The Study Of The Underlying Mechanism Of Betulinic Acid On Gastric Cancer Cell Metastasis And Invasion Via The Inhibition Of Skp2

Objective:Gastric cancer(GC)is one of the most common malignant cancers,the cancer incidence rank the fourth worldwide,metastatic invasion of gastric cancer is the main cause of cancer deaths.Skp2(S phase kinase-associated protein 2)is a component of the SKP1-CUL1-F-box protein(SCF)E3 ubiquitin ligase complex.Skp2 has a high expression level in many type of cancers.Skp2 overexpression is closely related to the development of gastric cancer and negatively correlated with the prognosis of gastric cancer.Down-regulation of Skp2 can inhibit tumor metastasis.Betulinic acid(BA),which is a natural pentacyclic triterpenoid compound,is mainly distributed in the bark of birch,and has potent anti-tumor effect and selective effect towards cancer cells,demonstrating its potential for cancer treatment.At present,whether betulinic acid down-regulates Skp2 and inhibits metastasis and invasion of gastric cancer has not been reported.In this study,SGC7901 and BGC823 gastric cancer cell lines were used to investigate the underlying mechanism and effects of betulinic acid on the inhibition of migration and invasion gastric cancer cells.Methods:In this study,the effects of betulinic acid on Skp1-Skp2 complex were evaluated by molecular docking and co-immunoprecipitation;cell growth analysis were used to detect the proliferation inhibition of betulinic acid;The wound-healing experiment and Transwell assay was used to analyze the inhibitory effects of betulinic acid on metastasis and invasion of gastric cancer cells of SGC7901 and BGC823;The effect of betulinic acid on the stability of Skp2 protein in SGC7901 and BGC823 cells was evaluated by CHX assay;Western blot analysis was used to examine the effect of betulinic acid on Skp2 and its related proteins in SGC7901 and BGC823 gastric cancer cells;RT-PCR and gel electrophoresis experiments was used to confirm the effect of betulinic acid on Skp2 mRNA in SGC7901 and BGC823 gastric cancer cells at the transcriptional level;Skp2 overexpression was used to validate the effect of betulinic acid on the metastasis and invasion of gastric cancer cells.Results:The results of cell viability and growth showed that betulinic acid could selectively inhibit proliferation of gastric cancer,and has no apparent toxicity to normal GES-1 gastric mucosal epithelial cells at the established concentration;Wound-healing and Transwell experiments results showed that betulinic acid inhibited the metastasis and invasion of SGC7901 and BGC823 gastric cancer cells in a concentration-dependent manner,and Skp2 overexpression reversed the effect of betulinic acid on the metastasis and invasion of gastric cancer cells;Results of Molecular docking,CHX protein stability test and immunoprecipitation confirmed that betulinic acid can bind to the Skpl-Skp2 complex binding interface,interfere Skpl-Skp2 interaction,reduce the protein stability of Skp2,and reduce Skp2 expression.Western-blot results indicate that BA can significantly decrease the protein level of Skp2,Spl and MMP2/9;and upregulate the protein level of p27;RT-PCR and gel electrophoresis experiments demonstrated that betulinic acid can concentration-dependently inhibit Skp2 expression at the Transcription level;Skp2 overexpression can significantly reverse the above pharmacological effects of betulinic acid.Conclusion:This study demonstrated that betulinic acid inhibits the metastasis and invasion of SGC7901 and BGC823 gastric cancer cells by the inhibition of the Skp2 signaling pathway.The experimental results indicated that betulinic acid directly interacted with Skpl-Skp2 complex and decrease the protein stability of Skp2.Further studies showed that betulinic acid inhibited the proliferation of SGC7901 and BGC823 gastric cancer cells by inhibiting Skp2-p27 signaling pathway,and that betulinic acid inhibited the metastasis and invasion of SGC7901 and BGC823 gastric cancer cells via the attenuation of Skp2-Spl-MMP2/9 axis*However,this study did not test the effect of BA on gastric cancer metastasis in vivo.Next,we will study whether betulinic acid inhibits gastric tumor metastasis by inhibition of Skp signaling in vivo.