| Picropodophyllin（PPP）is a cyclolignan alkaloid derived from Podophyllum peltatum and Sinopodophyllum Emodi.PPP can inhibit tumor cell proliferation,promote cell death and induce cell cycle arrest in vitro.Tumors regressed significantly after PPP treatment in a variety of xenograft tumor models.At the same time,the result of human phase I trial of non-small cell lung cancer also showed that PPP is well tolerated.These results indicate that PPP is a safe,low-toxic and highly effective anti-cancer active substance with multiple mechanisms of action.However,the key molecules and signal transduction pathways that mediate its anti-tumor activity have not been discovered so far.PLK1 is a highly conserved serine/threonine protein kinase,which plays an important role in cell cycle regulation.It can promote the maturation of the centrosome and the polarization of spindles,control the initiation of mitosis,promote chromosome alignment,and promote mitotic exit.The studies have found that PLK1 is highly expressed in many different types of tumors and its expression level is closely related to the clinical stage and poor prognosis of tumors,thus leading to be considered as an oncogene.However,a number of recent studies have shown that PLK1 plays a role in inhibiting tumor progression in breast and colon cancer induced by oncogenes such as K-Ras,Her2 or APCmin,suggenting that PLK1 might also be a tumor suppressor gene under certain conditions.Whether PPP exerts anti-tumor effects through PLK1 has not been reported yet.To this end,this thesis comprehensively explored the most significant genes and signal pathways affected by PPP through high-throughput RNA sequencing methods,and then found that PLK1 was significantly up-regulated after PPP stimulation,suggesting that it is related to the effect of PPP-mediated cell death of Sup-b15 cells.Subsequently,further studies were carried out from the following three aspects:1.To explore the effect of PLK1 kinase inhibitor BI6727 on PPP-induced cell death;2.To explore the effect of overexpression of PLK1 on tumor cell death;3.To explore the upstream and downstream molecular mechanisms of PPP-mediated cell death through PLK1.Objective:Using the tumor cell lines as the research model,this thesis explore the effect of PLK1 in the process of tumor cell death inducted by PPP and the underlying molecular mechanism in vitro,so as to lay a good theoretical and experimental foundation for the further development and application of PPP.Method:1.To explore the effect of picropodophyllin on the expression profile of the geome-wide transcriptome of Supb15 cells.By analyzing the transcriptome data of Supb15 before and after the picropodophyllin treatment,PLK1 is not only related to cell division and cell cycle regulation,but also closely related to cell death among the genes with significant changes.Subsequently,qPCR and Western blot were performed to verify the above sequencing results.2.The role of PLK1 in PPP-induced tumor cell death2.1 Flow cytometry was used to detect the effect of BI6727 on PPP-induced Sup-b15 cell death and cycle arrest.2.2 Western blot was used to detect the effect of PPP and BI6727 on the expression of PLK1,phosphorylated PLK1（Thr210）,cleaved PARP and N-DFNA5 protein in Sup-b15.To detect the effect of PPP on the expression of PLK1,p-PLK1,cleaved PARP,N-DFNA5 and GSDMD protein in tumor cell lines from different tissues and 293T.2.3 Caspase-Glo?chemiluminescence method was used to detect the effect of BI6727 and PPP on Caspase activity in Sup-b15 cells.2.4 Cyto Tox96?method detected the effects of PPP and BI6727 on the cytotoxicity of different histoma cell lines and 293T cells.3.Explore the effect of overexpression of PLK1 on different cells3.1 Lenti-Pac?HIV Lentivirus Packaging Kit was used to construct and package Lenti-CON-GFP and Lenti-PLK1-GFP lentiviruses for overexpression of PLK1 in U87-MG,U118-MG,SH-SY5Y and 293T.3.2 Flow cytometry was used to detect the apoptosis of U87-MG,U118-MG,SH-SY5Y and 293T after overexpression of PLK1.3.3 Western blot was used to detect the levels of PLK1 and cleaved PARP proteins in U87-MG,U118-MG,SH-SY5Y and 293T after overexpression of PLK1.4.PPP enhances the expression and activity of PLK1 to trigger tumor cell death related molecular mechanisms4.1 Western blot was used to anlyze the protein expression of PLK1,MCL1,Bcl-2,Bcl-xl,survivin,cleaved PARP,N-DFNA5 and N-GSDMD in Sup-b15 cells treated with DMSO,PPP,PTX,VCR and with or without BI6727（pretreatment for 2 h）for 24 h in Sup-b15 cells.Western blot was used to anlyze the protein expression of FOXM1,PLK1,p-PLK1,cleaved PARP and DFNA5 in Sup-b15 cells treated with DMSO,PPP and with or without FDI-6（pretreatment for 2 h）for 24 h.4.2 Cyto Tox 96（?）methodwas used to assess the LDH release in Sup-b15 cells treated with DMSO,PPP,PPT,ETO,PTX,VCR and with or without BI6727（pretreatment for 2 h）for 48h.To detect the LDH release in OVCA433,Heyand 293T cells treated with DMSO,PPP and with or without FDI-6（pretreatment for 2h）for 48 h.Results1.The effect of PPP on the whole genome transcript expression profile of Sup-b15The results of geome-wide transcriptome analysis show that the genes with significant changes mainly affect the four signal pathways,and the genes