The Effect Of Calycosin On Colorectal Cancer And Therelated Mechanisms

Objective:Colorectal cancer has a high incidence and a poor prognosis worldwide.Amounting evidences have shown that Traditional Chinese Medicine(TCM)exerts potent antitumor activities against various tumors Calysosin is a single component extracted from Astragalus membranaceus.Its antitumor activity has been proved by many studies.Using the well-established colorectal cancer cell lines HCT-116 and SW480,this study aims to evaluate the effect of calycosin on colorectal cancer and to explore the related molecular mechanisms in vitro.Furthermore,a SW480 xenograft mouse model was constructed to evaluate the effect of calycosin on colorectal cancer in vivoMethods:In vitro colorectal cancer cells HCT-116 and SW480 were cultured to~ 80%confluency,seeded on cell culture plates,and treated cells for 48h with different concentrations(6.25?M?12.5?M?25?M?50?M?100?M?200?M?400?M)of calycosin.The effect of calycosin on the proliferation of HCT-116 and SW480 was determined by methyl thiazolyl tetrazolium(MTT)assay.48h after cells treated with different concentrations(50?M?100?M?200?M)of calycosin,the effects of calycosin on the apoptosis and cell cycle of HCT-116 and SW480 were detected by flow cytometry.The effect of calycosin on the migration of HCT-116 and SW480 was determined by scratch assay.The effect of calycosin on the intracellular signaling protein of HCT-116 and SW480 was measured by western blot analysis.In vivo,Balb/c nude mice were subcutaneously injected with a single cell suspension of 100 ?l of SW480 cells to construct the xenograft mouse model.When the tumor volumes reached 80-100 mm3(ie.1 week after cell transplantation),it indicates that the modeling was successful.After successful modeling,20 nude mice were divided into the control group and the low,medium,and high doses calycosin treatment groups,with 5 mice in each group.The calycosin treatment group was intraperitoneally injected with calycosin(20,40,60 mg/kg),while the control group was intraperitoneally injected with the corresponding dose of saline.The time when the tumor volumes in the control group reached 1000 mm3 was set as the end of the experiment.The effect of calycosin on tumor growth was assessed by measuring the tumor mass.The effect of calycosin on tumor differentiation was analyzed by tumor sections and hematoxylin eosin(HE)stainingResults:1.The inhibitory rates of 6.25?M?12.5?M?25?M?50?M?100?M?200?M?400?M calycosin on the proliferation of HCT-116 cells were 7%,12%,15%,22%,35%,40%,and 72%,respectively,and on the proliferation of SW480 cells were 6%,9%,12%,20%,28%,40%,and 62%,respectively,suggesting that calycosin inhibited the proliferation of HCT-116 and SW480 cells in a dose dependent manner in vitro.The higher the concentrations of calycosin were,the higher the proliferation inhibition rates were2.The percentages of apoptotic cells in HCT-116 cells treated with 0 ?M,50?M,100?M,and 200?M calycosin were about 10%,18%,25%,and 45%,respectively,and that in SW480 cells were about 15%,26%,35%,and 44%,respectively.The percentage of apoptotic cells was significantly higher in 50?M calycosin group than in the untreated group(P<0.05),and the percentage of apoptotic cells increased as the concentrations of calycosin increased(P<0.01),suggesting that calycosin induced the apoptosis of HCT-116 and SW480 cells in a dose dependent manner3.The percentages of G0/G1 phase cells in HCT-116 cells treated with O?M,50?M,100?M,and 200?M calycosin were about 41%,50%,55%,and 65%,respectively,and that in SW480 cells were about 52%,58%,68%,and 72%.The O?M calycosin group was used as the negative control group,respectively.The percentage of cells at G0/G1 phase was significantly higher in 50?M calycosin group than in the negative control group(P<0.05),and the percentage of cells at G0/G1 phase increased as the concentrations of calycosin increased(P<0.01),suggesting that calycosin arrested the cell cycle of HCT-116 and SW480 cells at G0/G1 phase in a dose dependent manner4.Calycosin inhibited the migration of HCT-116 and SW480 cells in a dose dependent manner,the higher the concentrations of calycosin were,the stronger the migration inhibition ability was5.The relative expressions of estrogen receptor ?(estrogen receptor?,ER?)in HCT-116 cells treated with O?M,50?M,100?M,and 200?M calycosin were about 0.31,0.39,0.60,and 0.80.That of phosphatase and tensin homolog(PTEN)were about 0.32,0.38,0.57,and 0.78,and that of phosphorylated protein kinase B(p-Akt)were about 0.8,0.6,0.4,and 0.3.The 0?M calycosin group was used as the negative control group,the levels of ER? and PTEN were significantly higher in 100?M and 200?M calycosin groups than in the negative control group(P<0.01).The levels of p-Akt were significantly lower in 50?M,100?M,and 200?M calycosin groups than in the negative control group(P<0.01).The relative expressions of ER? in SW480 cells treated with 0?M,50?M,100?M,and 200?M calycosin were about 0.28,0.36,0.52,and 0.68 That of PTEN were about 0.32,0.40,0.60,and 0.82,and that of p-Akt were about 0.75,0.62,0.32,and 0.21.The levels of ER? and PTEN were significantly higher in 50?M calycosin group than in the negative control group(p<0.05),and the level of p-Akt was significantly lower in 50?M calycosin group than in the negative control group(P<0.05).As the concentrations of calycosin increased,the corresponding ER? and PTEN levels increased(p<0.01),and the p-Akt levels decreased(p<0.01).These results suggested that calycosin up-regulated ER? and PTEN levels of HCT-116 and SW480 cells,and inhibited the phosphorylation of Akt in a dose dependent manner6.The average weight of tumors in the control group,low-,medium-,and high-doses calycosin treatment groups were about 1.5g,1.3g,1.1g,and 0.9g Medium dose of calycosin significantly inhibited transplanted tumor growth(P<0.05),and the inhibitory effect was stronger in high dose of calycosin group(p<0.01),suggesting that calycosin suppressed tumor growth in a dose dependent manner in vivo7.Calycosin promoted tumor differentiation in a dose dependent manner The heteronuclear cells in tumor tissues gradually decreased as the doses of calycosin increased.Obvious adenoid structures were found in the 60mg/kg treatment groupConclusion:The above results demonstrate that calycosin palys antitumor activity against colorectal cancer through inhibiting cell proliferation,inducing cell apoptosis,causing cell cycle arrest,and suppressing cell migration in vitro In vitro,it may exert these effects through interacting with ER?,up-regulating ER? and PTEN levels,and inhibiting the phosphorylation of Akt.Calycosin suppresses tumor growth and promotes tumor differentiation in vivo.This study lays the experimental basis for the development of calycosin as an anti-colorectal cancer agent.