Expression Of Id1 In Human Prostate Cancer And Effects Of Id1 Inhibition On Prostate Cancer Cells In Vitro And In Vivo

Inhibitor of DNA binding-1(Id1) is a dominant-negative regulator of basic helix-loop-helix transcription factor,which control malignant cell behaviors in several types of carcinomas.Recent studies have shown that Id1 over expressed in some types of human cancer.Id1 has much wider tumor biological roles that impinge on tumor differentiation,invasion and angiogenesis.Chetcuti A et al(2001) first identified differentially expressed Id1 gene in human organ-confined prostate cancer by gene expression array.Id1 overexpression was further confirmed in animal prostate cancer models and human prostate cancer specimens.However,there was only little clinical study on the relationship between Id1 and tumor differentiation,tumor stage, metastasis or prognosis on human prostate cancer.The relationship between Id1 and androgen,which is closely related to prostate cancer,remains unclear.In the present study,using immunohistochemistry and real time RT-PCR assay, we firstly observed Id1 expression in human prostate cancer and also their relationship to some clinical parameters.Androgen has closely relationship with the development of prostate cancer.To study if Id1 was regulated by androgen,we also observed the effects of androgen and androgen inhibitor on Id1 expression in androgen-dependent and independent prostate cancer cell lines.In order to confirm the in vitro and in vivo effects of Id1 gene in prostate cancer,we further observed the down regulation effect of Id1 gene by small interferon RNA(SiRNA-Id1) in androgen-independent PC3 cells and its combination effects with curcumin.We also observed the subcutaneous injection effects of SiRNA-Id1 on nude mice tumors transplanted with PC3 cells.The results were as follows:1.The levels of Id1 mRNA and protein were observed increased significantly in all prostate cancer specimens,while no Id1 expression in normal prostate cancer. Only weak expression in some benign prostate hypertrophy(BPH) samples was observed.The increased levels of Id1mRNA and protein correlated well with Gleason grade in prostate cancer specimens(r=0.9695,P＜0.05 or r_s=0.63,P＜0.01).Id1 expression was obviously higher in some samples that serum prostate specific antigen (PSA) level was more than 10ng/ml(P＜0.01),but no straight line relation between them.Id1 overexpression had no significant association with TNM stage and tumor size.2.Androgen-dihydrotestosterone(DHT) could up-regulate Id1 expression in androgen-dependent LNCaP cells(P＜0.05).Flutamide,the inhibitor of androgen receptor,not only blocked the up-regnlated Id1 expression by DHT,but also blocked the basic Id1 expression in LNCaP cells.The same concentration of flutamide did not have same block effect on Id1 expression in androgen independent PC3 cells.3.Down-regnlation of Id1 gene by small interferon RNA(SiRNA-Id1) in PC3 cells decreased cell viability,increased cell apoptosis and senescence rate.Synergistic effects of decreased cell viability and increased cell apoptosis were observed when the cells were treated by SiRNA-Id1 combination with curcumin.4.Down-regulation of Id1 gene by intratumor injection of SiRNA-Id1 in prostate carcinoma PC3 transplanted tumor in nude mice was confirmed by real time PCR and immunohistochemistry assay.The tumor volumes were smaller in SiRNA-Id1 treated mice than that of nonspecific SiRNA control(P＜0.05).The mRNA expression of proliferating cell nuclear antigen(PCNA) and matrix metalloproteinase2(MMP2) in the tumors were also decreased significantly in SiRNA-Id1 treated mice(P＜0.01).This study showed that overexpression of Id1 positively correlated with cell malignancy in human prostate cancer.Id1 expression,Gleason grade and serum PSA could be used together in the prognosis of prostate cancer.This study also identified that androgen and androgen receptor pathway might be great importance for Id1 expression in androgen-dependent prostate cancer cells.In androgen-independent cancer cells,down-regulation of Id1 gene could inhibit prostate cancer cell proliferation in vitro and in vivo,and had a synergistic effect on cell suppressor when combined with curcumin.This study implied that Id1 gene inhibition could be used as a new potential target of gene therapy on prostate cancer.