involved include:CDC20/CCNB1/PLK1/CCNB2/PTTG1/CCNE2/CCNE1.The results of qPCR analysis showed that they were completely consistent with the sequencing results.Western Blot results showed that,except for CDC20,the protein expression levels of other genes were consistent with the sequencing results.2.The role of PLK1 in PPP-induced tumor cell death2.1 The results of flow cytometry showed that:percentage of cell death was increased from（13.9±1.3）%to（73.3±0.5）%after 1μmol/L PPP treatmen for 48 hours in Sup-b15 cells,indicating that PPP induces significant cell death.The percentage of cell death of PPP treatment after 2h pretreatment with 1μmol/L BI6727 was derease to（17.81±4.3）%,indicating that BI6727 completely inhibited cell death induced by PPP.1μmol/L BI6727 and 1μmol/L PPP treatment alone resulted in significant G2/M cell cycle arrest,however,inhibition of PLK1 had no effect on the cell cycle progression induced by PPP,suggesting that PLK1 is not involted in PPP-mediated G2/M arrest.2.2 Western blot results showed that PLK1,p-PLK1,cleaved PARP and N-DFNA5 protein expression levels were significantly increased after1μmol/L PPP treatment of Sup-b15 cells for 24 hours;BI6727 can reduce the above protein levels to be equivalent to the DMSO group;except for SH-In addition to SH-SY5Y,A549,He La,U87-MG,Lo Vo and Jurkat cells were treated with PPP and the protein expression levels of PLK1,p-PLK1,cleaved Caspase3,GSDMD,cleaved PARP and N-DFNA5 all increased to varying degrees,indicating that PPP activates PLK1 kinase Activity causes cell apoptosis or pyrolysis is universal.2.3 The results of Caspase-Glo（?）chemiluminescence method showed that:the activity of Caspase 3/7,Caspase 2 and Caspase 4 was increased after 1μmol/L PPP treatmen for 24 hours in Sup-b15 cells.The activity of Caspase3/7,Caspase2 and Caspase4 after 2h pretreatment with 1μmol/L BI6727 was derease to 4095±742.4 and 2758±475.5 and 1946±318.1μmol/L BI6727 alone or in combination with PPP has no significant effect on cell Caspase1 activity.2.4 The results of Cyto Tox 96?methodwas showed that:percentage of cell death was increased after 1μmol/L PPP treatmen for 24,48 or 72hours in A549,SH-SY5Y and Lo Vo cells.The percentage of cell death was increased after 1μmol/L PPP treatmen for 24 or 48 hours in U87-MG and He La cells.That indicating that PPP induces significant cell death.The percentage of cell death of PPP treatment in A549,SH-SY5Y,He La,U87-MG and Lo Vo after 2h pretreatment with 1μmol/L BI6727 was derease to（19.36±9.1）%and（56.32±20.0）%and（22.19±0.3）%and（23.8±3.2）%and（22.57±10.7）%,indicating that BI6727 completely inhibited cell death induced by PPP.1μmol/L BI6727 and PPP alone or in combination had no significant effect on LDH release in 293T.3.The effect of overexpression of PLK1 on tumor cell death3.1 Lenti-CON-GFP and lenti-PLK1-GFP infection rates were 64%and 90%in U87-MG cells;the infection rates were 37.5%and 6.2%in U118-MG cells;the infection rates were 7.2%and 8.3%in SH-SY5Y cells;the infection rate is 39%and 10.1%in 293T cells;green fluorescence can be observed under a fluorescence microscope after the above cells are infected with Lenti-CON-GFP and lenti-PLK1-GFP.3.2 The results of flow cytometry showed that:percentage of GFP positive cell death was（12.38±3.3）%vs（60.98±10.7）%after Lenti-CON-GFP or lenti-PLK1-GFP treatmen in U87-MG cells.The percentage of GFP positive cell death was（7.143±0.2）%vs（37.6±4.1）%after infection with the two lentiviruses in U118-MG;the percentage of GFP positive cell death was（3.347±0.8）%vs（21.47±5.3）%after infection with the two lentiviruses in SH-SY5Y,indicating that overexpression of PLK1 on tumor cell completely inhibited cell death.In 293T cells,there was no significant difference in the apoptotic rate of the GFP-positive cell population between the Lenti-CON-GFP group and the lenti-PLK1-GFP group.3.3 The results of Western blot showed that the expression of PLK1protein and cleaved PARP protein was increased in SH-SY5Y and U87-MG cells,indicating that overexpression of PLK1 can trigger cell death.4.Explore the upstream and downstream molecular mechanisms of PPP inducing cell death through PLK14.1 Western blot results showed that BI6727 inhibited the increase of PLK1,Bcl-2,p-Bcl-2,cleaved PARP and N-DFNA5 protein expression induced by PPP,PTX and VCR in Sup-b15 cells;FDI-6 inhibited the increase of PLK1,cleaved PARP and N-DFNA5 protein expression induced by PPP in Sup-b15 cells.4.2 The results of Cyto Tox 96（?）methodwas showed that:the rate of cell death in Supb15 cells was increased after 1μmol/L PPP,0.25μmol/L PPT,0.2μmol/L PTX,0.05μmol/L VCR and 10μmol/L ETO treatmen for 48 hours,indicating that PPP,PPT,PTX,VCR and ETO induces significant cell death.Pretreatment with 1μmol/L BI6727 for 2 h can inhibit the increase of LDH release rate caused by PPP,PTX and VCR treatment.There was no significant difference in the LDH release rate between PPT and ETO before and after BI6727 pretreatment.FOXM1inhibitor FDI-6 can inhibit the increase of LDH release caused by PPP in OVCA433 and Hey,and this phenomenon does not occur in 293T.Conclusion:PPP selectively induces pyrotosis and/or apoptosis in tumor cells by modulating FOXM1/PLK1/MCL1 axis. |
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