Regulatory Mechanism Of EB1089 For Hepatocarcinoma Cell Proliferation And P27Kipl

Hepatocellular carcinoma is one of the commonest neoplasms in China. It ranks third in incidence among malignant carcinomas, and second mortality. Hepatocarcinoma has a high recurrence if surgically ablated, and has a low sensitivity to radiotherapy and chemotherapy. Immunotherapy and intervention therapy cannot achieve ideal result. With the development of molecular biology, gene in combination with drugs will be an ideal way for treatment of carcinoma.1,25-(OH)2D3, the active form of vitamin Da, regulates the gene expression by mediation of specific receptors and interaction between vitamin D receptors. The bioactivity of 1,25-(OH)2D3 can regulate the body's phosphorus and calcium balance and maintain homeostasis. Additionally, it has a role of regulation of cell growth and differentiation, e. g.. in clinic vitamin D can be used for long-term treatment of psoriasis and has a good curative effect. But at the same time it can cause hypercalcinemia. This has limited its clinical application. EB1089 is an analog of vitamin 03. Mediated by vitamin D receptors, it has a strong activity against cellular proliferation of carcinomassuch as breat cancer, prostatic cancer and colon carcinoma, and can induce apoptosis of many kinds of carcinoma cells. Though hypercalcinemia cannot be totally avoided, the probability drops about 50% compared with 1,25-(OH)2D3. Therefore, EB1089 is a vitamin D analog under extensive research. Cell cycle is mainly regulated by cyclin, cyclin dependent kinase(CDK) and cyclin dependent kinase inhibitor (CDKI). P27Kipl, a CDK inhibitory protein of cip/kip family discovered in recent years, mainly through inhibiting the kinase activity of cyclinE/CDK2, cyclinD/CDK4, CDK compound, inhibits the transition of cell cycle at GI and Gi-S. It can also produce inhibition at G2-M through inhibiting cyclin B. Its overexpression produces cytotoxicity to target cells. P27Klpl, a member of CDKI, can inhibit cellular proliferation, promote apoptosis, induce differentiation and be taken as marker of degree of carcinoma malignancy. It is a suppressor gene. The research on its carcinoma-inhibiting mechanism can provide some basis for differential diagnosis, judgment of prognosis and gene treatment, and has become an important topic in the biomedical field.The present study is aimed to discuss:(1) Relations between p27K'pl and VDR receptors and the occurrence and development of hepatocellular carcinoma.(2) Possible mechanism of EB108 9 's and overexpressed p27Klp' 's inhibition of hepatocarcinoma.( 3) EB 1089's regulation of p27Klpl expression and its mechanism. Methods(1) p27 lp mKNA and its protein expression in tissues of cirrhosis and tumor-surrounding and hepatocarcinoma tissues were determined using hybridization in situ and immunohistochemistry(IHC); VDR expression was determined using RT-PCR.(2) Plasmid mediation was used to transfer p27Klpl into HCC-9204 cells; Western blot was used to detect whether p27K'pl was transferred into HCC; MTT, the plate clone forming test and the soft agar clone forming test were used to determine inhibitory effects of 1,25-(OH)2D3, EB1089 and overexpressed p27 Klpl on HCC-9204; Nude mice were used to prepare a hepatocarcinoma animal model; Through computing the rate of nude mice's graft tumor's inhibition of drugs, EB1089's and overexpressed p27Klpl's inhition was determined; Possible mechanism of EB1089's and p27Klpl's inhibition of hepatocarcinoma cells was investigated using flow cell device, EM, TRAP-PCR-ELISA and TRAP-PAGE.(3) RT-PCR and Western-blot were used to detect EB1089's regulation of p27Klpl expression and its mechanism.Results(1) Hybridization in situ was used to examine the p27Klpl expression in 69 specimens. p27KlplmRNA was found mainly located in cytoplasm and distributed granularly and sporadically. The rate of positive expression in cirrhosis, tumor-surrounding and HCC tissues of different grades was similar to the positive cell index. There was...