A Preliminary Study On The Androgen-independent Prostate Cancer

Prostate cancer is one of the most common malignant tumors of men.Themortality has reached the second highest position among all male cancers.Althoughthe morbidity of prostate cancer in China is far lower than that of westerncountries,the morbidity in recent years is markedly increasing.Duration and spread of prostate cancer depend on signal transduction ofandrogen receptor.Therefore,the androgen deprivation therapy is the third standardtherapies of prostate cancer except surgery and radio-therapy.Most patients of prostatecancer gain a markedly curative effect after undergoing the first androgen deprivationtherapy.While almost all of the patients will relapse and their androgen-dependentprostate cancer will transform into highly deteriorated androgen-independent prostatecancer with widespread metastasis.In this case, the conventional androgen deprivationtherapy has no longer effect.Once ADPC transform into AIPC,cancer cells areuncontrollable,very high mortality rate,therefore become the major diseases that harmthe health of elderly male people.AIPC transformation mechanism is the hotspot in the research field of prostatecancer all over the world. The contents mainly include the amplification,over-expression and mutation of androgen receptor (AR) gene,the abnormity of AR signalpathway etc.The primary reasons for ADPC becoming AIPC are gene expressionvariation,signal pathway abnormity, and disorder of proto-oncogene,cancer suppressorgenes and growth factors.Researchers have discovered a large number of relevantgenes and signal pathways of prostate cancer. The tumor genesis and development ofprostate cancer are very complex to involve multi-steps and poly-genes.In addition,the majority of AIPC studies are based on androgen dependent cell PC3,DU145as themodel,since it doesn’t express endogenous androgen receptor and induces androgenresistance as a result.But the occurrence and development of prostate cancer,as well asfrom hormone dependent to independent changes,are inevitable to experience thecombination of the androgen and receptor,through the androgen receptor signaling pathway,translating signals into the cell specific target genes to effect.Therefore,theabove theories can’t reasonably explain the mechanism of AIPC and androgenindependent prostate cancer still need further research.This research chooses LNCaP human prostate cancer cells maintaining thedifferentiation feature of human prostate cancer and being extremely strong dependent,to establish model to simulate the process of clinic prostate cancer change fromandrogen-dependent to androgen-independent.Using the modern cell biology,molecular biology,bioinformatics technology to explore the changes of expression ofhuman prostate cancer gene,we may further reveal the possible transformationmechanism from androgen dependent prostate cancer to androgen independentprostate cancer,to provide a new theoretical basis for treatment to androgenindependent prostate cancer.1. The establishment of androgen independent prostate cancer sub-cellular modelThis research chooses LNCaP human prostate cancer cells maintaining thedifferentiation feature of human prostate cancer,to establish androgen independentprostate cancer cell model through the androgen decrement method.After LNCaPcells being cultured in the androgen deprivation medium and androgen with flutamideenvironment respectively and continuously sub-cultured, changes in cell morphologywere observed and the ability of cell proliferation, invasion and migration ability ofPSA secretion,changes in biological activities were detected.Results can be seen that after androgen deprivation being induced and continuousculture of the30generation of LNCaP-A cell morphology changed obviously: the cellbody became small,the morphology of the cells from the triangle,long spindle,circularshape into irregular shape;cells no longer uniform distribution, more single cellindependent growth were viewed,aggregation growth phenomena occur at the sametime. Increased cell proliferation can grow quickly in the androgen deprivationenvironment and produce resistance to androgen competitive inhibitor offlutamide.Invasion and migration of cancer cells also greatly enhanced and can stillrecover part of the secretion of PSA in androgen free environment,increasing with thetime.Indicating that description of the cell model from the morphological andbiological behavior are very close to the AIPC formation process of clinical endocrinetreatment, preliminary study on occurrence mechanism can be used for prostatecancer in cytology and molecular biology. 