Periplocymarin Induces Colon Cancer Cells Apoptosis Via Impairing PI3K/Akt Pathway

Part One The effects of periplocymarin on proliferation,apoptosis andcell cycle of colon cancer cellsObjective:Periplocymarin(PPM),a cardiac glycoside isolated from Periploca sepium,is a latent anticancer compound.In this study,the effects of PPM on the proliferation,apoptosis,and cell cycle of colon cancer cells were investigated.Methods:1.Effect of different concentrations of PPM on cell viability of colon cancer cells(HCT 116、SW480、RKO、HT-29)was determined by the CCK-8assay.2.Flow cytometry(FCM)and TUNEL fluorescence staining were used to detect the effects of different concentrations of PPM on the apoptosis ratios of HCT 116 and RKO cells.3.The effects of PPM on the expression of apoptosis-related proteins in HCT 116 and RKO cells were detected by Western blot.4.FCM was used to detect the effects of different concentrations of PPM on the cell cycle distribution of HCT 116 and RKO cells.5.The effects of PPM on the expression of cell cycle-related proteins in HCT 116 and RKO cells were detected by Western blot.Results:1.The results indicated that,PPM could inhibit the viability of colon cancer cell lines(HCT 116,SW480,RKO,HT-29)in a dose-dependent manner within the concentration of 12.5～100 ng/m L(P<0.05).2.PPM increased the proportion of Annexin V-FITC positive cells and TUNEL positive cells in a dose-dependent manner within the concentration of12.5～100 ng/m L(P<0.05),suggesting the effect of PPM on inducing cell apoptosis.3.The Western blot results showed that the expression of pro-apoptotic protein of Bax,cleaved caspase-9 and cleaved caspase-3 were increased(P<0.05),and the expression of anti-apoptotic protein including Bcl-2 and survivin were decreased in HCT 116 and RKO cells under PPM treatment(P<0.05).4.The FCM results showed that 12.5～50 ng/m L PPM treatment could significantly increase the proportion of cells in G0/G1 phase(P<0.05),while decrease the proportion of cells in S phase(P<0.05)with a dose-dependent manner in HCT 116 and RKO cells.5.Western blot results showed that,compared with controls,PPM treatment might cause significantly increased p21 protein expressoion(P<0.05)while decreased cyclin D1 protein expression(P<0.05)in HCT 116and RKO cells.Summary:PPM exerts the anti-colon cancer effect by inducing cell apoptosis and cell cycle arrest at G0/G1 Phase in vitro.Part Two The underlying mechanism of PPM effects on colon cancercellsObjective:To explore the underlying mechanism of the PPM effects on colon cancer cells by proteomics analysis.Methods:1.TMT quantitative proteomics analysis was taken after proteins were extracted from HCT 116 cells treated with 50 ng/ml PPM and untreated cells respectively.A fold-change<0.83 or>1.2 with a P<0.05 was selected as the cutoff criteria for the identification of differentially expressed proteins(DEPs).2.Bioinformation analysis was used to analyze the functions of DEPs.The gene ontology(GO)annotation of DEPs was performed by Blast2GO software.Pathway annotation was based on the online Kyoto Encyclopedia of Genes and Genomes(KEGG)to screen out the key signaling pathways that involved in PPM function.3.Western blot was used to detect the effects of PPM on the expression of IRS1,PI3K,Akt,p-PI3K and p-Akt in HCT 116 and RKO cells.Results:1.The total number of proteins identified by TMT quantitative proteomics technology was 6645,and the number of DEPs was 539,including286 up-regulated proteins and 253 down-regulated proteins.2.Bioinformatics was used to analyze the functions of DEPs.The most enriched GO terms were annotated as transmembrane receptor protein tyrosine kinase activity,signal transduction receptor activity,molecular transformer activity,and transmembrane receptor protein kinase activity in the molecular function category,low-density lipoprotein particle in the cellular compartment category,and animal organ morphogenesis in regard to the biological process category.KEGG pathway enrichment