| Prostate cancer(CaP) is one of the most common cancers and also thesixth leading cause of cancer-related mortality in men, accounting for903,500new diagnoses and258,400deaths every year all over the world.Due to the improvement of diagnostic technology and the valid CaPscreening programs, increasing numbers of men had been recentlydiagnosed and treated for clinically localized CaP both in Asia and Europe.Although antiandrogen therapy is initially effective, resistance inevitablyoccurs within a few years. There are limited ways for hormone-refractoryprostate cancer(HRPC), which has become a burning issue in clinical.PI3K/Akt/mTOR signaling pathway is an important therapeutic targetof CaP. mTOR is an evolutionarily highly conserved Ser/Thr kinase, that is amember of the phosphoinositol3-kinase(PI3K) family and a downstreameffector of PI3K/Akt signaling pathway. mTOR is widely expressed in cells,and regulates a diversity of cellular functions of different cells, such asproliferation and survival. The mTOR regulates translation of5’ terminaloligopyrimidine (TOP)-containing mRNAs in response to extracellular signals. And it is also the central to a signaling cascade that regulatesinitiation of cap-dependent RNA translation.4EBP1is a crucial target ofmTOR signaling pathway, and also a negative regulatory factor for proteinsynthesis.4EBP1inhibits the combination of eIF4G and eIF4E, andeventually inhibits the initiation of cap-dependent translation.4EBP1isphosphorylated by mTOR, and dissociates from eIF4E. Then eIF-4E isdisinhibition, and consequently promotes the cap-dependent translation.Additionally, the p70ribosomal protein S6kinase (p70S6K) thatphosphorylates the S6protein of small ribosomal subunits is alsophosphorylated and activated by mTORC1, along with other kinases,thereby enhancing protein translation. In prostate cancer and other solidtumors, mTOR is a key protein associated with tumor generation andtreatment resistance. And in prostate cancer, mTOR signaling pathwayintegrates the cell survival signaling, as well as the critical downstreamproteins are always activated.As well as cell proliferation, apoptosis is a complicated processregulated by multiple signaling pathways and many genes. Cells startorganized self-destruction as a protective biological response to maintainautoallergic homeostasis when cells meet harmful or abnormal stimuli.Disorders of the apoptotic process may promote the proliferation ofabnormal cells and finally lead to cancer. So the apoptosis signalingpathway is a key target of cancer treatment. Ligusticum chuanxiong Hort is a plant classified to Genus Artemisia,Family Apiaceae. It is known as an herb that promotes the circulation ofblood in traditional Chinese medicine. Its major active component istetramethylpyrazine (TMP,2,3,5,6-Tetramethylpyrazine). TMP couldimprove microcirculation through its effects of anti-platelet aggregation andarteriolar regulation. In addition, TMP exhibits antioxidant, antifibrotic, andcalcium antagonism effects. While TMP has been used as a supplement toprevent and cure vascular diseases in China, recent studies suggest that TMPstill has various anti-tumor effects, such as suppresses tumor growth, MDA,and tumor metastasis.In this research, firstly we tested the effect on the proliferation activityof PC-3cells by the MTT assay. Then probed into the mechanism of thisproliferation inhibition of TMP：(1) The expression of p-mTOR, p-p70S6,p-S6, p-4E, and p-4E-BPP1were detected by Western blot analysis. Andfurther more, the expression changes of the translation initiation proteinswere estimated by pull-down assay with7-methyl-GTP Sepharose4B beads.Finally, the translation assay was confirmed by luciferase gene, and theluciferase mRNA expression was examined by RT-PCR.(2) We detected theapoptotic cells by flow cytometry, then the nuclear alteration because ofapoptosis was revealed by the fluorescence microscope. And the expressionchanges of apoptosis related proteins were tested by Western blot analysis.This research will provide theoretical basis for the PCa therapy of TMP. Methods1. The PC-3cells were treated with different concentrations (0、0.6、1.2、2.4、3.6、4.8、6.0、7.2、8.4mM) TMP for48h and72h. The HCT-116cellsand the RWPE-1cells were treated with different concentrations(0、0.6、1.2、2.4、3.6、4.8、6.0、7.2、8.4mM) TMP for48h. Then the growth inhibitionrates were assessed using the MTT assay.2. PC-3cells were treated with TMP at the indicated concentration(0、4.8、7.2mM) for48h:(1) Detected the effect on mTOR signaling pathway.The expression of mTOR, p70S6K, S6,4E,4E-BP1, p-mTOR,p-p70S6K, p-S6, p-4E, and p-4E-BPP1were detected by Western blotanalysis (the treatment time of phosphorylation is45min).The interaction of4E-BP1and eIF4E was detected by the pull-downassay with7-methyl-GTP Sepharose4B beads (the treatment time is24h).The translation assay was estimated by luciferase gene.The expression of luciferase mRNAwas examined by RT-PCR.(2) Detected the effect on apoptosis.Detected the apoptosis of PC-3by flow cytometry.The fluorescence microscope was applied to reveal the nuclear alterationbased on apoptosis.The changes of apoptosis related proteins expressions were tested by Western blot.3. All the data were analyzed with the SPSS17.0software, and comparisonsbetween groups were tested by One-Way ANOVA analysis and LSD test.Result1.PC3cells, HCT-116cells and RWPE-1cells were treated with TMP at theindicated concentration(0、1.2、2.4、3.6、4.8、6.0、7.2、8.4mM) for48h,the proliferation of the cancer cells (PC-3, HCT-116) were significantlyinhibited(p<0.05), and resulted in a dose-and time-dependent growthinhibition of PC-3cells(p<0.05), while there was no obvious change in theRWPE-1cells(p＞0.05).(MTT)2. PC-3cells were treated with TMP at the indicated concentration(0、4.8、7.2mM) for48h:(1) The effect on mTOR signaling pathwayTMP treatment decreased the activation of mTOR and the relateddownstream targets(p<0.01), but had no significant effect on the totalproteins(p＞0.05).(western blot)The binding of4E-BP1to eIF4E was remarkably increased(p<0.05), andthe availability of eIF4E for translation initiation was decreased(p<0.05).(pull-down with7-methyl-GTP Sepharose4B beads)The translation of the luciferase reporter gene was suppressed(p<0.05).(relative activity of luciferase) But there was not a decrease in mRNAlevel of the luciferase gene (p＞0.05).(RT-PCR) (2) The effect on apoptosisPromoted apoptosis of the PC-3cells(p<0.05).(flow cytometry)The nuclear alteration because of apoptosis was induced by TMP.(fluorescence microscope)The expression of Bcl-2was significantly reduced(p<0.05) and theexpression of the pro-apoptotic protein, Bax and Caspase-3, wereincreased(p<0.05).(western blot)ConclusionTMP could inhibit the proliferation of human prostate cancer PC-3cell line, and the potential mechanisms can be divided into two broadcategories. On the one hand TMP promotes the dephosphorylation ofmTOR and the related downstream targets, and inhibits the translationinitiation, thus inhibits the proliferation of PC-3cell. On the other, TMPpromotes the apoptosis of PC-3cells by increasing the expression of thepro-apoptotic protein, Bax and Caspase-3, and decreasing the expression ofBcl-2. In summary, TMP could inhibit the proliferation of PC-3, andpromote its apoptosis. Consequently TMP is a safe anticancer Chinesetraditional medicine. |
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