Study On The Mechanism Of Interleukin-8in Progression Of Prostate Carcinoma

Prostate cancer is the most prevalent urological malignant tumor in men in America and Europe. In our country,the incidence of prostate cancer is also going up year by year. Androgen deprivation therapy (ADT) is a regular therapy for prostate cancer.However, ADT ultimately results in the emergence of a hormone-refractory disease that is invariably fatal.There are multiple ways mediate the transition from hormone sensitive to hormone refractory prostate cancer.Recently,we found that Interleukin-8expression in prostate cancer was higher than that in benigan prostate tissue.Furthermore,the level of IL-8is increased with the progression of prostate cancer.It showed that the progression of androgen independent prostate and migration of tumor cells may request the activion of IL-8.To date,there is no report about the therapy of androgen independent prostate cancer through knockdown IL-8gene.Objective:To study the effect of IL-8in the process of progression of prostate cancer.Further confirm whether IL-8may promote androgen dependent to androgen independent prostate cancer transition via activation of AR.And it will provid theory basis for the gene target therapy on IL-8in androgen independent prostate cancer.Content:(1) The expression and significance of IL-8in tissues and cells of ADPC and AIPC.(2) The effect of IL-8gene on the proliferation and apoptosis of androgen dependent and independent prostate cancer lines in Vitro using RNAi and rhIL-8.(3) To confirm IL-8may regulate activation of AR by using human prstate cancer cell line LNCaP.Methods:Immunohistochemistry was used to detect the expression of IL-8in PCa (n=64) and BPH (n=40); Immunohistochemistry result were analysis by Image-pro Plus software and the expression result was indicated by integrated optical density (IOD). IL-8mRNA was investigated using RT-PCR and Western blot analysis in LNCP and DU145cell. We used different concentration and action time of rhIL-8to stimulate human prostate cancer cell line LNCaP and DU145.The survival and proliferation condition was determined by MTT study. Cell cycle and apoptosis of AGS cells were detected with Flowcytometry (FCM). RT-PCT and Western blot was used to determine if rhIL-8can upregulate the expression of AR mRNA and protein.Recombinant plasmids pGCsilencer-U6-siIL-8were transfected with Lipofectamine2000into human prostate cancer cell lines DU145and PC-3and the transfection efficiency can be acquired by observing red fluorescence of cells. RT-PCT was used to determine if recombinant plasmids can downregulate the expression of IL-8mRNA and which shRNA has better result. The silencing effect can also be detected by Western blot in protein levels. The change of cell morphology and IL-8protein expression level after transfection can also be detected by immunocytochemistry and light microscope. The cell survival and proliferation condition was determined by MTT study. Cell cycle and apoptosis of AGS cells after transfection were detected with Flowcytometry.We used different concentration and action time of rhIL-8to stimulate human prostate cancer cell line LNCaP.Results:Immunohistochemistry indicated that the expression rate of IL-8in Pea was higher than BPH (P<0.01). The expression level of IL-8was strongly correlative with pathological grade and clinical stage (P<0.01). RT-PCR and Western blot indicated that there were the expression of IL-8in DU145cell and were not in LNCaP cell. MTT results showed rhIL-8was able to promote proliferation of LNCaP and DU145cell(P<0.01). Cell cycle analysis also showed that rhIL-8induced decrease in the percentage of cells in G1phase by (32.77±3.78)%、(44.75±2.26)%(P<0.01)with a significant increase in the percentage of cells in S-phase by (25.44±4.55)%、(9.63±2.35)%(P<0.01) in LNCaP and DU145cells relative to control. rhIL-8(100ng/ml) could upregulate level of AR mRNA and protein at24h、48h and72h treatment in LNCaP cells (P<0.01). MTT results showed that the cell survival rates of PC-3and DU145cells96h after transfecting with pGCsilencer-U6-siIL-8-2were (46.15±5.06)%and (43.12±5.94)%respectively when comparing with blank. Cell cycle analysis showed that pGCsilencer-U6-siIL-8-2induced accumulation of cells in G1phase by (26.53±4.55)%and (29.57±2.35)%with a significant decrease in the percentage of cells in S-phase by (27.56±3.78)%and (26.99±2.26)%in PC-3and DU145cells relative to control.Conclusion:Results indicated that the expression of IL-8was strongly correlatated with pathological grade of Pca. Furthermore rhIL-8could promote proliferation of ADPC and AIPC cell line. And IL-8could upregulate level of AR mRNA and protein in LNCaP cells (P<0.01).Knockdown IL-8could inhibit proliferation of AIPC cell line and promote apoptosis of them. Therefore,we presume that IL-8may become a new therapy for androgen independent prostate cancer.