2. Androgen independent prostate cancer gene expression changesUsing gene chip technology on androgen dependent LNCaP cells and androgenindependent sub-cellular model LNCaP-A to scan full gene expression spectrum,screen gene expression differences of AIPC formation1,integrate KEGG andPANTHER learning tool online analysis and MAS biological information at the sametime, the genes relating the AIPC were analyzed in many aspects of biologicalinformation pathway, gene regulation network and biological function, to understandthe changes in gene expression after ADPC into AIPC, and based on these to screenkey genes and the possible signaling pathway from ADPC into AIPC.Through the analysis and processing of the original data of gene chip test, a totalof12207expression genes were compared. With the standard of difference fold>2.0,347differential expression genes were acquired from androgen-independentLNCaP-A cells and LNCaP cell control. Among those genes,156were up-regulatedand191down-regulated which indicates androgen dependent prostate cancer celloccurred significant changes in gene expression.comparing the androgenindependent LNCaP-A cells with LNCaP-A+F cells which is androgen deprived thentreated with flutamide, the expression of two groups of cell gene had no obviousdifference,which also shows that growth of androgen independent prostate cancercells induced by androgen deprivation is no longer affected by androgen inhibitors,being resistant to androgen inhibitor.With application of ToppGene tools differences in gene expression are classifiedand analyzed,the results of which showed down-regulated genes mainly involved inmembrane protein,trans-membrane protein,ion binding, intracellular signaling,regulation of gene expression and so on,and function of gene up-regulated areinvolved in cell internal gene regulation,metabolism,expression,variable shear.Participation of intracellular gene regulation and the expression,metabolism and so onincreased. Participation of membrane protein, exogenous substances inside the cellsignal transduction,and expression of intercellular adhesion and the level ofrecognition of genes reduced,which are in good agreement with the experimentalphenomena androgen independent prostate cancer cells are protected from the controlof external androgen deprivation.Combining the functions of differentially expressed genes,biological process andpublished literature,with a preliminary analysis of the signal pathway of key genes in the process and in the up-regulation of the expression of genes,there are4main genesinvolved in the signal transduction pathway of4types, and the down regulated genesin the7genes involved in the signal transduction pathway of various types.The set upof these genes and signal pathways provides a direction for further study on themechanism of AIPC.3. A preliminary discussion on the mechanism of androgen independent prostatecancerUsing RT-PCR and Western blot method to verify the apoptosis pathwaymediated by TLR2, PI3K/AKT pathway, MAPK pathway and other signaling pathwaykey protein, we explored the possible role of transformation process from androgendependent prostate cancer to androgen independent prostate cancer.Western blot results shows that Caspase3, FADD protein expression level didnot change significantly, and there were no activation of Caspase3bands, suggestingthat apoptosis pathways in the Toll-like signaling pathway not involved in thetransformation of androgen independent prostate cancer. The expression of AKT,p-AKT was not significantly increased, but NF-kappa B protein expressionup-regulated, the expression level of I kappa B alpha protein down-regulated. Theseindicate Tip NF-kappa B may be one of the key sites in the signal path, but in theLNCaP-A cell line PI3K/AKT pathway is not activated, there may be other activationpathway, lowering the inhibitory protein I kappa B alpha expression may affect NF-kappa B expression changes.When detecting MAPK signaling pathway related proteins, ERK, MEK, JNKprotein expression showed no significant change, but p38, HSP27, IL-8expressionwas significantly increased, suggesting when LNCaP cells transformed intoLNCaP-A cells, the ERK pathway, JNK pathway is not involved in MAPK/p38signaling pathway, but play an important role, and IL-8, HSP70may all be importantmolecules in the pathway of transmission information.Studies have shown that MAPK/p38plays an important role in breast cancer,ovarian cancer, endometrial cancer resistant mechanism, but there is no report inandrogen resistant prostate cancer. The findings of the present study lay thefoundation for the study of AIPC transformation mechanism and development ofAIPC targeting drugs.