Studies Of Prostate Cancer Chemotherapy:Dissecting Molecular Mechanisms Of Anacardic Acid-induced Cell Apoptosis And Identifying Similar Drugs Modifying P53Level In Prostate Caner Cells

ABSTRACT:Prostate cancer is a common malignant tumor and prostate cancer has the highest morbidity among males in the Europe and America. In our country the incidence rate of prostate cancer is also increasing with the improvement in domestic living standards, increased intake of animal foods. In Shanghai, Prostate cancer has become the most common tumor of the urinary system. The main treatment of patients with metastatic prostate cancer is endocrine therapy, also named androgen depletion therapy (ADT). Androgen depletion therapy is effective for80-90%of patients with metastatic prostate cancer. But after the average duration time of22months, there will be a progression of the disease, the hormone-dependent prostate cancer will gradually progress to androgen-independent prostate cancer (AIPC) and at last to hormonal refractory prostate cancer (HRPC), Castrate-resistant prostate cancer(CRPC). Most research about prostate cancer focused on the effective drug treatment for advanced prostate cancer.We have studied a potential widely used drug named anacardic acid (AA), which has lethal effects on prostate cancer cell lines. We have found anacardic acid could lead to obvious cell proliferation inhibition and induce apoptosis on the LNCap and Du145cell lines and the cell proliferation inhibition was dose-and time-dependent with AAWe have verified the new mechanism of anti-tumor effect of AA in prostate cancer LNCap cells through silencing of p300or p53gene using RNA interference. First, AA can inhibit p300expression and AR expression by p300/AR signal path in LNCap cell lines.Second, AA can increase expression of p53through increasing phosphorylation of p53gene on ser15sites and increase p21expression by p53/p21signal path in LNCap cell lines. The important anti-tumor effect of AA. is to adjust the protein expression of p53.For conveniently screening a large number of Chinese traditional herbal medicine and small molecules to find drugs with similar anti-tumor effect; we have finally constructed a p53-fused dual luciferase reporter plasmid, p53FL/IRES/RL and tested its function. The plasmid can find out target drugs with simplified and shortened experiments in a high-throughput screen fashion and has a broad prospect of application.The innovations of this study are as follows:1) We have firstly reported that AA can exert anti-tumor role through p53/p21and p300/AR signal path in LNCap cells;2) We have firstly figured out that AA can regulate multiple prostate cancer related oncogenes in prostate cancer cells including c-myc,VEGFA and Cox2;3) We have constructed a new p53-fused dual Luciferase reporter plasmid(p53FL/IRES/RL) and verified its biological function. There are26figures,3tables, and81references. Objective:To verify the anti-tumor role of anacardic acid on prostate cancer LNCap cells on aspects of cell proliferation, cell cycle and apoptosis, getting the optimal dose and action time of AA. Methods:We have detected the inhibition of cell proliferation after different dosages of AA acting on prostate cancer LNCap cells using MTT assay. The apoptosis and cell cycle distribution of LNCap cell lines were examined by flow cytometry after dealing with AA. Results:According to the MTT assay results, AA inhibits cell proliferation of LNCap cells. The optimal action time of AA is from24to48hour, optimal dose of AA is125umol/L. AA induces apoptosis of prostate cancer LNCap cells (p<0.05) and blocks cell cycle arrest in Gl/S phase. Conclusion:Anacardic acid has obvious lethal effect on prostate cancer LNCap cells. AA inhibits cell proliferation, induces apoptosis and blocks the cell cycle. In our study, the optimal action time of AA is24hours and the optimal dose125umol/L. Objective:To investigate the possible anti-tumor mechanism and signal path of anacardic acid on prostate cancer LNCap cells. Methods:The mRNA and protein expression of p300, p53, p53-ser15, p21, AR were detected by RT-PCR and Western Blot after dealing with different dosage of anacardic acid in LNCap cells. The p300-siRNA was transfected into prostate cancer LNCap cells silencing p300gene before dealing with AA