Plumbagin Inhibit Proliferation, Invasion And Metastasis Of Renal Cell Carcinoma Through PI3K/AKT Signaling Pathway And MCM5

Renal cell carcinoma(RCC)is one of the most common urinary system cancer,accounting for about 2-3% of human malignant tumors.Due to the lack of typical symptoms and there are no routine tests used to screen early RCC,20-30% of renal cell carcinomas have metastasis at the time of diagnosis,so that the prognosis of RCC is extremely poor.Metastatic renal cell carcinoma is insensitive to current treatment methods(radiotherapy,chemotherapy,interferon immunotherapy),and most patients develop resistance to targeted therapy after 6-15 months,resulting in the 5-year survival rate less than 10%.Therefore,it is of great value to reveal new approaches for RCC treatment.Plumbagin is a quinonoid constituent derived from the roots of the medicinal plant Plumbago zeylanica.A variety of pharmacological activities of PLB,including anti-inflammatory,neuroprotective,anticancer,hypolipidemic,antiatherosclerotic,antibacterial,and antifungal effects,have been reported in vitro and in vivo models.Recently,Plumbagin showed potent anti-tumor activity in various tumor,including Prostate cancer,breast cancer,liver cancer and so on.It could inhibit the proliferation,invasion and metastasis of cancer.Plumbagin exhibits these anticancer effects via interaction with multiple targets and modulation of various molecular signaling pathways,including AMPK,CDK1/CDC2,cyclin B1,cyclin D1,NF-kappa B,p53,p21,PI3K/AKT/m TOR,Ras,STAT3/PLK1/AKT,and Wnt,resulting in induction of apoptosis and autophagy,cell cycle arrest,inhibition of invasion,and metastasis.Up to date,the effect of PLB on proliferation,invasion and metastasis of renal cell carcinoma and its mechanism are not clear.Microchromosome maintenance proteins(MCMs)are thought to function as regulatory components in the S-phase of the cell cycle,presenting weak helicase activity by binding to chromatin long before the initiation of DNA synthesis.MCM5 is located on the chromosome 22q13.1 and it is a member protein of the minichromosome maintenance(MCM)protein family,which is over expressed in gastric cancer,lung cancer,oral squamous cell carcinoma,skin malignant tumors,cervical cancer and bladder cancer.In addition,MCM5 is closely related to tumor size,stage,pathological grade and prognosis.In RCC,there is a significant association between MCM2 and tumour grade,angiogenic phenotype,overall survival,but not with patient age,patient sex,tumor size or stage.However,the influence of MCM5 protein expression on the progression and prognosis of RCCs has not been investigated.In this study,in order to further understand whether PLB has therapeutic effect on renal cell carcinoma and the role of MCM5 in the occurrence and development of renal cell carcinoma,we investigated:(1)The effect of PLB on proliferation,invasion and metastasis of renal cell carcinoma.(2)The influence of PLB on PI3K/AKT signaling pathway in renal cell carcinoma.(3)The role of MCM5 in the occurrence and development of renal cell carcinoma.(4)Whether MCM5 participates in the inhibition of PLB in renal cell carcinoma.Part 1 Plumbagin inhibits proliferation,invasion and metastasis of renal cell carcinoma through PI3K/AKT signaling pathwayObjective: To investigate the effect of Plumbagin on proliferation,invasion and metastasis of renal cell carcinoma,and to confirm the influence of Plumbagin on PI3K/AKT signaling pathway in renal cell carcinoma.Methods:(1)According to the concentration gradient of Plumbagin,the experiment was divided into five groups: 0,1,5,10 and 20 μmol/L.MTT and plate clone formation assay were used to detect the effect of Plumbagin on the proliferation of renal cancer cells,and flow cytometry was used to evaluate the effect of Plumbagin on the apoptosis of renal cancer cells.Western blot was used to examine the effects of Plumbagin on apoptosis-related proteins such as Bax,Bcl-2,Cleaved-Caspase-3 and so on.(2)According to the concentration gradient of Plumbagin,the experiment wasdivided into four groups: 0,1.25,2.5,5.0 μmol/L.Scratch test and Transwell were used to evaluate the effect of Plumbagin on invasion and metastasis of renal cell carcinoma.Tumor xenograft in nude mice was used to evaluated the influence of Plumbagin on the growth of tumour.Western blot was used to examine the effects of Plumbagin on invasion and metastasis-related proteins such as TGF-beta 1,Smad3,E-cadherin,N-cadherin,MMP2 and so on.