Research Of The Cell Apoptosis Mechanism Induced By Epoxomicin Via Endoplasmic Reticulum Stress On Prostate Cancer Cells

Backgroud:The new cases of prostate cancer in American had the first incidence rate in males. Prostate cancer is the second death cause of males in cancer deaths. The incidence rate of prostate cancer in china is increasing obviously with the aging of the population. The morbidity and mortality of prostate cancer in our country focuses on the age of 60-84 and 75-85, which means men over the age of sixty are priority control object. Prostate cancer is an androgen-receptor-dependent disease, and the blocking of androgen receptor signaling is a hallmark of prostate therapeutics. Most patients initially benefit from androgen deprivation as a first-line therapy. Hormonal therapy eventually leads to remissions for 12 to 48 months, but in most men metastatic prostate cancer ultimately progresses to an androgen independent state, depending on the disease burden, host factors, and inherent tumor biology. After progression, the disease evolves into a new clinically and molecularly heterogeneous known as castration-resistant prostate cancer (CRPC), which is invariably fatal.Although the multi-disciplinary comprehensive treatment including new second-line endocrine therapy, chemotherapy drugs and biological treatment and so on make the treatment effect of CRPC have improved, but these characteristics such as narrow scope, higher cost make benefit from the limited number of patients, the overall survival rate is still not optimistic. Therefore, looking for new targets for anticancer drugs becomes a research hotspot.Proteasome inhibitors play a role of antitumor via the ubiquitin-proteasome pathway. Proteasome inhibitors increase the protein levels of promoting apoptosis and reduce the protein levels of resistance to apoptosis, which results in the stagnation of the cell cycle and cell apoptosis. Importantly, it was suggested that the test found in a variety of human tumor cells, protease inhibitors, relative to standard cytotoxic agents showed more effective ability of apoptosis induction.Proteasome inhibitors are effective sensitization of chemotherapy/radio-therapy through selective strategy and overcome its resistance via targeting on cancer cells selectively with minimal collateral damage in normal tissue. Therefore, these research results provide convincing evidence that suggests ubiquitin-proteasome targeted ways is one of the effective treatment of human malignant tumors.A new highly selective, irreversible epoxy ketone epoxomicin (EPO, epoxy ketone tetrapeptide proteasome inhibitors) analogues-Carfilzomib were developed and different with bortezomib,is a new natural compounds of proteasome inhibition activity and chemotherapy/radiotherapy sensitization agent at the same time.In 2012 it was approved by the FDA for the treatment of refractory multiple myeloma. It has a distinct effect mechanism that distinct functional groups of epoxy ketone and THR1 forme a unique morpholine ring system to have inhibitory effect.It can inhibit the activity of protease body trough the irreversible combination to end of the 20s proteasome threonine active site. Because there is no other protease N-at the end of the nucleophilic residues as part of its active site, it reflects the epoxomicin unique specificity to the proteasome. Unlike other proteasome inhibitors, such compounds of proteasome inhibition is the specificity, not inhibiting protease body other than the protease, which is determined by the inhibition of its special mechanism. Compared with boric acid drugs, the combination is more specific and more stability, avoiding the peripheral neurotoxicity. Due to the particularity of the drug, it is a new generation of proteasome inhibitors and has become a hot spot in the treatment of solid tumor. Proteasome inhibitors can induce tumor cell apoptosis through a variety of mechanisms, which may activate endoplasmic reticulum stress tumor cells such as the head and neck cancer and myeloma cell, however, the mechanism caused by a proteasome inhibitor and its relationship with apoptosis is still unclear. It is still further not reported in prostate