| Polyphyllin are extracted from the dry roots of Lilium or Aesculus in Yunnan,which can effectively inhibit the proliferation of tumor cells.Existing studies have found that the anti-tumor effect of polyphyllin mainly relies on apoptosis.Recently,with the deepening of research,it has been found that polyphyllin can cause autophagy of many kinds of tumor cells and can cause autophagic death of tumor cells.In this study,SW480 cells were used as a model to investigate the role of autophagy in death of SW480 colon cancer cells induced by polyphyllin Ⅰ(PPI)and related pathways.To understand the regulatory mechanism of polyphyllin Ⅰ induced autophagy in colon cancer cells,so as to provide sufficient and reliable theoretical support for the study of polyphyllin Ⅰ in the treatment of colon cancer.The main findings are as follows: 1.polyphyllin Ⅰ can inhibit the proliferation activity of SW480 cells.In the experiment,SW480 cells were cultured in L-15 medium containing 10% fetal bovine serum.When the cells were grown to about 80% of the culture,they were given different concentrations of polyphyllin Ⅰ(0μmol/L,1μmol/L,2μmol/L,3μmol/L,4μmol/L,5μmol/L)treatment for 12 h,24h,MTT assay was used to detect the change of cell viability,and the change of cell morphology and quantity after treatment with different concentrations of polyphyllin Ⅰ for 24 h was observed by inverted microscope.The results showed that the effect of polyphyllin Ⅰ on the proliferation of human colon cancer SW480 cells was significantly inhibited in a time-and-concentration-dependent manner.At the same time,under the microscope,it was found that there were obvious changes in the morphology and density of cells stimulated by polyphyllin Ⅰ,and it was confirmed that polyphyllin Ⅰ can effectively promote the death of SW480 cells.2.The effect of polyphyllin Ⅰ on autophagy in SW480 cells.The effect and pathway of autophagy on the occurrence of autophagy in colon cancer SW480 cells is still unknown.Western blot analysis and acridine orange(AO)staining method was used to analyze the effect of polyphyllin Ⅰ on the expression of autophagy marker protein LC3-II/I and intracellular acidic autophagic vacuoles.The results showed that polyphyllin Ⅰ can indeed induce the accumulation of orange-red acidic autophagic vacuoles in SW480 cells,and the expression of LC3-II/I,an autophagy marker protein,was significantly increased,and autophagy was induced in SW480 cells.To further verify the effect of polyphyllin Ⅰ on autophagy induction in SW480 cells,pretreatment of cells with autophagy inhibitor CQ for 1 h,determination of cell proliferation activity and expression of autophagy marker protein LC3-II/-I by different concentrations of polyphyllin Ⅰ influences.The results showed that the autophagy induced by polyphyllin Ⅰ in SW480 cells played different roles with the change of drug concentration and time.When 12 hours,PPI+CQ(1-4 μmol/L),the cell viability of the polyphyllin Ⅰ alone group was significantly reduced,indicating that the autophagy of SW480 cells induced by polyphyllin Ⅰ is a protective autophagy at this time.After autophagy was blocked by the autophagy inhibitor CQ,it was induced by polyphyllin Ⅰ.Cell death increased significantly and anti-tumor effects increased.However,when treated for 24 h(including 5 μmol/L PPI at 12 h),the cell viability measured by MTT was significantly higher than that in the polyphyllin Ⅰ alone group,indicating that the autophagy of SW480 cells induced by polyphyllin Ⅰ was increased.Promote the role of cell death,block the anti-tumor effect of polyphyllin Ⅰ after autophagy is weakened.3.Polyphyllin I induced autophagy of ROS pathway and AKT/mTOR pathway in SW480 cells.To further investigate the mechanism of autophagy induced by polyphyllin Ⅰ in SW480 cells,the change of intracellular ROS(12 h,24 h)was measured after DCFH-DA binding assay was applied to IC50 concentration of polyphyllin Ⅰ and pretreatment with NAC for 1 h.Western blot was used to analyze the influence of the expression levels of p-mTOR,m-TOR,p53,p-AKT,AKT,p-JNK and JNK in the polyphyllin Ⅰ autophagy pathways.The changes of LC3-II/I expression and cell proliferation activity in cells treated with NAC and SP600125 were compared.The results showed that polyphyllin Ⅰ induced a rise in ROS in SW480 cells.Compared with the control group,IC50 concentration of PPI induced intracellular ROS increased by 42% and 81% at 12 h and 24 h,respectively,the difference was extremely significant(P<0.01).The level of LC3-II/-I protein was decreased in the NAC group;at 12 hours,the survival rate of the pre-addition NAC group was not significantly different from that of the group alone;however,at 24 hours,the survival rate of the NAC group was significantly increased.High(P<0.01).The results showed that polyphyllin Ⅰ induced autophagy in ROS pathway in SW480 cells and promoted cell death.The polyphyllin Ⅰ induced a significant decrease in the expression of p-AKT and p-mTOR,and the expression of p-JNK was significantly increased.The expression of m-TOR,AKT,p53,and JNK did not change significantly.There was no significant difference in the level of LC3-II/-I between the JNK inhibitors treated with SP600125 and the group treated with SP600125.The survival rate of cells treated with JNK inhibitor was significantly higher than that treated with PPI alone at 12 h and 24 h after combined treatment.High(P<0.05).The increase of JNK protein expression induced by polyphyllin-I is not related to autophagy,but may be related to non-autophagic cell death.In summary,the experimental results of this experiment show that polyphyllin Ⅰ has a strong growth inhibition on SW480 cells,and for the first time found that polyphyllin Ⅰ can induce autophagy in SW480 cells,and its induced autophagy at different concentrations and Time has played a different role,initially discussed the regulation mechanism of polyphyllin Ⅰ-induced autophagy in SW480 cells. |