analysis was performed to check pathways associated with the DEPs.The results showed that 14 KEGG pathways were significantly enriched based on the number of proteins with P<0.05.Among them PI3K/Akt pathway associated with the largest number of DEPs,suggesting that the pathway may be involved in the effect of PPM on colon cancer cells.3.Compared with controls,PPM treatment could lead to significantly decreased expression of IRS1,p-PI3K and p-Akt(P<0.05)without altering total protein expression of PI3K and Akt in HCT 116 and RKO cells,indicating PPM may inhibit PI3K/Akt signaling pathway.Since this pathway is closely related to cell proliferation and apoptosis,it is speculated that PI3K/Akt pathway is involved in the effect of PPM on the proliferation inhibition and pro-apoptosis of colon cancer cells.Summary:The effect of PPM on colon cancer cells is related to the inhibition of PI3K/Akt signaling pathway activity.Part Three The study of the anti-cancer effect of PPM in vivoObjective:To investigate the effect of PPM on viability of colon cancer cells in vivo.The drug safety was also assessed to explore the clinical prospect of PPM in the treatment of colon cancer.Methods:1.BALB/c-nu mice were used as tumor growth assay.HCT 116 cells were subcutaneously injected into the right flank of each mouse.When the tumors’volume reached 100 mm~3,the mice were randomly divided into 4groups(n=6 per group).Control group(CON):mice were intraperitoneally injected with 0.9%physiological saline every two days.PPM group(PPM):mice were intraperitoneally injected with 3 mg/kg PPM every two days.Fluorouracil group(5-FU):mice were intraperitoneally injected with 10 mg/kg fluorouracil every two days.Fluorouracil group+PPM(5-FU+PPM):mice were intraperitoneally injected with a combination of PPM(3 mg/kg)and fluorouracil(10 mg/kg)every two days.21 days after treatment,mice were sacrificed by spinal dislocation.2.The volume of tumors was observed every three days,and the tumors were harvested and weighed at the end of study.3.Immunohistochemistry was used to detect the expression of Bax,cleaved caspase-3,IRS1,p-PI3K and p-Akt in tumor tissues of each group.4.Blood samples were collected from vena angularis and used for blood routine test,liver and renal function test.HE staining was performed to check the tissue morphological alterations.Results:1.The volume and weight of tumors were significantly lower in the 5-FU and PPM groups and more magnificent in 5-FU+PPM groups when compared with those in control group(P<0.05).PPM may perform similar tumor inhibiton effects with 5-FU(P>0.05).2.The expression of pro-apoptotic proteins of Bax and cleaved caspase-3were increased while the expression of IRS1,p-PI3K and p-Akt decreased significantly in PPM group,5-FU group and 5-FU+PPM group(P<0.05),indicating that PPM might induce apoptotsis and impaire PI3K/Akt pathway in vivo.3.PPM treatment led to no impairment on blood cells,liver and renal functions.5-FU treatment caused decreased number of leukocytes and increased alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels(P<0.05).Increased blood urea nitrogen level was found in5-FU+PPM treated animals(P<0.05),suggesting that 5-FU combined with PPM may cause renal damage and PPM may alleviate the damage of 5-FU on liver function and leukocytes.4.PPM or 5-FU+PPM treatment caused no obvious morphological changes in the heart,lung,liver,kidney and brain tissues of nude mice,indicating that PPM caused no apparent organ damage.Summary:PPM can induce apoptosis,inhibit the PI3K/Akt pathway in vivo,and perform anti-cancer effects with less toxic and side effects.Conclusion:1.PPM can inhibit the viability of colon cancer cells by inducing cell apoptosis and cell cycle arrest at G0/G1 Phase in vitro.2.Impaired PI3K/Akt pathway might be involved in PPM effect on cancer cells.3.PPM can inhibit the growth of tumor in vivo,with less damage to blood system,liver and kidney function.Combination of PPM with 5-FU can enhance anti-tumor effects.