The Role Of Prohibitin In The Cholesterol Regulate PC-3 Cells Cycle

Prostate cancer is one of the most prevalent malignant tumors threaten male health.It is the most common form of non-cutaneous cancer and second most lethal cancer in American men with an incidence of 234,460 new cases, and nearly 30,000 deaths in 2006 alone.The incidence rate of prostate cancer in China is going up recently,and reported cases already up to the third in urinary malignant tumor.Although in many cases this cancer is treatable through hormone therapy and/or surgery,these 'cures' are associated with a host of medical problems(e.g.increased likelihood of osteoporosis and heart disease),and often provide only temporary relief,as prostate cancer reoccurs in part of cases as a far less treatable and much more malignant disease -androgen independent prostate cancer(AIPC).Recent studies showed high fat/high cholesterol 'Western-type' diets have been linked to PCa incidence and progression.High levels of serum cholesterol contributes to prostate cancer progression and upon stimulation with androgen.Modification of cholesterol levels through diet,exercise and the use of pharmaceuticals may contribute to slowing prostate cancer progression.In our Studies we demonstrate that prohibitin protein expression increased after treatment in cholesterol depletion media(CDM).We further examined the prohibitin gene promoter sequence homology with SREs.This analysis identified a number of sites with high homology to sterol response element(SREs).SREs consist in cholesterol-sensitive gene indicate prohibitin may sensitive to cholesterol activity.Prohibitin is ubiquitously expressed in the cytoplasm,nucleus,and mitochondria where it named for anti proliferative and tumor depression activity.Prohibitin gene express product prohibitin protein is an amino acid structure conserved,new-style mitochondria molecule parter which involves in the progresses of cell cycle regulation.Prohibitin protein involves in the key biological processes of cell cycle regulation,cell signaling mediation,aging,apoptosis and proliferation.It's still unclear whether prohibitin is a cholesterol-sensitive regulator of cell cycle progression in prostate tumor cells and whether the effect of antiproliferation in CDM is associated with prohibitin.ChapterⅠThe prohibitin expression in PC-3 under different cholesterol level conditions.Objective:To study the prohibitin mRNA and protein different expression in PC-3 cells under NM and CDM condition.Analyze cholesterol affect prohibitin expression and the relationship with prostate cancer process. Methods:PC-3 cells were cultured in NM and CDM,added/or not PDGF or EGF. Cell number count under microscope,mRNA were assayed by RT-PCR and Real-time PCR.Using 2-DE and Western-blot assayed prohibitin protein level.Results:1.PC-3 cells number of cultured in CDM was significant less than the number of cultured in NM.Added PGDF or EGF into NM promoted PC-3 cells proliferation,and added into CDM resulted in cells death.(p＜0.05).2.PHB mRNA in PC-3 cells of cultured in CDM was significant higher than cultured in NM(p＜0.05).3.Contrast with PC-3 cells cultured in NM,PHB protein expression in PC-3 cells cultured in CDM was significant upon modulation(p＜0.05).Conclusions:1.Cholesterol depleted conditions can inhibit the proliferation of PC-3 cells in vitro.2.Cholesterol depleted conditions can advance the PHB mRNA and protein expression in PC-3 cells.3.The inhibit prohiferation effection of PC-3 in cholesterol depleted conditions is connect with the overexpression of PHB. ChapterⅡThe cholesterol-sensitive point situation in prohibitin promoterObjective:To affirm the cholesterol-sensitive point situation in PHB promoter. Discuss the molecular mechanism of prohibitin as cholesterol-sensitive regulator of cell cycle progression in PC-3 cells.Methods:Transfected PGL3 expression plasmid and PHB promoter conjoint report plasmid construction into PC-3 cells and then cultured in LPDS and LPDS+CHO.Luciferase activity analyzed the difference of PHB promoter expression.Processed PCR proliferation and made series of successive promoter truncations and isolated within 200bp the cholesterol-sensitive promoter elements.After that,we indentified the most likely SREs in this region and made point mutation.We indentified the specific SRE situation response for the cholesterol sensitivity of prohibitin promoter.Results:1.Luciferase activity analyzed showed that whole prohibitin promoter transfected PC-3 cells cultured in LPDS luciferase expression were significant higher than PC-3 cells cultured in LPDS+CHO.2.Compared with whole prohibitin promoter transfected PC-3 cells, luciferase expression in PPHB-179 were significant decreased(p＜0.05). luciferase expression was not statistic different between LPDS group and LPDS+CHO group in PPHB-179.(p＞0.05)3.After point mutation most likely SRE located between -117 to -108bp in prohibitin promoter.Compared with PPHB-1192,point mutation group (SREMUT) were significant decrease in luciferase expression(p＜0.05). luciferase expression was not statistic different between LPDS group and LPDS+CHO group in SREMUT(p＞0.05).Conclusions:1.Prohibitin gene expression is regulated by cholesterol.2.The cholesterol-sensitive in prohibitin gene promoter located between -117 to -108bp.3.The cholesterol-sensitive point situation in prohibitin promoter is sequence homology with SREs.ChapterⅢThe effect molecular mechanism of PHB in PC-3 cellsObjective:To observe the effects of RNAi inhibit PHB on cell growth and apoptosis in Pc-3 cells.Explore the possible molecular mechanism of PHB on human prostate cancer. Methods:SiRNA knockdowned PHB express in PC-3 cells.PC-3 cells were treated with NM or CDM added/or not PDGF/EGF,and apoptosis was determined by Annexin V/PI double staining technique and flow cytometry.The proliferation of Pc-3 was measured by MTT assay.Results:1.Annexin V/PI double staining and flow cytometry discovered that after using siRNA knockdowned PHB in PC-3 cells,PC-3 cells apoptosis rate of cultured in CDM was significant higher than cells cultured in NM(p＜0.05).Addred PDGF or EGF into CDM can upon apoptosis rate(p＜0.05).2.MTT assay discovered that PHB RNAi can significant increase PC-3 cells number in NM team(p＜0.05).The cells number in group of stimulated with 40ng/ml PDGF or EGF was higher than stimulated with 20ng/ml, However in CDM team,cells number was decrease in PHB RNAi groups after stimulated with PDGF or EGF(p＜0.05).The proliferate cells number has downtrend along with the increase of PDGF or EGF concentration.Conclusions:1.Cholesterol depleted conditions add growth factors can promote apoptosis in PC-3 cells.2.The effect of cholesterol depleted conditions inhibit the proliferate activity of PC-3 relate with the higher expression of PHB. 3.PHB can use as a target of gene therapy in prostate cancer.