cancer furturly.Anticancer drugs can be induced apoptosis of tumor cells through endoplasmic reticulum stress. Three ways inducing tumor cell apoptosis currently had been confirmed:recognized death receptor mediated apoptosis, also known as the extrinsic apoptotic pathway; Mitochondrial dependent on apoptosis, also known as endogenous cell apoptosis and endoplasmic reticulum mediated apoptosis.The process of apoptosis induced by endoplasmic reticulum stress is complex, in which endoplasmic reticulum, mitochondria and other organelles participate, involving multiple signaling pathways. Endoplasmic reticulum stress(endoplasmic reticulum stress, ER stress) is due to the lack of oxygen, heat shock and other physical or chemical factors lead to abnormal protein aggregation of endoplasmic reticulum.The folded protein aggregation can be detect through the endoplasm retinal receptors which is folded proteins reaction(unfolded protein response, URP).This complex reaction in the cell receptor is mediated through three receptors of endoplasm retinal:PERK, ATF6, IRE1.GRP78 free from these three receptors in the initial stage of endoplasmic reticulum stress, causing a lot of free GRP78, result in higher express clearly, which is also the ER-stress activation performance. Three UPR reaction can activate the CHOP which also is the center of the endoplasmic reticulum stress adjustment. Under being the state of continuous endoplasmic reticulum stress, its expression level is obviously increased. Endoplasmic reticulum stress induced apoptosis mainly through three ways:CHOP approaches and ways of ASK1 (apoptosis signal-regulating kinasel)-JNK(ciun N-terminal kinase) and Caspase-12. Thus CHOP is one of the main way to induce apoptosis via endoplasmic reticulum stress.Based on the above, this topic studied the effect of proteasome inhibitors epoxomicin (EPO) on the hormone independent DU-145 cells of prostate cancer and the relationship between the drug and the endoplasmic reticulum stress. Meantime, we tried to find whether the apoptosis protein CHOP affects cell apoptosis endoplasmic reticulum stress or not. To explore and explain the influence and the mechanism of action of epoxomicin on hormone independence cells DU145 of prostate cancer, providing laboratory basis for us to our clinical treatment.Objective1. Research epoxocimin in prostate cancer cell proliferation and apoptosis.2. Explore the mechanism of epoxocimin preliminary through studying molecular markers of endoplasmic reticulum stress GRP78, CHOP, XBP-ls of gene transcription and GRP78, CHOP protein on prostate cancer cells.3. Explore the blocker influence of 4-PBA on epoxocimin inducing cells endoplasmic reticulum stress.4. Obsever the impact of epoxocimin on prostate cancer cell growth and the impact on the cell apoptosis and explore the mechanism of apoptosis induced by epoxocimin after silicing the gene CHOP.Method:1. Using MTS method and morphological observation to test the effect of proteasome inhibitors EPO on DU-145 of prostate cancer:Cells of EPO group were incubated in vitro with EPO incubating concentration(0.8mM,4 mM, 10mM, 20mM,100mM)and different incubating time(24,48,72h).The influence on the number of viable cells and cell proliferation on DU-145 cells of prostate cancer were inspected by MTS after treatment with different dosage and time of proteasome inhibitors EPO at the end of the expriment; The influence of the DU-145 cells were observed from morphology by inverted microscope.2. Detection cells apoptosis on DU-145 by Annexin V/PI:The cells were harvested after 24 hours with the treatment of EPO 10 and 20nM. Cells apoptosis were tested by Annexin V/PI after treatment with EPO10,20 nM for 24 hours on DU-145.3.CHOP,GRP78, XBP-1 and XBP-1s genetic changes by Real-time fluorescent quantitative PCR:CHOP, GRP78, XBP-1s genetic changes were tested after treatment with EPO10 and 20 nM for 24,48,72 hours; XBP-1 and XBP-1s genetic changes were detected after 6、12、24、36 hours treated with EPO20nM.4. CHOP, GRP78 protein level changes by western blotting method:The changes of protein levels of CHOP and GRP78 were determined by western-blotting after 24、 48、72 hours with EPO 10、20nM.5.4-PBA