| Objective:The present study aims to investigate the effects of Petasin on the proliferation,apoptosis,invasion and migration of colon cancer cells and the growth of transplanted tumors through the establishment of a model of cell lines for colon cancer in vitro and a model of nude mice xenografts model in vivo.The mechanism was also discussed by detecting PI3K-Akt-mTOR signaling pathway related factors,and a new experimental basis was provided for the clinical application of Petasin.It also laid a preliminary foundation for the development of Petasin into a new anti-tumor drug.Methods:In vitro,MTT assay was used to detect the effects of Petasin compared with control on the proliferation inhibition of human colon cancer cell lines,Caco-2 cells,LoVo cells,SW-620 and HT-29 cells.The apoptosis rate of cells was measured by flow cytometry with AnnecineV-FITC/PI staining.The cell cycle distribution was analyzed by flow cytometry with PI staining.The changes of nuclei were observed by Hoechst 33258 fluorescence staining.The cell migration was detected by scratch experiment.Mitochondrial membrane depolarization(MMP)was analyzed by JC-1dye staining.The invasive ability of cells was detected by transwell experiment.The expression levels of PI3K-Akt-mTOR signaling pathway related mRNA(Akt,mTOR,P70S6K,MMP-3,MMP-9,Caspase-3,Caspase-9,Bcl-2)were detected by real time PCR(RT-PCR).Further the changes of expression level in PI3K-Akt-mTOR signaling pathway related proteins were measured by Western blot.In vivo,the median lethal dose(LD50)of the complex was calculated by Bliss method through acute toxicity experiment in mice.A nude mouse transplantation tumor model was established by subcutaneous injection of colon cancer SW-620 cells to observe the effect of Petasin compared with blank control and positive control(5-FU)on the growth of transplantation tumors.H-E staining was adopted to detect the effect of Petasin on tumor cells in transplanted tumor tissues.TUNEL fluorescence staining was used to detect apoptosis of tumor cells in transplanted tumor tissue.Expression of Ki-67 and p53 in transplanted tumor tissue cells were detected by immunohistochemical staining method.Expression of PI3K-Akt-mTOR signaling pathway related proteins were measured by western blot.Results:In vitro,Petasin inhibited the proliferation of four human colon cancer cell lines,Caco-2 cells,LoVo cells,SW-620 and HT-29 cells.Compared with the control group,the inhibition rate of cell proliferation in Petasin treatment groups(1,5,25μmol/L)was significantly increased(p<0.05).And 25μmol/L Petasin showed the largest inhibitory effects against the SW-620 cells with the proliferation inhibition rates of(21.16±3.59)%(24h),(38.52±4.55)%(48h)and(47.15±7.65)%(72h),respectively.Compared with the control group,Petasin(25μmol/L)induced a significant increase in the apoptosis rate of SW-620 cells[(6.01±1.56)%vs(31.03±3.52)%,p<0.01],decreased the mitochondrial membrane potential[(3.22±0.49)%vs(0.25±0.09)%,p<0.05]and indicated changes in the morphology of cell nuclear[(2.31±0.13)%vs(26.13±5.62)%,p<0.05).There was no significant effect of Petasin on cell cycle of SW-620 cells.The control group had a strong ability to wound,with a 24 h wound ratio of(68.1±9.6)%,while Petasin group was only(21.1±2.3)%.It was suggested that the invasiveness of SW-620 cells was significantly inhibited by Petasin(p<0.01).The transwell experiment showed that the number of invasive cells was significantly reduced by Petasin(Control:268.01±35.51,Petasin:61.33±10.97)and the difference was statistically significant(p<0.01).The result of RT-PCR showed that Petasin significantly inhibited the level of mRNA of MMP-3(1.25±0.19 vs 0.71±0.14),MMP-9(1.17±0.12 vs 0.75±0.11)and Bcl-2(0.72±0.09 vs 0.43±0.08)(p<0.05,p<0.05,p<0.05),while induced increased expression of Casepase-3(0.98±0.13 vs 1.78±0.33)and Caspase-9(1.09±0.31 vs 1.95±0.36)(p<0.05,p<0.05).and there was no significant effect on the level of mRNA of Akt,mTOR and P70S6K.Western blot showed that the phosphorylation levels of Akt(1.01±0.16 vs 0.74±0.06),mTOR(0.71±0.12 vs 0.32±0.11),and P70S6K(1.23±0.21 vs 0.85±0.14)proteins in SW-620 cells were significantly inhibited(p<0.05,p<0.05,p<0.01),and there was no significant effect on the total Akt,mTOR and P70S6K.The expression levels of MMP-3(1.51±0.31 vs 0.82±0.11)and MMP-9(1.56±0.32 vs 0.94±0.15)proteins were significantly inhibited by Petasin(p<0.05,p<0.05),the expression of cleaved Caspase-3(0.41±0.09 vs 0.74±0.12)and cleaved Caspase-9(1.10±0.27 vs1.98±0.22)were increased(p<0.05,p<0.01)and the expression of apoptosis inhibition gene Bcl-2(2.75±0.47 vs 1.51±0.36)was decreased(p<0.01).In vivo,acute toxicity tests in mice showed that the LD50 of Petasin was 68.65mg/kg.Compared with the control group,the tumor volume was decreased by Petasin(10mg/kg)and 5-FU(20mg/kg)[21d:Control(488.90±48.60mm3),Petasin(289.22±22.59mm~3),5-FU(192.89±21.33mm~3),p<0.05,p<0.05;28d:Control(924.18±101.23mm~3),Petasin(577.67±75.13mm~3),5-FU(577.67±75.13mm~3),p<0.01,p<0.01].The H-E staining and TUNEL showed that Petasin inhibited the proliferation of SW-620 cell and significantly increased the apoptosis rate of SW-620 cells of xenografts in nude mice[Petasin:(35.72±0.73)%vs(3.63±0.73)%,p<0.01;5-FU:(49.67±2.70)%vs(3.63±0.73)%,p<0.01].Compared with the control group,Petasin significantly reduced the percentage of ki-67 positive cells[(63.50±10.05)%vs(43.75±4.51)%,p<0.05],increased percentage of p53 positive cells[(23.75±2.22)%vs(36.26±6.55)%,p<0.05].Western blot showed that the phosphorylation levels of Akt(0.35±0.04 vs 0.15±0.01),mTOR(0.84±0.16 vs 0.32±0.09),and P70S6K(0.63±0.17 vs 0.26±0.05)in xenografts in nude mice were significantly inhibited(p<0.05,p<0.05,p<0.05),and there was no significant effect on the total Akt,mTOR and P70S6K.The expression levels of MMP-3(0.46±0.12 vs 0.16±0.06),MMP-9(0.52±0.17 vs 0.25±0.04)and Bcl-2(0.91±0.24 vs 0.71±0.15)were significantly inhibited by Petasin(p<0.05,p<0.05,p<0.05),and the expression of cleaved Caspase-3(0.19±0.09 vs 0.59±0.16)and cleaved Caspase-9(0.32±0.13vs 0.98±0.32)were increased(p<0.05,p<0.01).Conclusion:Petasin has significant anti-cancer effect on colon cancer SW-620 cells both in vitro and in vivo.It can inhibit SW-620 cell proliferation,induce apoptosis and inhibit its invasion and metastasis.The underlying mechanism may be related to the modulation of PI3K-Akt-mTOR signaling pathway. |
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