| SignificanceHepatocellular carcinoma (Primary liver cancer, PLC, referred to as HCC) is one of the common malignant tumors in China. The majority of HCC patients have been diagnosed with locally advanced or distant metastasis, so it is a serious threat to people’s health and lives. Traditional Chinese medicine plays an important role in treatment of liver cancer. It can control the disease progression and has a better security, objective response and survival benefit. Arsenic trioxide (arsenic trioxide, As2O3) is the main active ingredient in arsenic. In recent years, enormous researches have demonstrated that arsenic trioxide has a palliative role in the treatment of advanced liver cancer. Therefore, we explore the mechanism of arsenic trioxide on hepatocellular carcinoma cell to provide experimental evidence for its better efficiency in the clinical treatment to liver cancer.ObjectiveThis issue studies human hepatoma cell line HepG2, SMMC-7721 and constructs human hepatocellular carcinoma cell bearing nude mice model on this basis. We investigate the mechanism of arsenic trioxide on liver cancer cells mainly from the cell proliferation, autophagy and apoptosis, etc to provide the experimental basis and new ideas for comprehensive treatment model of liver cancer.Methods1. MTT was used to detect the proliferation inhibition of hepatoma HepG2 cell in different concentration (0μM, 1μM,2μM,5μM, 10μM,15μM,20μM,25μM) of As2O3 for 24h,48h, 72h respectively.2. Inverted phase contrast microscope was applied to observe the morphological change of hepatoma HepG2 cell in different concentration (OμM,5μM, 10μM,15μM) of As2O3 for 24h.3. Hoechst33258 staining was used to detect the apoptotic morphological change of hepatoma HepG2 cell in different concentration (0μM,5μM, 10μM,15μM) of As2O3 for 24h.4. TUNEL was used to detect the apoptotic morphological change of hepatoma HepG2 cell in As2O3 15μM for 24h.5. Annexin V-FIFC/PI double staining was used to detect the apoptosis rate of hepatoma HepG2 cell in As2O3 15μM for 24h.6. Transmission Electron Microscope was applied to observe the apoptotic and autophagic change of hepatoma HepGz cell in As2O3 15μM for 24h.7. Immunofluorescence was used to assess expression of autophagic protein LC3 A/B, Beclin 1 of hepatoma HepG2 cell in As2O3 15μM for 24h.8. Western Blot was used to test expression of LC3 A/B and p62 of hepatoma HepG2 cell in different concentration (OμM,5μM, 10μM,15μM) of As2O3 for 24h and As2O3 15μM for Oh, 2h,4h,8h,12h,24h respectively.9. Western Blot was used to test expression of P-JNK, JNK, P-p38, p38, P-Akt, Akt, P-mTOR, mTOR, Caspase-3, Caspase-7, Caspase-8, Caspase-9, PARP, p27 of hepatoma HepG2 cell in different concentration (0μM,5μM,10μM,15μM) of As2O3 for 24h and As2O315μM for Oh, 2h,4h,8h,12h,24h respectively.10. Western Blot was used to test expression of P-Akt, Akt, LC3 A/B, p62 of hepatoma HepG2 cell in the pretreatment of PI3K/Akt inhibitor LY294002.11. Western Blot was used to test expression of LC3 A/B, p62, Caspase-3, P-JNK, JNK, P-p38, p38 of hepatoma HepG2 cell in the pretreatment of PI3K inhibitor 3-MA.12. We used 18 BALB/C mice to establish human liver cancer nude mice model treated with inoculating SMMC-7721 cells at right lower extremity subcutaneously.13. Nude mice were divided into three groups randomly:negative control group (0.9% sodium chloride injection), As2O3 low-dose treatment group [2mg/(kg·d)], As2O3 high-dose treatment group [4mg/(kg·d)] once abdominal tumor formed. We injected 0.2mL As2O3 once a day for two weeks. Tumors were measured every three days about long diameter (a) and minor axis (b), according to the formula V=ab2/2. After stopping doesing, We executed mice and peel tumors.At last we photographed and calculated the inhibition rate.14. We collected tumor tissue samples and made HE staining to observe morphological structure.15. TUNEL was used to detect the apoptotic hepatocarcinoma cells in As2O3.Results1. At the same time, with the increase of As2O3 concentration, the inhibition rate of HepG2 cells proliferation was gradually increased. Compared with the control group, except As2O3 1μM, 2μM,5μM for 24h and As2O3 1μM,5μM for 48h, other concentrations were significant (P<0.05); In the same concentration, with the extension of As2O3, except As2O3 1μM,2μM. the inhibition rate of HepG2 cells proliferation was gradually increased. Compared with the control group, except As2O3 1μM for 24h,48h and As2O32μM,5μM for 24h, others were statistically significant (P<0.05).2. Inverted phase contrast microscope was applied to observe the morphological change of hepatoma HepG2 cell in different concentration of As2O3 for 24h. Microscopically, compared with the control group, cells in As2O3 became in bad state, edge enhanced and irregular. With