blockers and EPO on DU-145 cells:The effect of CHOP, GRP78 gene transcription and the influence of cell growth on DU-145 were detected after pretreatment with 4-PBA for one hour following with EPO.6. Silencing gene CHOP and EPO on DU145 cells:The influence of gene CHOP, CHOP protein, cell growth, cell apoptosis were examed after treatment with EPO20nM on DU-145 cells when silencing CHOP gene with different doses of CHOPsiRNA genes.7. Statistical method:All datas were analyzed by SPSS 17.0 statistical softwire. The results of experiment were expressed by the way of mean±SD. General linear models of univariate was used to test the effect of interaction between group and time.Using 95% confidence interval to compare with the control group on gene transcription and protein level;Using one-way ANOVA followed by Student-Newman-Keuls for other comparison test when the data was equal variance to test others. Multiple comparison test used F test(Welch) and Dunnett’S T3 test if the data Was not equal variance. P-Values were considered to be significant at<0.05.Results:1.The effect of cell proliferation inhibition of EPO on DU-145:The differences of living cells number in different groups were statistically significant (F=160.640, P <0.001); The difference of viable cells in different time were statistically significant (F=1.335,P=0.267); The interaction between time and groups was statistically significant (F=22.784, P<0.001); There was no significant statistically difference compared with normal group of corresponding time when treated with EPO0.8 nM (P>0.05); The number of viable cells of the group treated by EPO 4 and 10 nM were declined significantly compared with normal group on the second and third day (P<0.05); Treated with EPO 20 and 100 nM resulted in the decrease of the living cells number compared with normal group, having significant difference statistically on 1,2,3 days (P<0.05). Inverted microscope observation found that spindle cells morphological changes, becoming a polygon or circular.2.The impact of cells apoptosis on the DU-145:The cell apoptosis rate of EPO 10 and 20 nM groups were (25.04±3.21)% and (38.81±4.94)% respectively, having significant differences compared with normal group(5.51±0.61)%(P<0.05); The apoptosis rate in EPO20 nM is also higher than EPO 10 nM group (P< 0.05).3.The changes of CHOP、GRP78、XBP-1 and XBP-ls gene:Each gene expression difference after treatment with different dosage of EPO was statistically significant (CHOP:F=15.669, P<0.01; XBP-ls:F=12.724, P<0.01; GRP78:F=31.624, P<0.01); The differences of each gene expression after treatment with EPO for different time were statistically significant (CHOP:F=241.982, P<O.001; XBP-ls: F=91.382, P<0.001; GRP78:F=101.657,P<0.001); The interaction between time and groups was statistically significant for CHOP and GRP78 (CHOP:F=9.635, P=0.003; GRP78:F=15.521, P<0.001); but was no statistically significant for XBP-1smRNA (F=3.569, P=0.061)Using 95% confidence interval to compare with the control group, CHOP、 XBP-1s and GRP78mRNA expression showed an increasing trend, having the most obvious increase at 72 hours of treatment with EPO. The CHOP and XBP-1s gene expression of high dose group at 48h had significant differences statistically compared with low dose group (P< 0.05); The GRP78 gene expression at 48,72 h between low dose group and high dose groups had significant difference (P>0.05).XBP-1 and XBP-1s mRNA expression were statistically different after treatment with different dosage of EPO significant (F=724.225, P<0.001).The diffirences beteewn XBP-1 and XBP-1s mRNA were statistically significant (F=219.593, P<0.001), The interaction between time and XBP-1 and XBP-1s mRNA transcription level groups were statistically significant (F=202.587, P<0.001).The diffirences between XBP-1 and XBP-1smRNA from 6 to 36 hours were statistically significant. (P<0.01).Using 95% confidence interval to compared these two genes with control group,the results mean they were higher than the control group at 6、12、24、36h.4. The changes of the CHOP、GRP78 protein levels:CHOP、GRP78 protein levels in various EPO groups were significantly different (CHOP:F=46.148, P<0.001; GRP78:F=46.148, P<0.001); Each