Investigation Of Deguelin For Regulation Of CyclinD1 Expression In Zebrafish Embryos And Related Cancer Research

Deguelin is an natural isoflavonoids extraction from leguminous plants. Recent studieshave shown its chemopreventive and chemotherapeutic effect on cancer cells. Deguelin hasbeen reported as an potential anti-tumor drug. It has been demonstrated that deguelin restrainscell proliferation, induces cell apoptosis, causes cell cycle arrest and inhibits tumorangiogenesis by regulating many molecular pathways, but the specific anti-tumor mechanismis still vague. Cyclin D1, one of major cell cycle protein in G1 phase, is frequentlyoverexpressed in a broad range of human tumors, such as breast cancer, bladder cancer andpituitary adenoma, which indicates that the overexpression of Cyclin D1 contributesmaterially to the development of human cancer. Zebrafish as an cancer model system, theembryos has the similar characteristic with cancer cells like rapid cell proliferation and strongangiogenesis ability. In this study, cell growth inhibition of deguelin on breast cancer cellsMCF-7 and lung cancer cells H1299 was studied by MTT. Apoptosis induction and cell cyclearresting effect of deguelin on cancer cells was detected by flow cytometry on cell level. Inthe in-vivo experiments, we do research on the development inhibition effect of deguelin onzebrafish embryos, expression of Cylin D1 by the whole amount in situ hybridization. Forfurther study, the expression changes of Cyclin D1 m RNA in MCF-7, H1299 cells andzebrafish embryos were tested by real-time PCR.The research indicates that deguelin was capable of suppressing cell proliferation in bothhuman breast cancer cell lines MCF-7 and human lung cancer cell lines H1299.The effect ismore obvious with the increase of deguelin concentration and treatment time. By PI stainingand Annexinv-FITC/PI staining, we found that deguelin blocked MCF-7 and H1299 cells inthe G2/M phase and induced cell apoptosis. Low concentration of deguelin significantlydelayed zebrafish embryonic development in a dose-dependent manner. 400 n M deguelintreatment caused the embryo developmental stagnation after dosing period of 6-7 h. 600 n Mdeguelin treatment make embryos developmental stagnation after dosing period of 3-4 h.Thenegative regulatory effect of deguelin on Cyclin D1 gene in zebrafish embryos was detectedby the whole mount in situ hybridization. 400 n M deguelin almost completely inhibited theexpression of Cyclin D1 in shield phase zebrafish embryos(about 0.75 hour post fertilization).Expression of Cyclin D1 in brain, retina area,trunk, somites and tail of the 24 hpf zebrafishembryos were significantly reduced after 100 n M deguelin treatment. By real-time PCRanalysis, deguelin significantly reduced Cyclin D1 m RNA expression levels in zebrafishembryos and MCF-7, H1299 cells(P<0.05).We got the conclusion that eguelin can inhibit the proliferation of breast cancer cellsMCF-7 and lung cancer cells H1299, arrest the cell cycle in G2/M phase and induce cellapoptosis.Low concentration of deguelin can delay the development of zebrafish embryosand down-regulate the expression of Cyclin D1.Deguelin can down-regulate the expression ofCyclin D1 m RNA significantly in MCF-7, H1299 cells and zebrafish embryos.It provided atheoretical basis for the mechanism of the antitumor effects of deguelin.