(3)The experiment was divided into three groups:Control,Plumbagin,Plumbagin+IGF-1.MTT and plate clone formation assay were used to detect the effect of Plumbagin on the proliferation of renal cancer cells,Western blot was used to examine the effects of Plumbagin on PI3K/AKT-related proteins such as PI3 K,p-PI3 K,AKT,p-AKT and so on.Results:(1)Plumbagin can inhibit proliferation and induce apoptosis of renal cell carcinoma cells in a concentration-dependent manner.Plumbagin can also promote the expression of Bax,cleaved-Caspase-3,cleaved-Caspase-9 and inhibit the expression of Bcl-2,Cyclin D1.(2)Plumbagin can inhibit the invasion and metastasis of renal cell carcinoma in a concentration-dependent manner.Exposed to Plumbagin,the expressions of TGF-beta 1,Smad3,Vimentin,N-cadherin and MMP2 were down-regulated,which promoted the expression of E-cadherin.(3)Plumbagin can significantly inhibit the proliferation of renal cell carcinoma cells,and Plumbagin + IGF-1 group has less inhibition on the proliferation of renal cell carcinoma cells than Plumbagin group.Plumbagin can significantly inhibit the expression of PI3 K,p-PI3 K,AKT,p-AKT,and IGF-1 can partly reverse the inhibition of Plumbagin.Conclusions: Plumbagin can inhibit the proliferation,invasion and metastasis of renal cell carcinoma through PI3K/AKT signaling pathway.Part 2 MCM5 promotes the proliferation of renal cell carcinoma and is closely related to its progression and prognosisObjective: To explore the differential expression of MCM5 in renal cell carcinoma and its influence on the progression and prognosis of renal cell carcinoma.Methods:(1)The differential expression of MCMs in renal cell carcinoma was analyzed by Oncomine online data analysis.Furthermore,Linked Omics and Onco Lnc online data were used to analyze the correlation between MCM5 and clinicopathological characteristics and prognosis of renal cell carcinoma.(2)The expression of MCM5 in renal cell carcinoma was detected by immunohistochemistry and Western blot,and the correlation between MCM5 and clinicopathological characteristics of renal cell carcinoma was further analyzed.(3)The expression of MCM5 was up-regulated or down-regulated by virus transfection.MTT and plate clone formation assay were used to detect the proliferation of RCC cells.To observe the effect of MCM5 on the growth of renal cell carcinoma through Tumor xenograft in nude mice.Results:(1)Oncomine was used to analyze the differential expression of MCMs in renal cell carcinoma.The results showed that there was no significant difference in MCM3 and MCM4 expression between renal cell carcinoma and adjacent tissues.MCM2,MCM5,MCM6 and MCM7 were significantly higher in renal cell carcinoma tissues,and MCM5 had the highest fold change.Immunohistochemistry and Western blot showed that MCM5 was significantly up-regulated in renal cell carcinoma.(2)Linked Omics and Onco Lnc online data analysis showed that MCM5 was closely related to the clinicopathological characteristics and prognosis of renal cell carcinoma.Immunohistochemical analysis further confirmed the correlation between MCM5 and clinical characteristics of renal cell carcinoma.(3)The expression of MCM5 was up-regulated or down-regulated by virus transfection.Furthermore,MTT and plate clone formation assay showed that MCM5 could significantly inhibit the proliferation of RCC cells.Tumor xenograft in nude mice showed that down-regulation of MCM5 could inhibit the growth of renal cell carcinoma.Conclusions: The over expression of MCM5 in renal cell carcinoma can promote the proliferation of renal cell carcinoma cells and play an important role in the progress and prognosis of renal cell carcinoma.Part 3 Plumbagin inhibits the development and progression of renal cell carcinoma through MCM5Ojective: To investigate whether MCM5 participates in the inhibition of Plumbagin on renal cell carcinoma.Methods:(1)The effect of Plumbagin on MCM5 in renal cell carcinoma cells was detected by Western blot.(2)The experiment was divided into three groups: control group,Plumbagin,Plumbagin + MCM5 overexpression group.MTT and plate cloning were used to evaluate the proliferation of renal cell carcinoma cells in each group.Results:(1)Plumbagin significantly inhibited the expression of MCM5 protein in renal cell carcinoma cells.(2)Plumbagin can significantly inhibit the proliferation of RCC cells.Compared with Plumbagin group,Plumbagin + MCM5 share a stronger proliferation ability of RCC cells.Conclusions: Plumbagin can inhibit the proliferation of RCC cells through MCM5.