| Gastric cancer(GC)represents a health threat as the fourth most common cancer and the second leading cause of cancer death worldwide.Moreover,uncontrolled cell proliferation,invasion and metastasis are the main clinical features of gastric cancer.Most gastric cancer patients are diagnosed in the middle and advanced stages.Therefore,most patients do not die from primary cancer,but from metastatic cancer.The clinical treatment of gastric cancer is dominated by cytotoxic drugs(platinum and uracil),which leads to the clinical situation of single drugs,large toxic and side effects,secondary drug resistance and unsatisfactory effect.In addition,the research on gastric cancer metastasis is relatively lagging,and there is no specific anti-metastatic drug.Therefore,the discovery and development of effective drugs and the clinical application of more new drugs are extremely important and urgent,and the search for a new therapeutic drug that can inhibit the invasion,metastasis and proliferation of gastric cancer has become the focus and hotspot of gastric cancer research.Because natural products and their metabolites are characterized by novel structure,multi-target and unique activity,they have unique advantages in the field of innovative anti-tumor drug research and development.Zeylenone(Zey)is a polyoxygen-substituted cyclohexene compound isolated from the Uvaria griandiflora.Since its discovery in 1997,Zey has been a promising anti-tumor drug candidate worthy of further development and research value from the perspectives of production process(synthetic/semi-synthetic),pharmaceutical preparation(nano-slow-release preparation),pharmacodynamics and molecular mechanism.However,the inhibitory activity and mechanism of Zey on gastric cancer are still a blank,and it is still not clear.In particular,the effect of Zey on the invasion and migration of gastric cancer has not been reported and studied.Objective:In view of the above background,this study took gastric cancer as the research object,to explore whether Zey could inhibit the proliferation,invasion and migration of gastric cancer cells,and to clarify its potential mechanism of action.In addition,we revealed the tumor suppressive activity and related mechanism of Zey in vivo by establishing the model of gastric cancer transplantation.In a word,it laid a foundation for the development and application of Zey in the treatment of gastric cancer.Methods:First,MTT and plate cloning experiments were conducted to evaluate the effect of Zey on the survival rate and clone proliferation of different human gastric cancer cell lines.On this basis,on the one hand,the potential influence of Zey on the migration,motor ability,invasion and metastasis of gastric cancer cells was preliminarily explored through scratch test and Transwell test.Then,the effect of Zey on the expression of MMP-2/9 in gastric cancer cells was detected,and the upstream AKT and ERK pathway related proteins were also detected,in order to further clarify the main mechanism of Zey inhibiting the invasion and migration of gastric cancer cells.On the other hand,from the aspects of morphology,staining and apoptosis rate,we preliminarily determined the biological effect of Zey on reducing the survival of gastric cancer cells,and further clarified the main death mode of gastric cancer cells.Through the mitochondrial membrane potential detection,we preliminarily judged the possible signal pathway,and systematically studied and elucidated the molecular mechanism of the potential anti-tumor effect of Zey.In addition,the tumor suppressive activity of Zey in vivo was studied by establishing a xenograft model of BGC823 gastric cancer in nude mice,and its pharmacodynamics was evaluated in vivo.Then,the effect of Zey on the expression of AKT and other proteins in tumor tissues was studied by Western Blot,so as to clarify the mechanism of Zey's inhibition of tumor growth and metastasis in vivo.Results:1.Zey inhibits the proliferation and clone formation of gastric cancer cellsZey can dose-dependently inhibit the proliferation and colony formation of a variety of gastric cancer cells,but has a weak inhibitory effect on normal gastric epithelial cells GES-1,indicating that Zey is effective and selective for gastric cancer cells.2.Zey inhibited the invasion and migration of gastric cancer cellsWound-healing experiments showed that 24h after Zey treatment,the motor migration ability of SGC7901 and MGC803 cells decreased,and the wound healing rate decreased significantly.The statistical results of invasion rate and metastasis rate in the Transwell experiment showed that the number of gastric cancer cells passing through the artificial basement membrane in the Zey treatment group was significantly lower than that in the control group,and it was dose-dependent.These results indicate that Zey can significantly inhibit the invasion and migration of gastric cancer cells.3.Zey inhibits the invasion and migration of gastric cancer cells by regulating the secretion of MMP and the upstream AKT and ERK pathwaysActivation of AKT accelerates tumor progression and distant metastasis,and MMPs degrades extracellular matrix,leading to accelerated metastasis.Western blot results showed that Zey significantly down-regulated the expression of MMP2 and MMP9 in gastric cancer cells,and significantly inhibited the phosphorylation of AKT and ERK,indicating that Zey's inhibitory effect on invasion and migration was related to AKT/MMP2/MMP9 and ERK pathways.4.Zey induces apoptosis of SGC7901 and MGC803 cells in gastric cancerCell morphology and Hoechst 33258 staining revealed a series of typical apoptotic characteristics of gastric cancer cells in the Zey group.Annexin V/PI results show that Zey group,compared with the control group,had obvious apoptosis.The apoptosis rate of gastric cancer cells gradually increased with the extension of Zey's treatment time to 24h,indicating that Zey had a significant apoptosis-inducing effect on gastric cancer cells,and showed an obvious time-dependent and dose-dependent effect.5.Zey induces apoptosis of gastric cancer cells by acting on the endogenous mitochondrial apoptosis pathwayBy regulating Bcl2 family proteins,Zey reduced the mitochondrial membrane potential,further increased the permeability of mitochondrial membrane,activated the downstream effector caspase-3,further initiated the cascade reaction,and finally led to the apoptosis of gastric cancer cells.6.Zey suppresses tumor growth in a mouse xenograft modelThe tumor growth rate of the Zey high-dose group(30 mg/kg)and the paclitaxel group(15 mg/kg)was significantly slower than that of the control group,with the tumor inhibition rate of 51.2%and 51.4%,respectively,indicating that Zey had a significant inhibitory effect on the tumorigenesis of gastric cancer in vivo.Subsequently,the expression analysis of p-AKT,AKT,MMP9 and MMP2 proteins in tumor tissues also confirmed the findings of the previous three chapters:Zey can inhibit the activation of AKT,inhibit the expression of MMPs,and thereby affect the growth of tumor in vivo.Conclusion:Zey selectively inhibited the survival of various gastric cancer cell lines in vitro,as well as the clonal proliferation of gastric cancer cells.Zey also inhibited the growth of gastric cancer transplanted tumor in vivo.It was innovatively found that Zey inhibited the invasion,migration and metastasis of gastric cancer cells,and inhibited the secretion of matrix metalloprotein enzyme.We systematically studied the molecular mechanism of endogenous mitochondrial apoptosis,invasion and metastasis,and found that the inhibitory effect of Zey on gastric cancer was closely related to the AKT/MMP2/MMP9,ERK and Bcl-2/Bax/Caspase3 pathway. |
PDF Full Text Request