group was statistically significant diverse dealing with different time of EPO (CHOP:F=161.677, P<0.001; GRP78:F=80.982, P<0.001); There was interaction between different concentrations and different times on EPO treatment group for CHOP protein (F=21.941, P<0.05),but was no interaction for GRP78 protein (F=3.594, P=0.060).The protein of GADD153 and GRP78 were increased constantly on DU145 treated with EPO and had significantly difference with the control group at 24、48、72 hours Using 95% confidence interval to compare with the control group. CHOP protein levels comparative differences were statistically significant of EPO 20 nM and 10 nM at72 h(F=27.475,P=0.003).GRP78 protein levels went up to the highest and had a distinct difference between the low dose and the high dose group at 24 and72 hours(24h:F=24.332,P<0.05; 72h: F=17.824,P<0.05).5.4-PBA blockers and EPO on DU-145 cells:Compared with the control group using 95% confidence interval, CHOP, GRP78mRNA in PBA1、3 Mm alone had no significant change; Using the AVONA to compare with EPO group, PBA1 and 3 mM plus EPO20nM group had a significant reduction in the expression and the difference was statistically significant (P<0.05). The number of viable cells were increased significantly pretreatment with PBAlmM for one hour before EPO20nM treatment compared with EPO group (P<0.05), But the cell number was significantly lower than normal group (P<0.05); The cell number was no obvious difference after pretreatment with PBA 1mM plus EPO 4 nM compared with normal group (P>0.05), and the number of cells were higher significantly than EPO4nM group (P<0.05).Pretreatment with PBA3mM for 1 h plus EPO4 and 20 nM had no significant difference in cell number relative to the corresponding EPO group (P> 0.05), while lower than normal group (P<0.05)6. Silencing gene CHOP and EPO on DU145 cells:Silencing gene CHOP, CHOP mRNA expression and protein level were significantly higher than normal group in NS (that is the negative control group:(the non-specific siRNA group)+EPO group(P<0.01), having no significant difference compared with EPO group(P>0.05).CHOP gene transcription and protein level decreased significantly compared with EPO group in CHOPsiRNA10 and 50 nM plus EPO group (P<0.01).That confirmed our gene silencing is successful. Silence gene CHOP, CHOPsiRNA plus EPO20nM reduced the number of cells significantly compared to EPO20nM group (P<0.05), CHOPsiRNA 50 nM combination group is more apparent, but two groups of cells number are still lower than the normal control group (P<0.05). EPO treatment after CHOP gene silencing on DU-145 cells had (21.96±2.31)% of the apoptotic rate, which was significantly higher than that of normal group, but significantly lower than the EPO group (P< 0.01).Conclusion:1. EPO had obvious inhibitory effect of prostate cancer on DU-145 cells in time and concentration dependence; The lethal impacts by EPO can be manifested further on the cell morphology;Cells apoptosis rate of prostate cancer DU-145 by EPO was also increased with the rise of concentration of EPO.2. These molecular marker associated with endoplasmic reticulum such as CHOP, GPR78 and XBP-ls whose transcriptional levels increased significantly over time induced by EPO on DU-145 cells. XBP-ls gene expression was increased obviously by EPO on DU-145 cells with time, while XBP-1 gene expression did not change significantly. GRP78, CHOP protein levels can be stimulated to increase obviously by EPO over time, protein levels had obvious difference between high dose of 20 nM and low dose of 10 nM in 72 hours.3.The expression of CHOP and GRP78 gene transcription level of DU-145 induced by EPO via endoplasmic reticulum stress can be blocked by 4-PBA which also can block the cell proliferation inhibition induced by EPO. These two points is exactly proved that EPO produced endoplasmic reticulum stress on DU-145.4. CHOP gene expression and CHOP protein levels significantly decreased after silencing CHOP gene with siRNA, which means the method of silencing CHOP genes is successful; The effect of EPO on the cell growth inhibition and cell apoptosis of DU-145 were blocked partly by silencing CHOP gene.