| Hepatocellular carcinoma(HCC)is one of the most common digestive system tumors.As the third most common cancer in the world,the incidence and mortality of HCC are increasing year by year.At present,surgery,radiotherapy,drug therapy and gene therapy are the main treatment methods of hepatocellular carcinoma.And the most important methods is drug therapy.However,the disadvantages of drug treatment is poor tissue selectivity,serious systemic toxicity and side effects.Therefore,it is necessary to found low toxicity and high efficiency targeted drugs for hepatocellular carcinoma treatment.Our study found that the PI3K/Akt/mTOR signaling pathway is highly activated in HCC.It promotes the development and progression of liver cancer by promoting angiogenesis,cell proliferation,and protein translation.Meanwhile,the feedback activation of AKT is also closely related to the chemotherapeutic resistance of HCC.Therefore,searching for small molecule compounds both targeting PI3K/Akt/mTOR signaling pathway and inhibiting AKT feedback activation are expected to solve the problem of decreased efficacy of targeted drugs.Our previous study showed that IHZ-1,a novel indole hydrazide compound,inhibited the proliferation of HepG2 cells(IC50=3.54±0.38μM),and has no effect on MRC-5(IC50>100μM).In addition,IHZ-1 inhibitied the tumor growth in hepatocellular carcinoma xenograft tumor model mice(the inhibition rate = 72.6%),but had no effect on the weight of mice.These data suggested that IHZ-1 had good anti-hepatocellular carcinoma activity,drug selectivity and safety.Next,we used HepG2 and Huh-7 to study the anti-hepatocellular carcinoma mechanism of IHZ-1.Firstly,MTT results confirmed that IHZ-1 had significant cytotoxic effects on HepG2(IC50=5.08 ±0.14μM)and Huh-7(IC50=6.59±0.32μM)cells,Meanwhile,compared with control group,the size of cells became smaller,brighter and rounder,and separated from other cells.This phenomenon further indicated that IHZ-1 induced the death of HCC.Previous studies have found that IHZ-1 can induce cell cycle arrest and apoptosis in HepG2 cells.So we also identified the effect of IHZ-1 in Huh-7 cells.We found that IHZ-1 induced G2/M cell cycle arrest and apoptosis of Huh-7 in a dose dependent manner.It suggested that IHZ-1 could induce cell death by cell cycle arrest and apoptosis increasing.Our previous studies also found that PI3K/Akt/mTOR signaling pathway is highly activated in tissue samples from HCC patients.Therefore,we detected the effect of IHZ-1 on PI3K/Akt/mTOR signaling pathway using Western Blot.The results showed that IHZ-1 significantly inhibited the expression of p-PDK1(Ser241),p-Akt(Ser473),p-Akt(Thr308),p-mTOR(Ser2448),Raptor(mTORC1),Rictor(mTORC2).Moreover,CO-IP assay showed that the interaction between p-P70S6K(Thr389)and Raptor was inhibited,and the downstream protein translation activity of mTORC1 was inhibited.These suggested that the anti-hepatocellular carcinoma activity of IHZ-1 may be assocaited with inhibition of PI3K/Akt/mTOR signaling pathway.It is noteworthy that IHZ-1 does not induce feedback activation of AKT like traditional mTORC1 inhibitors.To investigate this question,we examined the phosphorylation level of IRS-1,a key factor in AKT feedback activation signaling pathway.The results showed that IHZ-1 increased the level of p-IRS-1,but the expression of p-P70S6 K was down-regulated.IRS-1 is regulated by P70S6 K and JNK.Therefore,we detected the expression of ROS and JNK using DCFH-DA probe and Western Blot.The results showed that IHZ-1 induced the accumulated of ROS and increased the expression of p-JNK.Further study showed that the NAC(ROS inhibitor)and SP600125(JNK inhibitor)could decreased the cell apoptosis which induced by IHZ-1,respectively.It suggested that IHZ-1 induced apoptosis of HCC was dependent on ROS/JNK signaling pathway.In conclusion,IHZ-1 can inhibit the cell cycle arrest and induce apoptosis of HCC.Its mechanism may be through inhibiting PI3K/AKT/mTOR signaling pathway and activating ROS/JNK pathway. |
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