The cell proliferation and apoptosis of LNCap cells were examined by MTT and flow cytometry after AA treatment. The mRNA and protein expression of p300, AR, p53in LNCap cells were detected by real-time RT-PCR and Western blot with relative quantity count. The p53-siRNA was transfected into LNCap cells silencing p53gene before dealing with AA The cell proliferation and apoptosis of LNCap cells were examined by MTT and flow cytometry after AA treatment. The mRNA and protein expression of p53, p21, puma in LNCap cells were detected by real-time RT-PCR and Western-blot. Results:The protein expression of p53, p53-ser15, p21have increased and the expression of p300, AR decreased in LNCap cells after AA treatment (P<0.05), which correlated with the dose of AA. With the transfection of p300-siRNA into LNCap cells before treatment of AA, we have found cell survival rate was (35.70±4.23)%and cell apoptosis rate (24.1±0.87)%in AA group, compared with cell survival rate (55.30±4.69)%and cell apoptosis rate (15.5±0.83)%in AA+p300-siRNA group. The anti-tumor role of AA in LNCap cells have lowered after transfection of p300-siRNA (P<0.05). On the other hand, contrasted with AA group, the mRNA and protein expression of AR have decreased in AA+p300-siRNA group (P<0.05). With the transfection of p53-siRNA into LNCap cells before treatment of AA, We have found cell survival rate was (34.85±4.41)%and cell apoptosis rate (22.2±0.91)%in AA group, compared with cell survival rate (52.17±4.50)%and cell apoptosis rate (12.4±0.72)%in AA-p53-siRNA group. The role of AA in LNCap cells have lowered after transfection of p53-siRNA (P<0.05). Contrasted with AA group, the mRNA and protein expression of p21decreased in AA+p53-siRNA group (P<0.05). Conclusions:Anacardic acid can exert anti-tumor role through increasing protein expression of p53, p53-ser15, p21and decreasing protein expression of p300, AR in LNCap cell lines. AA can down-regulate p300and down-regulate expression of AR through p300/AR signal path in LNCap cells. AA can upregulate expression of p21through p53/p21signal path in LNCap cells. The activation of p53by AA mainly depends on phosphoration of p53on ser15sites. The acetylation of p53by AA through p300/p53path is not obvious. Objective:To investigate the effect of AA in different prostate cell lines DU145. To study the change of some prostate cancer related oncogenes in LNCap and DU145cells after dealing with AA. Methods:After treatment with AA, we have studies the lethal effect of AA in Du145cells using MTT assay and flow cytometry test. The mRNA and protein expression of prostate cancer related oncogenes including c-myc, VEGF-A and COX2were detected by RT-PCR and Western-Blot in both LNCap and DU145cells after dealing with AA. Results:AA inhibits cell proliferation and induces apoptosis of Du145cells. The mRNA transcription and protein expression of c-myc, VEGFA and COX2have decreased in both LNCap and Du145cells (P<0.05). Conclusion:AA also has lethal effect on Dul45cells and can regulate multiple prostate cancer related oncogenes in different prostate cancer cell lines including LNCap and Dul45cells. Objective:To screen similar anti-tumor drugs like AA conveniently. We constructed a p53-fused dual Luciferase reporter plasmid and tested its function in order to identify drugs modulating p53protein level in a high-throughput screen fashion. Methods:Addition of a restriction endonuclease site to and removal of the stop codon of each terminus of a wild-type full-length p53open reading frame (ORF) were made by PCR. Removal of the start codon and addition of a restriction endonuclease site to each terminus of the Firefly Luciferase ORF were made by PCR. These two modified ORFs were inserted upstream of the IRES-induced Renilla Luciferase ORF in a CMV-derived vector. Thereafter, we demonstrated that the p53FL fusion protein is degraded by MDM2oncoprotein and majorly locates inside nucleus. Results:We successfully construct a p53-fused dual Luciferase reporter plasmid. We verify the plasmid mimics the wild-type p53protein satisfactorily and it is capable of being applied to high-throughput screen. Conclusion:The p53-fused dual Luciferase reporter plasmid (p53FL/IRES/RL) is capable of identifying similar drugs modulating p53protein level in a convenient high-throughput screen.