Mechanism Of The N-3 Polyunsaturated Fatty Acids Promoting Apoptosis In Prostate Cancer Cells

Prostatic cancer(PC) is a major cause of cancer deaths in men patients and it is very harmful to men life quality and health. However, clinical diagnosis and treatment of patients with prostate cancer is still a lack of more scientific and effective method.Moreover, pathological changes of prostate cancer is complex and high accurate identification and classification is difficult. Therefore, Multiplex ELISA method is also the basis for the auxiliary diagnosis of prostate cancer. Gray Level Entropy Matrix(GLEM) is a new texture analysis and it has a unique advantage in evaluating morphometric changes in stromal tissues. Recent studies show that native glandular tissue and the surrounding interstitial tissue play an important role in the progress of cancer.How to use GLEM to perform assistly stage and classification diagnosis of prostate cancer is the main content of this research.The traditional treatment and prevention of prostate cancer is androgen deprivation therapy(androgen deprition treatment, ADT), including antiandrogen drug and surgical castration. This way is to block androgen combined into androgen receptors in order to suppress tumor growth,which is the main treatment way of prostate cancer. However, the traditional antiandrogen treatment can not fully suppress the progress of prostate cancer, most prostate cancer patients relapsed after ADT treatment in the form of the hormone independent.Deep sea fish oil, which main composition is n-3 Polyunsaturated fatty acids(n-3 PUFAs), especially eicosapentaenoic acid(EPA; 20:5n-3) and docosahexaenoic acid(DHA; 22:6n-3) which enriched in fish oil. In recent years, many national population survey and meta analysis showed that the increasing intake of n-3 PUFA in normly diet could reduce the risk of prostate cancer. Base on these results, we want to know whether n-3 Polyunsaturated fatty acids in cell level play a role in anti-cancer and be directly impact on prostate cancer cells.In order to solve these problems, this study is divided into three parts: The first part of the study : Using GLEM to test prostatic adenocarcinoma reactive stroma and metastasis potential compared with Correlative ELISA. The second part of the study: Using n-3 Polyunsaturated fatty acids to treat prostate cancer cells and by this way we can observe which kind of n-3 PUFAs can affect the growth of prostate cancer cells. The third part of the study:To discuss the mechanism of n-3 Polyunsaturated fatty acids DHA changing gene expression and promoting apoptosis of prostate cancer cells. Given above all, we design this study to provide experimental basis and theoretical basis for clinical auxiliary staging classification of prostate cancer and to discuss weather using n-3 PUFAs as dietary supplement can prevent and treat prostate cancer. PartⅠGray Level Entropy Matrix in the Clinical Detection of ReactiveStroma and Metastatic Potential of High-Grade ProstaticAdenocarcinomaObjective: To compared Multiplex ELISA with Gray Level Entropy Matrix and to dicuss whether GLEM can be used in the detection of reactive stroma and metastatic potential.Methods:1 Prostatic adenocarcinoma samples were obtained for observing the pathological changes and kernel density analysis.2 GLEM gray entropy matrix texture analyses were carried out on the sample.3 Using Multiplex ELISA to evaluate the prostate cancer samples.Results:1 Analysis of tissue morphology and nuclear densityThe main characteristic of BPH is hyperplasia and hypertrophy of basal cell: Most of basal cells are cubic and short columnar and smooth muscle cells are thickly, densely, unevenly distributed in the stroma, which uclear shape have no obvious changes.Compared with BPH, the structure of advanced PA basal cells are disorder, the nuclei are hyperchromatic and particle number, size is differ, this is consistent with the analysis shows that in nuclear density(P<0.05).2 Texture analysis of GLEMUsing professional images processed for enhanced visualization of the stromal organization the interstitial tissue signals: There is higher interstitial proportion in BPH and changes in interstitial structure composition, as well as in smooth muscle of interstitial area.The result reveals the highly disorganized matrix in higher grades of PA and tissues obtained from treatment-resistant cases. Compared with BPH, there were higher loss of neighboring pixels contras in advanced PA and treatment-resistant PA.The result of GLEM shows these were correlated with loss of contrast between neighboring pixels and higher correlation on the GLEM texture parameter due to loss of heterogeneity between the pixels. Images processed for enhanced visualization of the stromal organization, which reveals the highly disorganized matrix in higher grades of PA. In every sample, GLEM showed lowest entropy in the sample of benign prostatic hyperplasia(P < 0.05) and the highest in androgen independent PA tissue samples(P<0.05). The discretion of entropy represents the image of reactive substrate.3 Analysis of Multiplex ELISAUsing multiplex ELISA to test the serum antigen of PA: neovasculogenesis, VEGF, E-M transition(beta1-integrin, E-cadherin, MMP3) and osteogenic metastasis(RANKL and osteoprotegerin). Correlation analysis shows: there is statistical difference in BPH sample and PA sample.This lack of correlation is shown in five samples.Conclusions:Multiplex ELISA method can be only used for distinguish between benign and malignant tumors, which can be not for auxiliary staging and grading of PA.GLEM entropy of gray level pixel in providing quasiquantitative estimate of a reactive stroma in PA and thus can be auxiliary diagnosis, staging and grading of PA. Part Ⅱ Promoting apoptosis effect of Polyunsaturated fatty acids inprostate adenocarcinoma cellsObjective: In this part, we detect whether deep sea fish oil, EPA and DHA in