A Preliminary Experimental Study Of Drug Resistance Mechanisms On Androgen Indepe Ndent Prostate Cancer Cell

Prostate cancer is the most frequently diagnosed cancer and the second-leading cause of cancer death in men in the United States and Europe. American Cancer Society estimated that in 2009 about 192,280 American men are diagnosed with prostate cancer, about 27,360 died of prostate cancer.At present, the incidence of prostate cancer, although far lower than western countries, but showed a significant growth trend in china with changing lifestyles, longer life expectancy and the improvement of diagnosis. our patients with newly diagnosed prostate cancer often have advanced, loss of opportunity for surgery and poor prognosis with compared to the West national prostate cancer. Androgen deprivation therapy is the standard methA for prostate cancer besides surgery and radiation therapy. However, after a median of 18 to 24 months, androgen dependent prostate cancer (ADPC) turned into androgen independent prostate cancer (AIPC), extremely high mortality rate of AIPC has become a major health hazard for older men.The studies for mechanism of AIPC pathogenesis is a hot research field, mainly: androgen receptor (AR) gene amplification; androgen receptor mutation; apoptosis gene abnormalities and so on. But the above theory can not reasonably explain the pathogenesis of AIPC because the biological behavior of prostate cancer is extremely complex.AIPC still need further study. To establish the cell mAel of prostate cancer from androgen dependent into androgen-independent can simulate gene changes and response to treatment in human prostate cancer. Mechanism of AIPC pathogenesis expected to be revealed by mAern cell biology, molecular biology and proteomics and metabolomics research tools and provide a new theory for AIPC treatment.Main contents:1. In the environment of free androgen hormones and androgen antagonist flutamide, prostate cancer LNCaP cells were transformed into androgen-independent cell line-LNCaP-AI cell line and LNCaP- AI + F cell line and their biological characteristics were identified.2. The changes of androgen receptor(AR) pathway were studied in LNCaP-AI and LNCaP-AI+F, including the AR receptor mutation, AR receptor expression (mRNA and protein level); AR receptor esponsiveness to androgen and PSA protein and PSAmRNA levels; the changes of the main protein in cell signaling pathway.3. The changes of the receptor tyrosine kinase RON in LNCaP-AI and LNCaP-AI+F. the RON gene expression was analysed by the methAs of PCR, Western Blot and immunohistochemical staining.4. Using high throughput Solexa sequencing, the profile of small RNA molecules was analysed in LNCaP-AI+F and LNCaP respectively. The miRNAs related to androgen-independent prostate cancer were screened and new miRNAs may be found.The main results:1. The morphological changes of LNCaP-AI and LNCaP-AI+F cell lines: its morphological changes as follows: the cell bAy became smaller, flat and round or spindle rare, simultaneous growth of aggregation; But LNCaP cells appeared in triangle or a long shuttle-type, the circular uniform distribution.2. Growth characteristics of LNCaP-AI and LNCaP-AI + F cell lines:2.1 In free androgen hormones environment, the growth rate of LNCaP-AI and LNCaP-AI+F cell lines were significantly higher than that of LNCaP cells; Growth inhibition of Flutamide on the LNCaP-AI and LNCaP-AI+F cell lines in vitro significantly reduced.2.2 The growth role of androgen responsiveness decreased in the LNCaP-AI cell lines, only the high concentration of androgen to stimulate cell growth, but in the LNCaP-AI+F cell line, different concentrations of androgen can not stimulate the cell growth. It indicated that LNCaP-AI+F might be different from LNCaP-AI cells in the cellcharacteristics.2.3 Cell invasion and migration activities of the LNCaP-AI and LNCaP-AI+F cell line were higher than the LNCaP cells, in particular, the activities were significantly higher in the LNCaP-AI+F cell line.3. The changes of AR signaling pathway in the LNCaP-AI and LNCaP-AI+F cell lines:3.1 AR mutation was not detected in the LNCaP-AI and LNCaP-AI+F cells, but AR expression was upregulated in LNCaP-AI cells, while downregulated in the LNCaP-AI+F cells in the transformation process of androgen dependent prostate cancer to androgen-independent prostate cancer.3.2 PSA expression was not difference between LNCaP-AI and LNCaP cells(p>0.05), but significantly reduced in the LNCaP-AI+F cells(p<0.01); flutamide, DHT and testosterone undecanoate can stimulat PSA expression and secretion in the LNCaP, LNCaP-AI and LNCa P-AI+F cells, but responsiveness of PSA expression to dihydrotestosterone and testosterone undecanoate in the LNCaP-AI and significantly decreased in the LNCaP-AI+F cells; the reactivity and sensitivity to the drug concentration decreased in the LNCaP-AI and LNCaP-AI+F cells. It indicated that AR signaling pathway is effective, but decreased in the responsiveness to androgen hormnones in the transformation process of androgen dependent to androgen independent.4. Expression of receptor tyrosine kinase RON in the LNCaP-AI and LNCaP-AI+F cells: The results showed that RONmRNA and RON protein were not detected in the LNCaP-AI and LNCaP-AI+F cells. It indicated that RON gene did not play role in the transformation process.5. The changes of miRNA expression profile in the LNCaP-AI+F cells:5.1 376 miRNAs were detected in the LNCaP-AI-F cells, while 357 miRNAs in the LNCaP cells. 94 miRNAs including 48 upregulated, 46 downregulated were differentially expressed in the LNCaP-AI-F cells compared to LNCaP cells.5.2 15 new miRNA were predicted, including 9 new miRNAs in the LNCaP-AI+F cells and 6 new miRNAs in the LNCaP cells.The main conclusions:1. Androgen-independent prostate cancer cell lines-LNCaP-AI and LNcaP-AI+F cell line were successfully established, their androgen responsiveness and growth characteristics significantly different from the LNCaP cells. It is a goA cellular mAel for studying the mechanisms for androgen-independent prostate cancer.2. In the transformation process of LNCaP cell line to LNCaP-AI and LNCaP-AI+F cell line, AR mutation was not detected and AR expression was decreasing; AR signaling pathway was not blocked, but reduced in the responsiveness to androgen.3. RON gene did not play role in the transformation process of LNCaP cell line to LNCaP-AI and LNCaP-AI+F cell line4. miRNAs might play important role in the transformation process of LNCaP cell line to LNCaP-AI and LNCaP-AI+F cell line, provided the foundation for further study of the incidence of AIPC.