Efficacy Of Spleen-Based Traditional Chinese Medicine Combined With Chemotherapy Followed By First-Line Chemotherapy For Advanced Gastric Cancer And Mechanistic Investigation

Objective:Evaluate the efficacy of spleen-based traditional Chinese medicine（TCM）formula combined with chemotherapy followed by first-line chemotherapy for advanced gastric cancer,and investigate the mechanism of spleen-based TCM formula weichang’an on inhibiting the metastasis of gastric cancer.Methods:In this study,186 cases with advanced gastric cancer that gained maintenance treatment after first-line standard chemotherapy meet filtering criteria.104 cases in the Chinese medicine group and 82 cases in the non-Chinese medicine group.Then,a retrospective cohort study was applied to evaluate the effect on TFST（Time to First Subsequent Therapy or death）,PFS（Progress Free Survival）and OS（Overall Survival）between the Chinese medicine group and the non-Chinese medicine group.Cox proportional hazards regression model was applied to evaluate the independent prognostic factors affecting the TFST,PFS and OS of patients with advanced gastric cancer.Kaplan-Meier approach was used to calculate the PFS,OS and TFST of the two groups.For a better comparison on the clinical efficacy between two groups,propensity score matching（PSM）was used to match factors,which includes gender,age,primary site,Lauren classification,peritoneal metastatic,first-line strategy,first-line cycles and maintenance strategy.MKN45 cells were cultured in vitro and incubated with different concentrations(600,900,1200,1500 mg·L^-1)of weichang’an formula for 24,48,72h.Cell viability was evalulated by counting kit-8（CCK-8）assay.The expression of RUFY3（RUN and FYVE domain containing 3）was detected in different cell lines by western Blot.Then the expression of RUFY3 was detected in the gastric cancer cells by Western blot.Lentivirus transfection technique was used to knockdown RUFY3 in gastric cancer MKN45 cells.Transwell assay was used to evaluate the influence of weichang’an formula and RUFY3expression on the migration and invasion in MKN45 cells.The expression of E-cadherin、N-cadherin、vimentin、SNAIL1 and SNAIL2 were detected by Western Blot.Results:A total number of 186 cases of advanced gastric cancer patients was enrolled,104 cases in the Chinese medicine group and 82 cases in the non-Chinese medicine group.As the results of Cox proportional hazards regression model shows,the TCM treatment（P=0.000）and maintenance protocols（P=0.027）are the independent prognostic factors that affect the TFST.The independent prognostic factors on PFS includes the TCM treatment（P=0.000）,the first-line cycles（P=0.002）and the maintenance strategy（P=0.016）.The independent prognostic factors on OS includes the TCM treatment（P=0.000）.The median TFST of the TCM group is 10.16 months,while that of the control group is 4.54 months,with statistical difference（P=0.001）.The median PFS of the TCM group is 16.77 months,while 11.18 months for the control,with statistical difference（P=0.001）.The median OS of the TCM group is 25.74 months,while 16.93 months for the control,with statistical difference（P=0.000）.After PSM,118 cases were enrolled,59for the TCM group and 59 for the other.As the results of Cox proportional hazards regression model shows,the TCM treatment（P=0.002）and the maintenance protocols（P=0.044）are the independent prognostic factors that influence the TFST.The independent prognostic factors on PFS includes the TCM treatment（P=0.001）and the maintenance protocols（P=0.008）.The independent prognostic factors on OS includes the TCM treatment（P=0.002）and the Age（P=0.007）.The median TFST of the TCM group is 10.22 months,while that of the control group is 4.34 months,with statistical difference（P=0.003）.The median PFS of the TCM group is 16.73 months,while 11.18 months for the control,with statistical difference（P=0.003）.The median OS of the TCM group is23.01 months,while 17.19 months for the control,with statistical difference（P=0.001）.The experiments,partly,elucidate the mechanism of spleen-based TCM formula weichang’an on inhibiting the metastasis of gastric cancer.RUFY3 was up-regulated in gastric cancer cells versus gastric mucosal cell.Western blot results showed that weichang’an formula significantly down-regulated the expression of RUFY3（P<0.01）in a dose-dependent manner in MKN45 cells（P<0.01）.The expression of RUFY3 was significantly inhibited by small hairpin RNA（sh RNA）（P<0.01）.The transwell experiments showed that both of weichang’an formula and knockdown RUFY3 can remarkably suppressed the migration and invasion in MKN45 cells（P<0.01）.The expression of E-cadherin was up-regulated,while N-cadherin,vinmentin,SNAIL1 and SNAIL2 were down-regulated under weichang’an treatment combined with RUFY3silencing.Conclusion:The TCM treatment can be an independent prognostic factor on advanced gastric cancer cases that gained maintenance treatment after first-line standard chemotherapy,which can extend the TFST,PFS and OS.The spleen-based TCM formula weichang’an could inhibit the migration and invasion of gastric cancer cells by targeting RUFY3.