The Role Of Cytochrome P4504Z1and4A Under Pathological Conditions And Drug Intervention Studies

Cytochrome P450(Cytochrome P450, CYP)4family of enzymes constitute a multigene superfamily of hemoproteins, which mainly involved in the endogenous (fatty acids and steroid hormones) metabolism and were closely related to the development of human tumor and a variety of metabolic diseases. In general, the CYP4family consisting of six subfamilies (A, B, F, V, X, and Z) in humans. CYP4Z1, a novel CYP4family member, was found to be frequently upregulated in primary mammary carcinoma. Furthermore, CYP4Z1overexpression was specifically associated with increasing tumor grade of breast cancer. It is still unclear that whether CYP4Z1is involved in progesterone-induced progression of human breast cancer. In recent years, studies have also shown that CYP4A has involved in the fatty acids catabolism in liver and has participated in the development of fatty liver. In the present study, we not only investigated the roles of CYP4Z1on the effects of progesterone on cell invasion of breast cancer and drug intervention, but also investigated the molecular mechanisms of CYP4A on tectoridin (an isoflavone glycoside from the flower of Pueraria lobata)protecting against ethanol-induced liver steatosis. It is expected to provide a new target and the candidate drug for prevention and treatment of tumors and alcoholic liver disease. The study includes two parts:Part I:The role of CYP4Z1on the effects of progesterone on cell invasion of breast cancer and drug intervention studiesBackground and objectives:The connection between progesterone and breast cancer is still a matter of debate. In recent years, there is growing evidence that progesterone promotes breast cancer invasion. However, the exact mechanism of progesterone-induced cell invasion of breast cancer is not well characterized. Cytochrome P450(CYP)4Z1, a novel CYP4family member, was found to be frequently upregulated in primary mammary carcinoma. Furthermore, CYP4Z1 overexpression was specifically associated with increasing tumor grade of breast cancer. Until now, it is still unclear that whether CYP4Z1is involved in progesterone-induced progression of human breast cancer. In the present study, we investigated the roles of progesterone and CYP4Z1in cell invasion of breast cancer and explored their possible underlying molecular mechanisms on breast cancer invasion.Methods:(1)In order to study progesterone promoting cell invasion and inducing CYP4Z1expression in breast cancer cell, PR-positive breast cancer cell lines T47D and BT-474treated with progesterone at doses of10nM-1000nM in the presence or absence of RU486for24h.(2)We constructed overexpression systems of CYP4Z1in T47D cells (T47-CYP4Z1) in order to further study the role of CYP4Z1induced by progesterone in cell invasion of breast cancer in the presence or absence of HET0016.(3)Cell invasion induced by progesterone was measured from T47D and BT-474cells co-treated with HET0016or after transfection with CYP4Z1siRNA for48h.(4)T47D cells were treated with100nM progesterone in the presence or absence of HET0016(100nM) or after transfection with CYP4Z1siRNA for48h. The cells were subjected to GC/MS analysis for fatty acid contents and20-HETE production.(5)T47D-CYP4Zl cells or T47D and BT-474cells treated with100nM progesterone in the presence or absence of RU486HET0016、NIP71or after transfection with CYP4Z1siRNA, then we detected the activity of NMT and the protein levels of NMT1.(6)When T47D and BT-474cells treated with100nM progesterone in the presence or absence of HET0016、NIP71or after transfection with CYP4Z1siRNA, we detected the protein levels of TIMP-1-. TIMP-2、c-src and pc-src. Results:Here, our results provide novel mechanistic views on the effects of progesterone on cell invasion of breast cancer. In T47D and BT-474breast cancer cells, progesterone efficiently induced cell invasion, and it also enhanced expression of CYP4Z1in a concentration-dependent manner. Stable expression of CYP4Z1in T47D cells significantly increased the cell invasion of breast cancer cell line T47D. On the contrary, HET0016treatment potently inhibited the progesterone-induced cell invasion. Similarly, NIP71, an inhibitor of N-myristoyltransferase (NMT), also inhibited cell invasion. Compared with cells treated with progesterone, CYP4Z1siRNA transfection or treatment with HET0016significantly increased progesterone-induced intracellular levels of lauric acid (C12:0) and myristic acid (C14:0), and also decreased contents of20-hydroxyeicosatetraenoic acid (20-HETE). Further experiments showed that cell invasion induced by progesterone was significantly inhibited after combined treatments of CYP4Z1siRNA. Moreover, progesterone increased the levels of phospho-c-src (pc-src) and downregulated the levels of the tissue inhibitor of metalloproteinases (TIMP)-2, which were reversed by HET0016and NIP71. Conclusions:Collectively, these data suggest that progesterone leads to up-regulation of CYP4Z1, increased cell invasion of human breast cancer through interaction with NMT1-induced downstream signaling. Part Ⅱ:Studies on the role of CYP4A on tectoridin preventing acute ethanol-induced liver steatosis in miceBackground and objectives:Ethanol-induced hepatic steatosis was probably the result of both impaired fatty acid catabolism and increased lipogenesis in the liver. Early studies focused on the role of CYP2E1in alcoholic liver disease. In recent years, studies have shown that the peroxisome proliferator-activated receptor a has regulated the expression of CYP4A in liver and involved in lipid storage, transport and fatty acid oxidation. In traditional Chinese medicine, the flower of Pueraria lobata (Puerariae Flos) has been used in therapy to counteract the problems associated with alcohol drinking and liver injury. In this study, we investigated the molecular mechanisms of CYP4A on tectoridin (an isoflavone glycoside from the flower of Pueraria lobata)protecting against ethanol-induced liver steatosis. Methods:Ethanol (5g/kg) was given orally every12h for a total of3doses.1h after the last dose of ethanol, tectoridin (25,50,100mg/kg) was given intragastrically five times in three consecutive days. The mice were sacrificed at4h after tectoridin treatment. Peroxisome proliferators-activated receptor a (PPARa) and its target genes such as acyl-CoA oxidase (ACO) and cytochrome P4504A (CYP4A), were evaluated by biochemical analysis and quantitative real-time polymerase chain reaction (qPCR). Mitochondria were isolated for the mitochondrial permeability transition (MPT) and membrane potential (△Ψm) assay. Results:Acute ethanol exposure resulted in the significant increase of the alanine aminotransferase (ALT), aspartate aminotransferase (AST) and triglyceride (TG) levels and hepatic mitochondria dysfunction shown as the increase of MPT and the decrease of△Ψm. However, tectoridin treatment dramatically attenuated these effects. In addition, tectoridin remarkably alleviated the over-production of thiobarbituric acid-reactive substance. Furthermore, tectoridin inhibited the decrease of PPARa expression and its target genes, including medium-chain acyl CoA dehydrogenase (MCAD), ACO and CYP4A at mRNA and enzyme activity levels. Conclusions:These data showed that tectoridin protected against ethanol-induced liver steatosis mainly through modulating the disturbance of PPARa pathway and its target genes such as CYP4A and ameliorating mitochondrial function.