Prostate Cancer Cell Lines Secreted Protein Group And Related Proteins Function

Among American male, prostate cancer is the most common cancer and the second leading cause of cancer deaths. Nearly 200,000 men in the United States each year are diagnosed with prostate cancer, nearly 30,000 of whom die from the disease. In China, prostate cancer has become the third leading cause of the urogenital tumor in male patients. With the aging tendency of population and the change of life style, the incidence of PCa among Chinese men is significantly higher than that in earlier years.Prostate is an androgen-dependent organ and androgen withdrawal therapy for early stage of prostate cancer is effective. But after about two years, androgen withdrawal therapy will become useless. The tumor will become more aggressive and acquire stronger metastasis capacity. LNCaP is a representative cell line of prostate cancer, C4-2 and C4-2B are sublines of androgen dependent LNCaP cells. Eight weeks after LNCaP cells were inoculated s.c. into male athymic nude mice, castration was executed to the mice. Four weeks later, the tumor was obtained from the mice and inoculated into new mice and castration was executed again. With the similar steps repeated, the new androgen independent C4-2 and C4-2B were obtained. They have the same genetic background as LNCaP, but acquire androgen- independent growth capacity and a stronger bone metastasis tendency. LNCaP, C4-2 and C4-2B cells mimic different clinical progression stages of prostate cancer patients. These cell lines are internationally recognized cell models of prostate cancer. In order to find useful markers that can indicate the aggressive prostate cancer and to clarify the molecular mechanisms of aggressive and /or androgen- independent development, This study compared the secretome of LNCaP, C4-2 and C4-2B to find differentially expressed proteins and studied their expression patterns in prostate cancer cell lines and tissues of patients, as well as the function and mechanisms involved in tumor progression.Two-dimensional electrophoresis and mass spectrometry based proteomics technologies were used to separate, compare and identify the extracellular proteins of LNCaP, C4-2 and C4-2B. A total of 16 proteins were successfully identified, 6 of which were higher in 2-DE gels of C4-2 and C4-2B than that of LNCaP, including PSA, IGFBP2, uMtCK, PGK1, PRDXⅠand DNM2 (isoform 4, variant). Among these 6 proteins, PSA is the most extensively used tumor marker, IGFBP2 is greatly concerned about its potential value of androgen-independent diagnosis. In all these 16 proteins, PPⅠA has been proved to be a growth factor that exists in extracellular medium. PEBP1, PRDX 1 and TPI can be easily detected in extracellular medium by methods based on proteomic technology. These are concord with our results. Furthermore, many tumor markers such as GLOⅠ, PGK1 and PRDXⅠwere also found in our experiments. These proteins are already known to have roles of promoting tumor development and/or can be used as tumor markers. Then this study compared the mRNA and protein expression levels of the corresponding genes. With commercialized antibodies, this study directly verified the differentially expressed uMtCK and PGK1 in LNCaP , C4-2 and /or C4-2B cells. After transiently transfecting eukaryotic expression vector with the conrresponding genes into 293 cells, culture medium of the cells was collected and western blot was performed by his-tag antibody. Exogenous PRDX1 protein can be detected in conditioned medium of 293 cells, suggesting that it may be a new secreted protein. Whether these proteins in extracellular medium of prostate cancer cells have the clinical values still need to be further explored and confirmed.By comparison, this study found that ubiquitous mitochondrial creatine kinase (uMtCK) was overexpressed in C4-2 and C4-2B cells as well as in tissues of prostate cancer patients. In LNCaP cells, uMtCK can be significantly induced by androgen antagonists Bic or in the condition of cell crowding. These results indicated that uMtCK may have specific functions in the above situations. Therefore, a stably over-expressed uMtCK LNCaP cells (LNCaP-uMtCK) and its control were constructed to explore the possible function of uMtCK involved in tumorgenesis and tumor development. LNCaP-uMtCK showed a relatively higher growth rate than LNCaP cells transfected with a mock vector (LNCaP-neo) under conditions of 8% FBS or plus 20μM Bic. Androgen insensitivity and the slightly but significantly resistance to anti-cancer drug cisplatin was also found in LNCaP-uMtCK cells. Cell cycle analysis showed that S phase of LNCaP- uMtCK is higher than that of LNCaP-neo, and cyclinD1 expression of LNCaP- uMtCK was also increased. This study also found that androgen receptor expression and activity level of LNCaP-uMtCK in 8% FBS culture condition were both reduced comparing with LNCaP-neo. After stimulation of 1 nM synthetic androgen R1881, androgen receptor activity of LNCaP-uMtCK is still lower than that of LNCaP-neo. Further exploration showed that, compared with the LNCaP-neo cells, the mitochondrial membrane potential and reactive oxygen species levels of LNCaP-uMtCK are both higher. The elevated reactive oxygen species do not induce apoptosis; on the contrary, it may facilitate tumor cells to grow by multiple signaling pathways. By evaluating Akt signaling pathway, this study found that phosphorylation of Akt (Ser473) and GSK3β(Ser9) of LNCaP- uMtCK cells were higher than LNCaP-neo. Although lacking of direct evidences, this study speculated that reactive oxygen species is likely to contribute to advantageous growth of LNCaP-uMtCK by the activation of a number of signaling pathways including Akt pathway. After passages of over 20 generations, LNCaP-uMtCK cells became aneuploid, which may be the long-term effect of increased reactive oxygen species. The mechanisms of aneuploidization and the characterization of LNCaP-uMtCK aneuploid cells should be further studied.To sum up, possibly by means of the increased ROS production, overexpression of uMtCK induced by Bic stimulation or cell crowding pressure is involved in growth and survival of prostate cancer cells.