Study On The Antitumor Effect And Mechanism Of Protosappanin B On Colon Cancer Cells

Objective:Several studies have demostrated that Lignum Sappan extract had anti-cancer activity.Protosappanin B（PSB）is a key active component of Lignum Sappan extract.However,relatively little is known about the anti-tumor effects and mechanism of PSB on colon cancer.The aim of this study was to explore the effects of PSB on the proliferation,migration and apoptosis,and investigate the anti-tumor mechanism of PSB regulating Golgi phosphoprotein 3（GOLPH3）expression and intracellular signal pathway activity in colon cancer cells in vitro and in vivo.This can provide evidence for PSB as a new potential agent against colon cancer.Methods:1.SW620,SW480 and HCT116 colon cancer cells were treated with concentration of PSB（0μg/ml,6.25μg/ml,12.5μg/ml,25μg/ml,50μg/ml,100μg/ml and 200 μg/ml）for 24-96 hours.CCK-8 assay was used to detect the proliferation of SW620, SW480 and HCT116 cells.The proliferation inhibition rate was used to study the sensitivity of different colon cancer cells to PSB and calculate IC₅₀.2.The proliferation viability of SW620,SW480 and HCT116 cells was detected by colony formation assay.3.Apoptotic（Annexin V-FITC/PI）assay was used to observe the apoptosis of SW620 and HCT116 cells.4.Transwell assay was used to detect the migration ability of SW620 and HCT116 cells.5.Western blot assay was used to analyse the expression levels of GOLPH3,p-AKT, AKT,p-p70S6K,p70S6K,β-catenin,p-ERK1/2,ERK1/2 in colon cancer cells.6.The activity of Wnt signaling pathway in SW620 cells was revealed by Topflash.7.Liposome transfection method:human colon cancer SW620 cells were transfected with lentiviral vector overexpressing GOLPH 3（LV-GOLPH3）and empty vector （LV-NC）respectively.8.Nude Mouse Tumorigenicity Assay:Balb/c nude mice were subcutaneously injected with different colon cancer cells to establish the transplanted tumor model.The tumor volume was observed and measured.After 27 days,H&E staining was used to observe the histopathological difference of the xenograft tissue.The expression of PCNA protein was analyzed by immunohistochemistry and GOLPH3 protein was detected by western blot.Results:1.PSB could inhibit the proliferation of SW620 and SW480 colon cancer cells.With the increase of PSB concentration and treatment time,the inhibition rate of PSB increased in a dose-dependent and time-dependent manner in SW620 and SW480 cells.However,the inhibitory effect of PSB on HCT116 cells was very poor.The IC₅₀value of PSB in SW620 and SW480 cells at 24 hours was 35.25μg/ml and 52.15μg/ml,respectively.2.With the increase of PSB concentration,the viability of SW620 and SW480 cells decreased gradually.The difference of colony number between control group and experimental group,various experimental groups was statistically significant（P<0.001）.However,the inhibitory effect of PSB on the viability of HCT116 cells was not significant,and there was no significant difference in the colony number between experimental group and control group（P>0.05）.3.The apoptosis rate of SW620 cells treated with 35μg/ml PSB was significantly higher than that of the control group（P<0.001）.However,there was no significant difference in the apoptosis rate between the control group and the experimental group of HCT116 cells（P>0.05）.4.The migration number of SW620 cells treated with 35μg/ml PSB was significantly lower than that of the control group（P<0.001）.There was no significant difference in the migration number of HCT116 cells between the control group and the experimental group（P>0.05）.5.Compared with the control group,the relative expression levels of p-Akt,p-p70S6K, p-ERK1/2 andβ-catenin in SW620 cells were significantly decreased after PSB treatment（P<0.05）.However,the relative expression levels of p-Akt,p-p70S6K, p-ERK1/2 andβ-catenin in SW620 cells were significantly elevated when agonists （IGF-1,AZD2858,and TPA）were respectively added to SW620 cells under the same concentration of PSB treatment（P<0.05）.6.Under the PSB treatment,the relative light units（RLU）value of SW620 cells was significantly lower than that of the control group（P<0.001）.However,the RLU value were significantly elevated while AZD2858 were added to SW620 cells under the same concentration of PSB treatment（P<0.001）.7.With the increase of PSB concentration,the relative expression level of GOLPH3 protein in SW620 cells decreased in a concentration-dependent manner（P<0.001）. There was no significant difference in the expression of GOLPH3 protein in HCT116 cells with different PSB concentrations（P>0.05）.8.The survival rate of SW620 cells transfected with LV-NC was significantly lower than that of SW620 cells transfected with LV-GOLPH3 at the concentration of 35 μg/mL PSB（P<0.05）.9.The volume of subcutaneous xenografts in LV-GOLPH3 group was larger than that in control group,but the difference was not statistically significant（P>0.05）.The tumor volume in PSB group was significantly lower than that in control group,and the volume in PSB+LV-GOLPH3 group was significantly larger than in PSB group （P<0.05）.Compared with the control group,the GOLPH3 expression of the transplanted tumor was significantly increased in LV-GOLPH3 group and decreased in PSB group（P<0.01）.Moreover,the GOLPH3 expression in the PSB+LV-GOLPH3 group was significantly upregulated than that in the PSB group（P<0.01）.H&E staining showed that degeneration and apoptosis of cancer cells were obviously observed in PSB group under the light microscope.In contrast,these features were inapparent in the PSB+LV-GOLPH3 group.Immunohistochemistry indicated that PCNA-positive cells of xenograft tissue in the PSB group was lower than in PSB+LV-GOLPH3 group.Conclusion:1.PSB can effectively inhibit the proliferation of SW620 and SW480 cells,suppress the migration of SW620 cells and induce apoptosis,but it has weak effect on HCT116 cells.2.PSB inhibits PI3K/AKT/mTOR,Wnt/β-catenin and MAPK/ERK signaling pathways by down-regulating the expression of GOLPH3 in colon cancer cells.