p21, apoptosis, tumour necrosis factor
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