Anticarcinogenic Effects Of Water Extract Of Sporderm-Broken Spores Ganoderma Lucidum(G.Lucidum) On Colorectal Cancer In Vitro And In Vivo

Objective:In this paper,we investigated the anti-cancer effect and potential mechanisms of sporderm-broken spores of G.lucidum by hot water extraction(BSGLWE)on colorectal cancer in vivo and in vitro.The effect of nonsteroidal anti-inflammatory drugs activated gene-1(NAG-1),a pro-apoptosis gene on inhibiting cell proliferation induced by BSGLWE was explored.In order to providing the experimental data and theoretical basis in clinical treatment of colon cancer,we pursued the anti-tumor effect of BSGLWE at the cellular and animal level,and explored the regulation of cell proliferation and apoptosis-related by BSGLWE on the basis of the molecular biology.Methods:1.Anti-tumor effect of BSGLWE in vitro To explore the growth inhibitory potential of BSGLWE against colon cancer,cell viability was examined by MTT.Morphological changes of HCT116 cells treated with BSGLWE were observed by Hoechst 33342 staining.The expressions of cyclinA2,cyclinB1,P21,Bcl-2 and Survivin were detected by qRT-PCR.The expression of Bcl-2,PARP,caspase-3 and caspase-9 were detected by Western blotting.Apoptosis and cell cycle distribution were measured by flow cytometric analysis.2.Anti-tumor effect of BSGLWE in vivo Four week old male BALB/C nude mice were kept in Specific Pathogen Free(SPF)environment.After 2 weeks of adaptation,mice were randomly divided by weight.HCT116 cells were injected subcutaneously(s.c.)into the left flank of each nude mouse(5x106 cells in 200 μl PBS).The mice were randomized into treatment and control groups:control group(Saline,n=18),low dose(150 mg/kg,n=18),high dose(300 mg/kg,n=18),5-FU treatment(20 mg/kg,n=8).The day after injection of tumor cells,the high and low dose group mice were treated with BSGLWE intraperitoneally per day,while the control group mice were injected saline per day,and 5-FU was given every two days through intraperitoneal administration.Tumor volume and body weights were measured twice a week.Six weeks after injection,all mice were sacrificed.the xenograft tumors were carefully excised and weighed.Calculate the tumor inhibition rate,and take the control group,low and high dose group to H&E and immunohistochemical staining.The expression of,PARP,foxo3a were detected by Western blotting.3.Expression of NAG-1 induced by BSGLWE.To explore the effect of BSGLWE on the expression of NAG-1 in inhibiting the growth of HCT116 cells in vivo and in vitro.We detect the expression of NAG-1 in HCT116 cell culture medium and serum of nude mice by enzyme-linked immunosorbent assay(ELISA),and the expression in cell and tumor tissues was detected by western blotting.Result:1.BSGLWE inhibits cell viability in a dose-and time-dependent manner,and inhibits cell proliferation through down-regulated cyclin A2,cyclin B1 and up-regulated P21 expression at mRNA to arresting cell cycle progression at G2/M phase.Hoechst33342 staining showed that HCT116 cells distinct feature of condensation,coagulation,and fragmentation of muclear chromation.BSGLWE also down-regulated the expression of pro-caspase-9,pro-caspase-3 and Bcl-2,and cleaved PARP at protein level.The results of flow cytometry also showed that BSGLWE induce apoptosis of HCT116 cells in a dose-and time-dependent manner.2.BSGLWE reduced tumor growth in colon cancer xenograft in vivo Compared with the control group,the tumor weights of each treatment group was significantly reduced,and the tumor volume was significantly decreased in the high-dose group.In the process of injecting positive control drug 5-FU,the weight of nude mice was significantly decreased,but the weight of the high dose group and low dose group had no significant change.The results of HE staining showed that the number of cancer cells were decreased and the necrotic area was increased,which investigated the BSGLWE induced necrosis in a dose-dependent maner in xenograft.The expression of foxo3a was significantly up-regulated by western blotting.The positive rate of Ki67,PCNA and Bcl-2 in high and low dose groups was significantly lower than that in control group.3.NAG-1 was induced by BSGLWE both in vivo and in vitro In HCT116 cell culture medium,NAG-1 concentration was increased dose-dependent upon BSGLWE treatment as compared to control cells.In vivo,the serum of nude mice was also induced upon BSGLWE treatment by ELISA assay,The expression of NAG-1 was significantly increased both in HCT116 cells and nude mice tumor by western blotting.Conclusion:the present study demonstrated that BSGLWE significantly inhibited colorectal cancer cell proliferation and tumor growth through deregulating expression of the key molecular of cell cycle,apoptosis and proliferation.BSGLWE also induce NAG-1 expression in HCT116 cancer cells and NAG-1 induction may be closely related to reduced cell viability and increased apoptosis and possibly cell cycle arrest.BSGLWE could inhibit cell proliferation in colorectal cancer cells,which may involve NAG-1 induction.