Xanthatin Suppresses Glioma Growth Through Autophagy Regulation Via PI3K/AKT/mT0R Pathway

Gliomas,as one of the most common type of intracranial tumors originating from neuroectoderm,has the characteristics of high incidence,high mortality,high recurrence rate and low cure rate.As the most aggressive primary brain tumor,glioma is characterized with extremely poor prognosis.It is very difficult to resect the tumors thoroughly by general surgical methods,and the treatment methods of post-operative radiotherapy and chemotherapy are relatively common.Autophagy is the process of transporting damaged proteins and organelles into lysosomes for digestion and degradation.Under normal physiological conditions,cell autophagy is beneficial to cell homeostasis.When stimulated,cell autophagy effectively reduces the accumulation of proteins and organelles and inhibits cell carcinogenesis.And when tumors are formed,cell autophagy provides nutrients for cancer cells,reduces the sensitivity of chemotherapeutic drugs and promotes tumor growth.Therefore,in the process of tumorigenesis and development,autophagy plays the role of "double-edged sword".Xanthatin(Xn),a sesquiterpene lactone extracted from xanthium sibiricum,has many pharmacological activities,including anti-inflammatory,anti-fungal,inhibition of cell proliferation and inhibition of angiogenesis.Xanthatin has been shown to have significant anti-tumor effects in many tumor cell lines,such as breast cancer and lung cancer cells.At present,the role of xanthatin in glioma is only seen in a paper published by our group.It is proved that xanthatin inhibits the growth of glioma by imaging method.The molecular mechanism of its anti-cancer effect needs further study.Objective: To observe the effect of Xn on the proliferation and apoptosis of glioma cells,and to explore whether Xn plays a role in regulating autophagy through PI3K/AKT/m TOR pathway.Methods: Human glioma cell line U251 and rat glioma cell line C6 were treated with Xn at different concentrations or at different times.Sulfonyl rhodamine B(SRB)assay was used to observe the effect of Xn on cell proliferation.Plate cloning assay was used to detect the effect of Xn on cell cloning.Tunel staining was used to observe cell apoptosis.Western blot(WB)was used to detect the expression of apoptotic-related protein and analyze the effect of Xn on the proliferation and apoptosis of glioma cells.On this basis,WB and immunofluorescence staining were used to detect the expression and intracellular localization of autophagy-related proteins P62,LC3 and Beclin1,and to evaluate the effect of Xn on autophagosome formation and autophagic flux.WB was used to detect the activation of regulatory proteins related to the upstream pathway of autophagy,such as phosphorylated AKT(p-AKT),phosphorylated m TOR(p-m TOR),phosphorylated ULK1(p-ULK1),and to evaluate the effect of Xn on the upstream regulatory pathway of autophagy.WB was also used to detect the expression of P62,LC3,Beclin1 and their upstream signaling pathway related proteins p-m TOR,p-ULK1 in the xenograft tumors of C6 cells in nude mice.With the cocultivation of autophagy inducer Torin1 or Rapa and Xn,WB and Tunel staining evaluate the effect of autophagy up-regulation on Xn inhibiting cell proliferation and inducing cell apoptosis.With the cocultivation of PI3 K and m TOR inhibitor NVP-BEZ235 and Xn,WB,tunel staining and plate clone formation assay were used to detect the effect of NVP-BEZ235 on Xn inhibiting cell proliferation and inducing cell apoptosis.Results: 1.Xanthatin suppresses glioma cells proliferation and colony formation.Xanthatin induces apoptosis of glioma cells.To observe the effect of xanthatin on the proliferation of glioma cells,SRB assay and plate clone formation assay were used.We found that with the increasing concentrations and action time of xanthatin,the survival rate of C6 and U251 cells gradually decreased,and the number of clone formation gradually decreased.Tunel staining and WB results showed that the number of apoptotic cells increased with the increase of xanthatin concentration,and the expressions of cleaved-PARP and cleaved-caspase 3 were also significantly up-regulated.These results suggest that xanthatin can inhibit the proliferation of glioma cells and induce apoptosis of glioma cells.2.Xanthatin inhibits autophagosome formation and decreases autophagic flux in glioma cells.To observe the effect of xanthatin on autophagy of glioma cells,we detected the expression of autophagy-related proteins.We found that with the increasing concentrations and action time of xanthatin,the expression of autophagy-related proteins Beclin1,LC3II/LC3 I,ATG5 and ATG7 decreased,the expression of P62 increased gradually,which indicated that xanthatin inhibited autophagy in a concentration-dependent and time-dependent manner.The results of immunofluorescence showed that compared with the control group,the number of P62 fluorescent spots in cells treated with Xn increased significantly,but LC3 did not show obvious punctate aggregation.The aggregation of P62 and LC3 increased significantly in the treatment of autophagy inhibitor Baf A1.Compared with the treatment of Baf A1 alone,the aggregation of LC3 II decreased after co-treatment of Xn and Baf A1,suggesting that Xn could inhibit autophagosome formation and autophagic flux.At the same time,we successfully established a glioma model in nude mice and randomly divided the nude mice into five groups: negative control group,positive drug control group(temozolomide,TMZ),xanthatin low-dose group,middle-dose group and high-dose group.WB assay in vivo showed that with the increase of xanthatin concentration,Beclin1,LC3II/LC3 I gradually decreased,P62 expression increased,which was consistent with the results of cell line experiment in vitro.These results suggest that xanthatin inhibits the formation of autophagosome and reduces autophagic flux.3.Xanthatin inhibits autophagy of glioma cells through PI3K/AKT/m TOR pathway.In order to explore the mechanism of xanthatin inhibiting autophagy of glioma cells,we detected the activation of upstream signal of autophagy.We found that expression of autophagy-related proteins p-m TOR and p-AKT increased,with the increasing concentrations and action time of xanthatin,and the expression of p-ULK1 decreased gradually,while the trend of p-ERK1/2,p-JNK was not obvious.It is suggested that xanthatin may inhibit autophagy by activating PI3K/AKT/m TOR pathway.At the same time,we detected the expression of these proteins in xenograft tumors of nude mice.The results were consistent with in vitro experiments,suggesting that xanthatin inhibits autophagy of glioma cells by activating PI3K/AKT/m TOR pathway.4.Upregulation of autophagy reduces xanthatin-induced apoptosis of glioma cells and promotes cell proliferation In order to prove that autophagy participates in the anti-cancer effect of xanthatin,we added autophagy inducer Torin1 and Rapa to observe the effect of xanthatin on autophagy,proliferation,apoptosis of glioma cells after autophagy up-regulation.The results showed that autophagy up-regulation could alleviate the effect of xanthatin on apoptosis of glioma cells,and promote cell proliferation.In order to further verify that PI3K/AKT/m TOR pathway participates in xanthatin inhibiting autophagy of glioma cells,we used PI3 K and m TOR double inhibitor BEZ235 to observe the expression of protein related to autophagy upstream regulatory pathway,cell proliferation and apoptosis.The results showed that BEZ235 could alleviate the apoptotic effect of xanthatin on glioma cells.These results suggest that xanthatin can inhibit autophagy by activating PI3K/AKT/m TOR pathway.Conclusions: Xanthatin inhibits autophagy by activating PI3K/AKT/m TOR pathway,thus inhibiting glioma cell proliferation and promoting glioma cell apoptosis.