n-3 PUFAs and Arachidonic acid(AA) in n-6 PUFA play a important role in BPH cells and different PA cells.We also study which category ofs PUFA could surpress the growth of PA cell. This part of the study could provide experimental basis for clinical application of PUFA, which be used as prevention and treatment of prostate cancer.Methods:1 Using MTT assay to test the survival rates of prostate cancer cells and prostate cells influenced by Fish oil, EPA, DHA and AA.2 Using Western blot and RT real- time PCR to test the gene expression level of Caspase.3 Using Hochest 33258 staining to observe the changes in apoptosis nucleus.Results:1 Fish oil reduce the survival rate of prostate cancer cells.Compared with BSA group, benign prostate cells were incubated in different concentrations of Fish oil for 24 h, survival rates have no statistical difference in these groups. Compared with BSA group, PA cells(PC3 and DU145 cells)were incubated in different concentrations of Fish oil for 24 h, survival rates of two PA cells decreased obviously.(P<0.05)Compared with BSA group, LNCa P PA cell was incubated in different concentrations of Fish oil for 24 h, survival rates have no statistical difference in these groups.2 The effects of EPA, DHA and AA on survival rates of prostate cancer cells.PC3 and DU145 cells were incubated in the presence of EPA or DHA for 24 h, the survival rates of two PA cells decreased obviously.(P<0.05)DU145 cell was incubated in the presence of AA for 24 h,survival rates had no changes. DU145 Cell was incubated in the presence of DHA 50μM for 12, 24, 48, 72 h, there was time-dependent between the survival rate of DU145 and DHA stimulus. 3 Using DHA can promote the expression of Caspase 3 in prostate cancer DU145 cell, as well as induce apoptosis.Compared with BSA group, DU145 PA cell was incubated in different concentrations of DHA for 24 h, the level of Caspase 3 m RNA increased obviously and be concentration dependence.Compared with BSA group, DU145 PA cell was incubated in different concentrations of DHA for 24 h, the expression of Caspase 3 at protein level increased obviously. DU145 cell apoptosis was determined by Hoechst 33258 staining. Compared with BSA group, cells were incubated in the presence of DHA(50 μM) for 24 hours, nuclear staining were darker and nuclei were fragmental thick dense in various places.Conclusions:Deep sea fish oil can reduce the survival rate of prostate cancer cells and has no effect on benign prostate cells. N-3 series polyunsaturated fatty acid DHA can reduce prostate cancer cell(DU145 cell)survival rate with concentration and time dependence. Using DHA can promote the expression of Caspase 3 in prostate cancer DU145 cell, as well as induce apoptosis. Part ⅢThe mechanism of signaling pathways how can DHA induce theapoptosis of prostate cancer cellsObjective: To use RT2 Profiler PCR Arrays method, which can detect 84 apoptotic pathways related genes simultaneously, and to use Real-time PCR as reliable tools for validation. To explore how DHA change apoptosis signaling pathways and further lead to prostate cancer cells apoptosis. In the end, this study can provide important scientific basis and theoretical basis for prevention and treatment of prostate cancer.Methods:1 RT2 Profiler PCR Arrays was used to screen the targets of DHA induced prostate cancer cells apoptosis.2 Using Real-time PCR as reliable tools for the result validation.Results:1 The results of RT2 Profiler PCR Arrays screenRT2 Profiler PCR Arrays was used to screen the m RNA level of 84 apoptosis related genes.The result showed that after DU145 PA cell was incubated of DHA 50μM for 24 h, the expression of 10 genes has raised 2 times while 5 genes cut less than or equal to 0.5 times.Compared with BSA, after incubated of DHA for 24 h, the transcriptional level of Caspase family: Caspase 1, Caspase 3 and Caspase 9 in DU145 cell raised 2.06, 4.88 and 12.10 times respectively; The transcriptional level of pro-apoptotic genes BAX raised 2.93 times and BCL2/BAX ratio increasesed.Cell death inducing related genes CIDEA and DFFA raised 2.34, 3.21 times respectively; Tumor necrosis factor related genes LTA and TNF raised 2.04 and 2.24 times respectively; The expression of TP53 gene m RNA increased 2.97 times. In addition, compared with the control group( which incubated of BSA), after incubated of DHA for 24 h, the transcriptional level of apoptosis in-ducing factor 1(AIFM1), AKT1, BID,BIRC6 and XIAP decreased.2 The results of RT real-time PCR screenThe effect of DHA treatment for 24 hour on the Caspase family m RNA expression in DU145 cells by using real-time RT-PCR:the m RNA expression of Caspase 1, Caspase 3, Caspase 9 and Caspase 12 raised respectively. Moreover, the expression of Caspase 3, Caspase 9 and Caspase 12 had dose-dependent relationship with DHA stimulates concentration.The m RNA expression of BAX, CIDEA, DFFA, TP53 and TNF normalized with TBP in 24 h DHA treated DU145 cells raised obviously, the expression of BAX, CIDEA and TNF had dose-dependent relationship with DHA stimulates concentration.The m RNA expression of AIFM1, AKT1, BID, BIRC6 and XIAP were incubated in different concentrations of DHA for 24 h dereased obviously, and theexpressio n of BAX, CIDEA and TNF had dose-dependent relationship with DHA stimulates concentration.Conclusions:DHA can change the related genes expression of apoptosis signaling pathways in Caspase family, by this way it can induce the apoptosis of prostate cancer cells.Conclusions:1 GLEM entropy of gray level pixel in providing quasiquantitative estimate of a reactive stroma in PA and thus can be auxiliary diagnosis, staging and grading of PA.2 DHA in n-3 PUFAs can induce apoptosis of prostate cancer cells and has no effect on normal prostate cells.3 DHA can change the related genes expression of apoptosis signaling pathways in Caspase family, by this way it can induce the apoptosis of prostate cancer cells.