| Objective: Prostate cancer is the most common malignancy diagnosed in males and ranks respectively first and second counting in morbidity and mortality in tumors of Western males. In China, prostate cancer is the third most common malignancy in males. Due to the concealed pathogenesis, prostate cancer is usually diagnosed with invasion and metastases, which contributes to its high mortality and recurrence after operation. Furthermore, 85%~100% cases died from prostate cancer are with remote metastases. The most remote metastasis destination of prostate cancer cells is bone and bone metastasis is very common in advanced prostate cancer. In general, 40–80% of prostate cancers respond to hormonal therapy, but most of these tumors will progress to androgen-independent states within 2–3 years, and then the tumors recur in an androgen independent form that is unresponsive to additional androgen withdrawal. Therefore, there is an urgent need to elucidate its molecular mechanisms to establish effective therapies and to allow early detection and treatment strategies. Ubiquitin–proteasome system dysfunction, with demonstrated contribution to the development of cancer, renders it a feasible and rational target for novel therapies. Numerous E3 ubiquitin ligases in the ubiquitin proteasome pathway have been implicated in cell cycle control and uncontrolled cell proliferation. The Smurf1, Smurf2 and WWP1 in Nedd4(neural precursor cell-expressed developmentally downregulated gene 4) family of ubiquitin ligases E3 s are characterized by a distinct modular domain architecture, with each member consisting of a C2 domain, 2–4 WW domains, and a HECT-type ligase domain, therefore, they are thought to possess similar functions. Recent studies suggested that ubiquitin ligases E3 s have very close relationship with oncogenesis, invasion and metastasis of prostate cancer. WWP1 is found highly expressed in some kinds of prostate cancer cell lines and its down-regulation may lead to inhibition of prostate cancer cell proliferation. Bortezomib is a new type of selective ubiquitin-proteasome inhibitor and mainly applied in clinic for treating multiple myeloma. Recently, bortezomib is implicated to regulate levels of Smurf1, Smurf2 and WWP1. Additionally, bortezomib has also been demonstrated to arrest cell cycle progression and induce apoptosis in cultured human prostate cancer cells as well as inhibit the growth of prostate cancer xenografts in a murine model. Nevertheless, the specific mechanism of bortezomib in treating prostate cancer and is still not fully understood. In the present study, we investigated effects of bortezomib in treating prostate cancer by regulating Smurf1, Smurf2 and WWP1, and suggested that ubiquitin ligases E3 s may serve as potential target and bortezomib may be a promising drug for prostate cancer treatment.In this study, we investigated the effects and its mechanism of bortezomib on prostate cancer PC3 cell line by a series of experiment which could test cell proliferation, m RNA and protein expression levels of Smurf1, Smurf2 and WWP1.Method: 1 Human prostate cancer PC3 cells cultureUnder the condition of strict aseptic, prostate cancer PC3 cells were cultured in a flask with DMEM medium and culture solution was replaced every 2 days. As soon as the confluence reached 90~95%, the cells were digested with 0.25% trypsinase and subcultured. After 3-5 generations, cells can be applied for the experiment. 2 Grouping 2.1 blank control group 2.2 bortezomib treatment group(bortezomib treating concentration: 0.5, 5, 15,25 μmol/L, treating time: 24, 48 and 72h). 3 Cell proliferation activity measurementBrdu ELISA was employed to measure proliferation rate of PC3 cellsin each group. 4 m RNA levels of ubiquitin ligases E3 s Smurf1, Smurf2 and WWP1 After treated by bortezomib for 72 hours, RNA was extracted from PC3 cells. Real time-PCR(Real Time-Polymerase Chain Reaction) was employed to detect Smurf1, Smurf2 and WWP1 m RNAs in each group. 5 Protein expression levels of ubiquitin ligases E3 s Smurf1, Smurf2 and WWP1 After treated by bortezomib for 72 hours, protein from PC3 cells were extracted. BCA method was used to measure protein concentration. Then the protein samples were sent for western blotting to get measurement of protein expression levels of ubiquitin ligases E3 Smurf1, Smurf2 and WWP1. 6 Statistical analysis SPSS 18.0 software was used for statistical analysis. Metric data was noted as “mean ± standard error”. Comparison between two groups was made by SNK-t test and that among more than two groups was made by One-way AVON. P<0.05 was regarded as significant difference.Results: 1 Brdu ELISA result shows: 1.1 After treated by bortezomib for 24 h, 48 h and 72 h, proliferation of PC3 cells in treatment groups decreased to significantly lower levels than that in control group(P<0.05) in a dose-dependent pattern. Furthermore, under the concentration of 25μmol/L, PC3 proliferation reached its lowest level; 1.2 Under the concentratin of 5, 15 and 25μmol/L, proliferation of PC3 cells in bortezomib treatment group decreased in a time-dependent pattern. 2 Real time-PCR result shows: After treated by bortezomib for 72 h, WWP1, Smurf1 and Smurf2 m RNA levels of PC3 cells in treatment group were obviously inhibited than those in control group. In addition, the inhibiting effects were added with the addition of the concentration of bortezomib within certain range and showed its dose-dependence whose most significant effects was at 25μmol/L(P<0.05). 3 Western blotting result shows: After treated by bortezomib for 72 h, Smurf1, Smurf2 and WWP1 protein expression levels of PC3 cells in treatment group were obviously inhibited in comparison to those in control group. In addition, the inhibiting effects were added with the addition of the concentration of bortezomib within certain range and showed its dose-dependence whose most significant effects was at 25μmol/L(P<0.05).Conclusion: 1 Within a certain range, proliferation of PC3 can be inhibited by bortezomib treatment in a dose-time dependent pattern with increased inhibiting effects according to treatment dose addition. 2 After treating for 72 h, bortezomib can decrease Smurf1, Smurf2 and WWP1 m RNA levels of PC3 cells in a dose-dependent pattern. 3 After treating for 72 h, bortezomib can decrease Smurf1, Smurf2 and WWP1 protein expression levels of PC3 cells in a dose-dependent pattern. |
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