| Gastric cancer(GC)is a globally important disease,with more than 1 million newly diagnosed cases every year,and it is the fifth most familiar diagnosed cancer and the third leading cause of cancer-related death in the world.Gastric cancer originates from the epithelium of the gastric mucosa.It can occur in any part of the stomach,but most gastric cancer occurs in the antrum of the stomach.Most gastric cancers are adenocarcinomas,and the early symptoms are not obvious.They are very similar to chronic gastric diseases such as gastritis and gastric ulcers,so it is difficult to attract attention.Therefore,its early diagnosis rate is still very low.At present,the main treatments for advanced gastric cancer are surgical resection,radiotherapy and chemotherapy,but the average 5-year survival rate is still lower than 20%.In recent years,although,the diagnosis and treatment technology of GC has improved,the poor prognosis is still one of the problems faced by the treatment of gastric cancer.Standard surgical resection is not an ideal treatment for advanced GC.More importantly,multidrug resistance and refractory treatment have become the most difficult obstacles to the success of chemotherapy and radiotherapy.Therefore,the research and developing new drugs and inhibitors is also imminent.In recent years,there have been more and more researches on the development of anti-tumor drugs from plant natural compounds.Demethylzelasteral,a natural compound extracted from Tripterygium wilfordii Hook F.It was originally thought to be a drug with immunosuppressive and anti-inflammatory effects.However,in recent years,more and more studies have shown that demethylasteral has a significant inhibitory effect on many tumors.In melanoma,demethylasteral inhibits MCL1 to induce cell apoptosis;it can also inhibit the growth of gliomas by regulating the mi R-30e-5p/MYBL2 axis.However,its anti-tumor mechanism in gastric cancer is still unclear.Therefore,it is necessary to study the effects of demethylasteral on GC and its underlying anti-tumor mechanisms.The c-MYC proto-oncogene,encodes the transcription factor c-MYC,which has been reported to bind to 15% of the promoters in the human genome.The c-MYC expression and activity are strictly regulated due to its important role in regulating the cellular pathways.Cell differentiation,cell proliferation,cell cycle,migration,invasion and apoptosis are regulated by c-MYC,which is of great significance in tumorigenesis and development.In most human tumor types,c-MYC not only its high expression is often correlated with poor prognosis,but the downstream of multiple oncogenic pathways.In this study,western blot experiments showed that after treating HGC-27 and SGC-7901 cells with demethylasteral,the expression level of c-MYC decreased in a dose-dependent and time-dependent manner.Therefore,this study focuses on the studying the demethylasteral regulates FBXW7/c-MYC pathway to affect the migration and invasion of gastric cancer cells.The main experimental results of this article are as follows:1.Demethylasteral inhibits cell proliferation in GC cellsIn order to study the effect of demethylasteral on the proliferation of gastric cancer cells,we conducted MTT and BrdU assays found that it can effectively inhibit the proliferation of gastric cancer cells.We used flow cytometry to detect the cell cycle and found that demethylasteral induced GC cells to block at G0/G1 phase,and the expression of cycle-related proteins CDK4,CDK6,cyclin D1 decreased significantly.The above results indicate that demethylasteral inhibits cell proliferation in GC cells.2.Demethylasteral inhibits cell migration and invasion in GC cellsIn order to explore the effect of demethylasteral on the migration and invasion ability of gastric cancer cells,we conducted wound healing assay and transwell assay.The results indicated that demethylasteral can effectively inhibits GC cells migration and invasion ability.And the expression level of migration and invasion related proteins N-Cadherin,MMP9 and Vimentin were significantly down-regulated,and the expression level of E-Cadherin was up-regulated.The above results showed that demethylasteral can inhibit the GC cells migration and invasion.3.Demethylasteral inhibits GC cells proliferation,migration and invasion by FBXW7/c-MYC axisAs a famous oncogene,c-MYC is related to cell proliferation,migration and invasion.In order to explore whether demethylasteral can inhibit the proliferation,migration and invasion of gastric cancer cells by regulating c-MYC.We found that the expression level of c-MYC protein was significantly down-regulated after demethylasteral treatment.Furthermore,we overexpress c-MYC in gastric cancer cells.Through the MTT and BrdU assays results showed that overexpression c-MYC can rescue the inhibitory effect of demethylasteral on the proliferation,migration and invasion of GC cells.Western blot showed that after overexpression c-MYC,the proteins CDK4,cyclin D1,E-Cadherin and MMP9 were also partially rescued.In order to further explore how demethylasteral acts on c-MYC,we conducted qRT-PCR experiment and found that the mRNA level of c-MYC didn’t decrease after demethylasteral treatment.Therefore,we speculated that demethylasteral may regulate the expression level of c-MYC protein through post-translational modification.The immunoprecipitation experiment proved that the decrease of c-MYC protein level was caused by the increase of c-MYC ubiquitination level induced by demethylasteral.And the results of qRT-PCR and western blot experiments showed that demethylasteral increases the ubiquitination level of c-MYC by up-regulating the expression of FBXW7.The above experimental results show that demethylasteral inhibits the proliferation,migration and invasion of GC cells through FBXW7/c-MYC axis.4.Demethylasteral inhibits self-renewal and tumorigenesis in GC cells through down-regulating the c-MYC expressionIn order to explore the effect of demethylasteral on the tumorigenesis ability of GC cells in vivo an vitro,we conducted soft agar assay an mouse subcutaneous tumorigenesis experiments.The results showed that overexpression c-MYC can rescue the inhibitory effect of demethylasteral on the tumrigenesis ability of GC in vivo and vitro.At the same time,the results of IHC expreiments also proved the same results.The above experiment results show that demethylasteral can inhibit the tumorigenesis ability of GC cells in vivo and vitro by down-regulating the expression of c-MYC.5.Demethylasteral enhances chemosensitivity of GC cells to doxorubicin and 5-Fluorouracil in vitroIn order to explore whether demethylasteral can enhance the chemical sensitivity of the chemotherapy drugs 5-Fu and DOX,we conducted an MTT experiment.The experimental results showed that demethylasteral can enhance the inhibitory effect of5-Fu and DOX on GC cells in vitro.BrdU assay and clone formation assay also proved the same result.At the same time,flow cytometry analysis found that demethylasteral can enhance the apoptotic intensity of 5-Fu and DOX in GC cells.And apoptosis-related proteins Cleaved-PARP and Cleaved-caspase3 have significant changes.The above results indicate that demethylasteral can enhance the chemotherapy sensitivity of GC cells to 5-Fu and DOX.In conclusion,in this study,demethylzeylasteral can reduce the accumulation of c-MYC in cells through the FBXW7/c-MYC axis,thereby inhibiting the proliferation,migration and invasion of gastric cancer cells.In addition,demethylzeylasteral can effectively inhibit HGC-27 cell tumor growth in the mouse model.Meanwhile,demethylzeylasteral can enhance the chemotherapy sensitivity of the chemotherapy drugs 5-Fu and DOX to greatly inhibit the growth of GC cells in vitro.In summary,the above results prove that demethylzeylasteral has a potential to target c-MYC to treat GC. |
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