| BackgroundProstate cancer is the most common malignancy generated from the male reproductive system. In the United States, it is the second cause of cancer mortality which is only less than lung cancer. During recent years, detection rate of prostate cancer has been increasing in China. Currently, endocrine therapy is a very effective method for ADPC, however, HRPC is still a therapeutic challenge not to overcome. Docetaxel and Mitoxantrone are the representative drugs for the advanced prostate cancer. Although these drugs, to some extent, can postpone the progression of the disease, it is still not an ideal, effective therapy for HRPC. Therefore, to explore and study novel therapeutic strategies for AIPC is of clinical significance.AckI(activated Cdc42associated kinase1) has been shown to be critical for malignant tumors growth, and to be a key transducer in several cancer-related signaling pathway such as integrins, EGF, PDGF pathways and so on. It regulates activity of a number of proteins by tyrosine phosphorylation especially proteins critical for cell migration, cell growth, and proliferation. P130CAS adaptor proteins play an important role during cellular signaling by mediating the formation of protein complexes. A new very efficient and special tool for gene knockdown, RNAi(RNA interference) technology, has developed rapidly and has a wide application prospect during recent years. Indeed, the detailed mechanism of AckI and p130cas in prostate cancer remain far from clear and is needed to be investigated, though many studies have shown some mechanism about it.. In view of this, we carried out the present study to investigate the expression of AckI and p130cas in Pca as well as the role of it in the growth and metastasis of Pca and the possible underlying molecular mechanism. By using plasmid transfection and RNA interference(shRNA), as well as a series of in vitro assays, we got results as follows.Methods and ResultsFirst of all, Western blot and RT-PCR methods were used to examine the expression levels of AckI and P130CAS in androgen-dependent prostate cancer(ADPC) cells LNCaP and andandrogen-independent prostate cancer(AIPC) PC-3cells. As a result, the expression levels of AckI and P130CAS was lower in LNCaP than PC-3with significant difference(P<0.05), indicating that AckI and P130CAS may play an important role in the development of prostate cancer.Second, by the aid of the Technology of RNA interference via lentivirus which Can transcribe a short hairpin RNA against AckI, we have successfully generated AckI knockdown PC-3cells lines. The interfection result was confirmed by western bolt and RT-PCR and fluorescence microscopy was used to determine the fluorescence.Third, Cell proliferation was quantitated by the3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl-tetrazolium Bromide (MTT) assay and Colony formation assay, Cell migration was measured by in vitro wound healing assay and transwell healing assay. As a result, Inhibition of the expression of Ackl lead to decrease of the invasion ablitiy of PC-3cells migration in vitro. The results of scratch healing showed that migration ability of PC-3after AckI knockdown was significantly impaired,24-hour scratch healing rates of PC-3cells before and after AckI knockdown were34%VS89%, the difference was significan(P<0.05). Different invasive ability were found in pc-3cells before and after AckI knockdown by Transwell assay. After24hours cell culture, cell count of permeate through Transwell(10w power field)were21.31±4.15VS12.15±3.13, difference was significant(P<0.05). These results indicate that in vitro inhibition of AckI expression in PC-3cells can inhibit the invasion and metastasis of prostate cancer.We further detected the expression of cdc42, RhoA, RhoC, Racl, P130CAS protein levels in PC-3cells respectively after AckI gene knockdown. Down-regulating the expression of AckI-silenced in PC-3cells lead to down-regulated expression of p130cas protein expression (0.94±0.13VS0.32±0.11), up-regulated expression of Rac1(0.29±0.08VS0.67±0.10),with statistically significant difference(p<0.05).No statistically significant difference was found in cdc42, RhoA and RhoC protein expression(cdc42,0.61±0.14VS0.58±0.19; RhoA,0.67±0.15vs0.58±0.15; RhoC,0.55±0.15vs0.54±0.06).In the end, we evaluated the influence of knocking down of AckI gene with shRNA on the chemosensitivity of Docetaxel in PC-3cell line. PC-3cell was divided into three groups as previous study, Docetaxel was given as final concentration of0.25μM at24h after transfection. MTT test was used to measure the proliferation of PC3cell. Flow cytometer was used to measure cell apoptosis, cycle distribution. Inhibition ratio of PC3+AckI-shRNA+DTX group is higher than PC3+DTX group and PC3+NC-shRNA+DTX group (P<0.05, at48h), while there was no difference between two control groups(P>0.05). Cell apoptosis rate of PC3+AckI-shRNA+DTX group, PC3+DTX group and PC3+NC-shRNA+DTX group was23.79%,14.96%and15.10%respectively at48h after docetaxe1given. The apoptosis rate of PC3+AckI-shRNA+DTX group was the highest (P<0.05). Flow cytometry analysis showed there was less G0/G1phase arrest in PC3+AckI-shRNA group than two control groups without chemotherapy.Conclusions Our experiments have demonstrated AckI was overexpressed in AIPC cell and played an important role in the invasion and metastasis of Pca. we eidentified that P130cas was involved in AckI and AckI may promote invasion and metastasis of human prostate cancer via regulating P130cas and Racl. What's more, AckI-shRNA can enhance chemosensitivity of docetaxel in PC-3cell in vitro by knocking down of AckI gene expression. The possible mechanisms involved in chemosensitization of AckI-shRNA include increasing apoptosis rate and releasing cell cycle arrest of G0/G1. Our findings suggest AckI as a novel prognostic marker and a potential therapeutic target for treatment of Pca.
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