Study On The Effect Of PI3K/AKT/mTOR Signaling Pathway In Sodium Arsenite Combined With Astragaloside ? On Anti-hepatocellular Carcinoma

Clinical reports show that sodium arsenite(NaAsO₂)has a good effect on solid tumors such as hepatocellular carcinoma,but it has toxic side effects.Studies have shown that Astragaloside ?(AS-?)has anti-cancer,anti-oxidative stress,enhancing immunity,liver protection effects and so on.In this study,it was hypothesized that the joint use of NaAsO₂and AS-? might produce a synergistic effect against cancer.The PI3K/AKT/mTOR signaling pathway is activated in various tumors such as hepatocellular carcinoma,and is related to cell proliferation,apoptosis,differentiation and autophagy.Through HepG2 cells and nude mice hepatocellular carcinoma model,this thesis intends to systematically explore the synergistic anti-hepatocellular carcinoma effect and mechanism of NaAsO₂combined with AS-? from multiple levels(body,tissue,cell,protein,gene level)in vivo and in vitro,by using toxicology,histrophology and molecular biology methods and taking PI3K/AKT/mTOR signaling pathway and key regulatory points as the entry point.Part one:screening of the best effective concentration of NaAsO₂and AS-? and transcriptome study of their inhibitory effect on the proliferation of HepG2 cells.(1)When hepG2 cells were treated with NaAsO₂and AS-? at five concentrations of 0.078,0.156,0.312,0.625 and 1.250?g/m L for 24,48,72 and 96 h.The optimal concentration of NaAsO₂was 0.5?g/m L and the optimal concentration of AS-? was 0.8?g/m L.The drug combination group was administrated with 0.5?g/m L NaAsO₂+0.8?g/m L AS-?.The optimal treatment time was 72 h.(2)The analysis results of gene expression differences between different treatment groups(drug group)and control group showed that the number of down-regulated genes were obviously higher than that of up-regulated genes,indicating that the effect of NaAsO₂and AS-? on genes was mainly down-regulated,and the co-expression of 181 differentially expressed genes among the treated groups and control group,suggested that there might be a common pathway of action between different treatments.In all treatment groups,the most genes were involved in the regulation of multicellular biological processes,developmental process regulation and anatomical structure development.The concentrated enrichment pathways of all treatment groups mainly include cytokine-cytokine receptor interaction,ferroptosis,hematopoietic cell lineage,estrogen signal pathway and so on.It was also found that there were differences in many signal pathways,such as PI3K-AKT,FOXO,c AMP,MAPK and so on(P<0.05).Suggesting that NaAsO₂and AS-? mainly played a role of resistance to liver cancer through regulating cancer related signaling pathways.Part two:The study on the inhibitory effect of NaAsO₂and AS-? on the proliferation of HepG2 hepatocellular carcinoma cells in vitro(1)The activity,migration and invasion ability of HepG2 cells treated with NaAsO₂and AS-? alone or in combination were detected by using CCK8 test,scratch test and Transwell test.In the scratch test,only the NaAsO₂+AS-? group showed reduced cell migration ability(P<0.05).Other tests showed that the cell viability,migration and invasion ability of the NaAsO₂group,AS-? group and NaAsO₂+AS-? group were significantly decreased(P<0.05),and the NaAsO₂+AS-? group was the most significant.With the treatment of GNGT1 siRNA,HepG2 cell viability was increased,and the migration and invasion ability was enhanced significantly(P<0.05).(2)Flow cytometry was used to detect the effects of NaAsO₂and AS-? alone or in combination on HepG2 cell cycle and apoptosis.The results showed that the percentage of G0/G1 phase cells in NaAsO₂group,NaAsO₂+AS-? group and GNGT1 siRNA group decreased significantly(P<0.05).The percentage of S phase cells in NaAsO₂group,AS-? group,NaAsO₂+AS-? group and GNGT1 siRNA group increased significantly(P<0.05).The apoptosis rate induced by NaAsO₂group,AS-? group and NaAsO₂+AS-? group was significantly increased(P<0.01).The apoptosis rate induced by NaAsO₂combined with AS-? was significantly higher than that by NaAsO₂and AS-? alone(P<0.01),and the apoptosis rate induced by NaAsO₂was significantly higher than that by AS-? alone(P<0.01).