| Background: Prostate cancer (PCa) is one of the most malignant tumour in menworldwide. Although PCa can be treated with surgery and radiation, there remains ahighly local recurrence and/or metastasis of the tumour. Therefore, development ofnew treatment strategies is urgently needed. As the development of medicines andmolecular biology, combined gene therapy has attracted great attention.A balance between oncogenes and tumor-suppressing genes largely determinesthe production and development of PCa. P53is a well-known tumor suppressor thatplays a master role in the prevention of tumors by regulating cell cycle, apoptosis,DNA repair, antioxidant defense and even metabolic homeostasis. In about50%human tumors, P53mutations have been occured, and more than2000kinds of P53mutation types have identified in all tumors. Loss of P53expression or p53genemutations are common in a number of tumors including PCa, and are also associatedwith increased resistance to chemo-and radiotherapy. Therefore, strategies toreactivate defective P53function will be an ideal therapy. P53inactivation can becaused by a few different mechanisms. For instance,(1) P53can be inactivated isthrough up-regulation of Murine Double Minute2(MDM2). MDM2is induced byP53, and P53-MDM2will form a negative feedback to regulate each other. MDM2functions both as an E3ubiquitin ligase to recognize the N-terminal trans-activationdomain of the P53for degradation by the proteasome and as an inhibitor of p53transcriptional activation. Therefore, disruption of the P53-MDM2association holdsthe promise as a mechanism to reactivate the P53tumor-suppressing pathway.(2) Azinc (Zn) deficiency may also affect P53function. Zinc is the second most abundanttransition metal in the human body. Nearly half of eukaryotic transcription factorsbind zinc and in most of these instances the metal is used to maintain structure. P53isa transcription factor that contains a Zn ion near its DNA binding interface forsite-specific DNA binding and proper transcriptional function. Treating cells with theZn chelator TPEN causes P53to accumulate in the mis-folded conformation, and lose transcription activity. In addition, zinc is one of the essential trace elements insynthesis of prostatic fluid. Zinc content in normal adult prostate fluid is upto720μg/ml, and other organizations accounted for only80μg/ml. Patients with PCa oftenhave low levels of systemic Zn, and importantly prostate Zn levels are decreased inthe developing and progressive prostate cancers. Therefore, Zn deficiency may be acausative of prostate cancer due to inactivation of p53and also a reason for prostatecancer resistant to radio and chemotherapy. These results suggest the potential use ofZn to inhibit tumor growth by reactivating p53normal function.In summary, On the basis of successful construction of Pmp53[a plasmidcontaining both mdm2small interfering RNA (Si-mdm2) and the wild-type p53gene].We have firstly proposed a combined treatment of Pmp53with zinc. The aim, On theone hand, is to restore defective P53activity and function in prostate cancer. On theother hand, is to improve prostate cancer serum and tissue low levels of zinc status.Objective:The co-expression plasmid pcDNA3.1-U6si-mdm2-p53was constructedusing recombinant DNA technology. To evaluate the antitumor effect of combinedtreatment of Pmp53with zinc and explore it’s antitumor underlying mechanism invitro and in vivo.Method: Based on our previous laboratory work, using the gene sequence of P53andMDM2and the principles design of siRNA, we constructed the Pmp53plasmid. ThemRNA and protein expression levels of MDM2, P53and related genes were detectedby RT-PCR and Western blot analysis. Cell apoptosis was detected by FlowCytometry (FCM) analysis.In vitro, we chose PC-3(p53null) and DU145(mutant p53) cells as researchobjects. The cells were divided into seven groups: the control group, Zinc group,TPEN group, Pmp53group, Pmp53+Zn group, Pmp53+TPEN group andPmp53+Zn+TPEN group. The cell proliferation of combined treatment of Pmp53withzinc to cells was evaluated by MTT assay. Cell cycle was detected by PI assay. Cellapoptosis was detected by FCM and TUNEL assay. Mitochondrial membranepotential was monitored by Rh123. RT-PCR, qPCR and Western blot analysis todetect the expression of P53related genes and proteins changes were performed. P53proteins conformation was detected by Immunoprecipitation. P53transcriptionactivity was detected by Luciferase reporter activity. The capacity of P53protein tobind the promoter of p21and bax was detected by Chromatin immunoprecipitation. To study the effects of combined treatment of Pmp53with zinc in vivo, wedeveloped a PC-3xenograft model. Pmp53plasmid was delivered by AttenuatedSalmonella Typhi Ty21a, with zinc or TPEN supplemented by gavage, was to observeits impact on tumor growth. Tumor-bearing mice were divided randomly into fourgroups: the control group, Pmp53+TPEN group, Pmp53group and Pmp53+Zn group.The purpose of grouping was to build a low, normal and high level of zinc status. ThemRNA and protein expression levels of P53and related genes were detected byQRT-PCR and Western Blot analysis. P53proteins conformation was detected byImmunoprecipitation. Tumor cell apoptosis was detected by FCM and TUNELanalysis. The morphology of tumor tissue was assessed by using H&E staining andimmunohistochemical staining for PCNA protein expression.Result: the co-expression plasmid Pmp53was constructed successfully and verifiedby restriction endonuclease digestion. The studies using plasmid-transfected cellsshowed that Pmp53plasmid reduced mdm2mRNA and protein expression andenhanced P53protein expression.The combined treatment of Pmp53with zinc in vitro significantly increasedPAB1620reactive phenotype and restored P53conformation compared with othergroups. Pmp53with Zn led to to a G1-phase cell cycle arrest, and may be related toup-regulation P21expression and down-regulation CDK4, CDK6and CyclinD1expression. The apoptosis was greatly induced by Pmp53with zinc, and may beassociated with up-regulation of P53, Bax, cleaved-Caspase8, Caspase9and Caspase3expression and down-regulation of Bcl-2, PCNA, MMP2and MMP9expression.Nude mice implanted with PC-3cells and treated with the combined treatment ofPmp53with zinc showed significantly reduced average tumor weights and volumes,and increased apoptosis compared with other groups. Wild-type P53conformation andfunction were also restored, consistent with the result in vitro. H&E stainingdemonstrated significantly increase in necrotic area, and IHC showed significantlyincreased expression of P53and decreased expression of PCNA and MDM2.Conclusion: Co-expression plasmid Pmp53was successfully constructed. In vitro andin vivo the combined treatment of Pmp53with zinc plays an significantly inhibitoryeffect on PCa. The combined therapy is better than single one. The mechanism on theone hand may be associated with P53function. Pmp53plasmid through reduced theexpression of oncogenes MDM2and increased the expression of tumor suppressor P53, play the inhibitory effect on PCa. On the other hand may be associated with P53conformation. Zinc supplementation further retained wild-type P53conformation andenhanced its transcriptional regulation of p21and bax gene expression, leading to thedecreased proliferation and increased apoptosis. |
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