Study Of NEO214 Inhibits The Development Of Glioma By Blocking Autophagy Via MTORC1/TFEB Signaling Pathway

Glioblastoma(GBM)is the most common primary malignant tumor,exhibiting a high rate of recurrence and poor prognosis.Some conventional surgery treatment methods cannot effectively improve the cure rate and prognosis of patients,the average survival time of patients is only 12-15 months.Temozolomide is currently the first-line chemotherapeutic drug in the current standard treatment regimen for the clinical treatment of glioma.The clinical efficacy of TMZ is not ideal due to the drug resistance and recurrence of tumor cells.Therefore,finding and developing chemotherapeutic drugs that can target the treatment of TMZ-resistant GBM tumors in the clinic,and exploring their possible molecular mechanisms of action is one of the important tasks in the oncology research process.NEO214 is a new glioma treatment drug independently developed by Dr.Chen’s research group at the University of Southern California.NEO214 is a new hybrid small molecule chemotherapeutic drug formed by covalently linking rolipram and perillyl alcohol through a carbamate bond.In the previous research of Dr.Chen’s team,it has been shown that NEO214 has the ability to penetrate the blood-brain barrier(BBB)and strong anti-tumor properties.At present,NEO214 has entered clinical trials and has the potential to become a new type of clinical treatment drug for glioma in the future.Therefore,exploring the effects of various drugs of NEO214 in glioma cells and the molecular mechanism of inhibiting glioma is very important for the clinical application of this drug.Autophagy is an evolutionarily conserved catabolic process that plays an essential role in maintaining cellular homeostasis by degrading unneeded cell components.In recent years,targeting autophagy as a way to strengthen current glioblastoma treatment has shown promising results.Inhibition and activation of autophagy for cancer treatment has been evaluated in the clinic with autophagy modulators.In the present study,we mainly discussed the relationship between NEO214 and autophagy.we demonstrated that NEO214 was a novel autophagy inhibitor,which inhibited the late stage of autophagy pathway by blocking the autophagosomes– lysosomes fusion and induces apoptotic glioma cell death.We found that the inhibition of autophagy by NEO214 was via the mammalian target of rapamycin C1(m TORC1)pathway.We found that NEO214 could significantly reduce the survival and metastatic invasion ability of TMZ-resistant glioma cells through targeted cell autophagy.Therefore,in this article,we mainly explored the specific molecular mechanism of NEO214 inhibiting autophagy and the effect and influence of drugs on the proliferation,migration of glioma cells.First,we found that NEO214 has a strong ability to inhibit cancer cell proliferation on a variety of glioma cells in vivo and in vitro.And within the experimental dose range,there was no drug toxicity side effect on normal nucleus mice.Moreover,the drug function of NEO214 was only related to its self-conjugated molecular structure,and its effect on glioma cells was significantly better than that of Rolipram,POH or the combined use of the two components.Secondly,we observed that LC3-II protein levels and p62 protein level markedly increased after NEO214 treatment.To provide more evidence about autophagy regulation by NEO214,we performed transient transfection of green fluorescent protein GFP-LC3 plasmid to U251,and we determined NEO214 inhibits autophagic flux in glioma cells.NEO214 down-regulated the expression of the lysosomal protease CTSD and reduced the co-localization of LAMP1 and GFP-LC3,indicating that NEO214 inhibited the autophagic flow of gliomas by affecting the pH of the lysosome cavity and the autophagosome-lysosome fusion process.Subsequently,through a series of experiments on the mechanism of NEO214 inhibited autophagy,we found that NEO214 could activate m TOR kinase activity,induced the enrichment of endogenous TFEB in the cytoplasm through m TORC1 complex,inhibited the translocation of TFEB to the nucleus,and reduced the phosphoric acid of TFEB.In this way,the expression of autophagy-lysosome genes was decreased,and the autophagy flow of cells was blocked in the autophagy-lysosome stage.Finally,a series of experiments showed that NEO214 as a new type of autophagy target small molecule drug could promote the drug effect of CQ and TMZ in the treatment of glioma.NEO214 combined with low-dose CQ and TMZ has a significant inhibitory effect on the growth of glioma cells and the ability to form colonies.It could also induce G0/G1 block in glioma cells and decrease the expression of related proteins cyclin D1 and cyclin D2.The expression of apoptosis protein PARP and P53 protein increased.And NEO214 could inhibit the high expression level of MDM2,thereby hindered the development of tumor cell EMT process,and reduced the proliferation and migration ability of glioma cells.This article explores the molecular mechanism of NEO214 as a new small molecule chemical drug and its effect on the proliferation of glioma cells,revealing that NEO214 inhibits glioma cells mediating m TORC1-TFEB-autophagy-lysosome signaling pathway.It demonstrates the potential of NEO214 for the treatment of glioma patients in the future,deepers the understanding of synthetic small molecule drugs in the treatment of gliomas,and provides more options for future clinical treatment of gliomas and improving the quality of life of patients after surgery.