(3)The effects of NaAsO₂and AS-? on the key genes and proteins(PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR)of PI3K/AKT/mTOR pathway were detected by immunofluorescence,real time PCR and western blot method in vitro.The results showed that GNGT1,PI3K,p-PI3K,AKT,p-AKT,mTOR and p-mTOR were all expressed in HepG2 cells.GNGT1m RNA expression levels were significantly increased in all drug groups(P<0.05),and GNGT1 m RNA and protein expression levels were significantly decreased in GNGT1 siRNA group(P<0.05).On the whole,the expression levels of PI3K,p-PI3K,AKT,p-AKT,mTOR and p-mTOR were obviously decreased with the treatment of NaAsO₂and AS-? alone or in combination.When the upstream gene GNGT1 was inhibited,the protein expression of the above genes increased significantly(P<0.05),suggesting that GNGT1 may be involved in the regulation of PI3K/AKT/mTOR pathway.In terms of the effects of each drug group on target genes and proteins,the NaAsO₂+AS-? group showed the most significant regulatory changes(P<0.05),showing the results of the NaAsO₂+AS-? group>NaAsO₂group>AS-? group.Part three:In vivo study of the anti-hepatocellular carcinoma effects of NaAsO₂and AS-?(1)The model of hepatocellular carcinoma transplantation were established in nude mice.The nude mice were treated as follows:control group(the model group,with equal physiological saline),NaAsO₂group(30 mg/kg),AS-? group(30 mg/kg)and NaAsO₂+AS-? group(15 mg/kg NaAsO₂and AS-? respectively)for 25 days(the administration frequency was once every three days).The results show that there was no significant difference in body weight,but the tumor volume of NaAsO₂+AS-? group decreased significantly on the 16th day after administration(P<0.05),and the tumor volume of NaAsO₂group,AS-? group and NaAsO₂+AS-? group decreased significantly on the 19th day after administration(P<0.05).(2)The effects of different treatments on serum tumor markers AFP,DCP,CEA and TNF-?in nude mice were detected by ELISA method.The results showed that the contents of AFP,DCP and TNF-?in serum of NaAsO₂group,AS-? group and NaAsO₂+AS-? group were significantly lower than those in control group(P<0.05).Serum CEA content in AS-? group and NaAsO₂+AS-? group were significantly decreased(P<0.05).The serum tumor markers of NaAsO₂+AS-? groups were the lowest among all groups(P<0.05).(3)HE staining and TUNEL staining were used to observe the effects of NaAsO₂and AS-? on the histrohistology of hepatocellular carcinoma xenograft in nude mice.The results showed that the NaAsO₂+AS-? group had the most obvious tumor cell nuclear pyknosis and cell apoptosis.(4)The effects of NaAsO₂and AS-? on key genes and proteins of PI3K/AKT/mTOR pathway were detected by real-time PCR and western blot in vivo.The results showed that the m RNA expression levels of PI3K,AKT and mTOR in NaAsO₂group,AS-? group and NaAsO₂+AS-? group were significantly lower than those in the control group(P<0.05),and the expression level of NaAsO₂+AS-? group was the lowest.Compared with the control group,the protein expression levels of PI3K,AKT,mTOR,p-PI3K and p-mTOR in NaAsO₂+AS-? group were significantly decreased(P<0.05).The m RNA expression of GNGT1 in NaAsO₂group,AS-? group and NaAsO₂+AS-? group were significantly increased than that in the control group(P<0.05).The combination of dispelling pathogenic and fuzheng traditional Chinese medicine was used to study the anti-hepatocellular carcinoma(anti-HCC)effects.The results showed that both NaAsO₂and AS-? had anti-HCC effects.In vitro studies showed that the joint use of NaAsO₂and AS-? had a significantly higher inhibitory effect on HepG2 cells than the separate use of NaAsO₂and AS-?,which preliminarily indicated the synergistic effect of NaAsO₂and AS-?.In vivo studies had shown that the anti-HCC effect of low-dose NaAsO₂combined with low-dose AS-? was still significantly better than that of high-dose NaAsO₂and AS-? alone,which further indicates that the joint use of NaAsO₂and AS-? had synergistic anti-HCC effect.PI3K/AKT/mTOR pathway key genes and proteins(PI3K,p-PI3K,AKT,p-AKT,mTOR,p-mTOR)are closely related to the carcinogenicity,and the results of in vitro and in vivo showed that NaAsO₂and AS-? downregulated their expression,indicating that NaAsO₂and AS-? can exert anti-HCC effects through the PI3K/AKT/mTOR signaling pathway,and that the combination of NaAsO₂and AS-? can regulate the target gene and protein more significantly.It showed that there was a synergistic anti-HCC effect between the NaAsO₂and AS-? at the molecular level,suggesting that PI3K,AKT and mTOR can be used as therapeutic targets for anti-HCC drugs.