the drug concentration increasing, cells floated losing their characteristics.3. After HepG2 cells in different concentration of As2O3 for 24h, Hoechst33258 staining showed:with the drug concentration increasing, the nucleus of As2O3 treatment group presented dense with the bright blue fluorescence intensity gradually becoming strong compared with the control group.4. After HepG2 cells in As2O3 15μM for 24h, TUNEL assay showed:a handful of green fluorescence was visible in the control group, while a large number of green fluorescent apoptotic cells were observed in As2O3 15μM group.5. After HepG2 cells in As2O3 15μM for 24h, flow cytometry showed:compared with the control group, early and late apoptotic cell percentage of As2O3 15μM group became increased significantly.6. After HepG2 cells in As2O3 15μM for 24h, transmission electron microscopy showed: compared with the control group, the phenomenon of reduced microvilli and rough surfaced endoplasmic reticulum, mitochondrial swelled, chromosome concentration, "vacuole" in cytoplasm was observed in As2O3 15μM group. In the cytoplasm, there were a lot of autophagic vacuole in the double layer and multilayers, lysosomal staining deepened and autolysosome appearing.7. After HepG2 cells in As2O3 15μM for 24h, immunofluorescence was used to assess expression of autophagic protein LC3 A/B, Beclin 1. Bright fluorescence of LC3 A/B and Beclin 1 in As2O3 15μM group could be observed underconfocal and the expression increased significantly compared with the control group.8. After HepG2 cells in different concentration of As2O3 for 24h, Western Blot results showed: with the drug concentration increasing, the expression of LC3 A/B gradually increased, while the expression of p62 decreased gradually; After HepG2 cells in As2O3 15μM for different times, with the extension of As2O3, the expression of LC3 A/B gradually increased, the expression of p62 decreased gradually.9. After HepG2 cells in different concentration of As2O3 for 24h, Western Blot results showed: with the drug concentration increasing, the expression of P-JNK, P-p38 increased gradually, the expression of Caspase-3, Caspase-7, Caspase-8, Caspase-9, PARP, P-Akt, P-mTOR decreased gradually, the expression of JNK, p38, Akt had little change. After HepG2 cells in As2O3 15μM for different times, with the extension of As2O3, the expression of p27 gradually increased, the expression of P-Akt, Caspase-7, Caspase-8, Caspase-9, Caspase-3 decreased gradually, the expression of Akt, mTOR had little change.10. After the pretreatment of PI3K/Akt inhibitor LY294002, Western Blot results showed: LY294002 significantly inhibited the expression of P-Akt. Combined with As2O3, LY294002 could significantly improve the expression of LC3 A/B and reduce the expression of p62.11. After the pretreatment of PI3K inhibitor 3-MA, Western Blot results showed:after 3-MA inhibitting autophagy, compared with As2O3 group, the expression of LC3 A/B decreased, and the expression of p62 increased in combined treatment group; The expression of Caspase-3 in combined treatment group was lower than the control group but higher than As2O3 group; Compared with As2O3 group, the expression of P-JNK, P-p38 were significantly reduced and the expression of JNK, p38 had little change in combined treatment group.12.18 nude mice model were successully made. After As2O3 injected for two weeks, the nude mice of physiological saline group had loss of weight and poor nutritional status significantly; while it was not obvious in As2O3 groups. The second one of As2O3 high-dose treatment group was unexplained dead in the tenth day.13. The effect of As2O3 on the growth of human hepatocarcinoma SMMC-7721 transplantation model nude mice:The tumor volume inhibition rate of As2O3 low-dose and As2O3 high-dose group were 23.24% and 37.90%. Compared with the saline group, the mean tumor volume change of As2O3 high-dose group was statistically significant (P<0.05).14. After the tumor sections stained with HE, we could find the saline group tumor cells in cluster distribution and organizational structure acceptable; The cell volume became small and apoptotic change could be observed partly in As2O3 low-dose group; As2O3 high-dose group appeared nucleus deeply stained and contracted, normal structure disappeared, large necrosis region.15. The effect of As2O3 on the apoptosis of human hepatocarcinoma transplantation model nude mice:TUNEL staining showed:Under the microscope nucleus appeared brown or tan was positive whlie negative control no results. Compared with the saline group, the proportion of positive cells in As2O3 low-dose and high-dose group increased. The degree of staining gradually deepened in brown precipitation, mostly weakly positive or positive.Conclusion1. As2O3 could inhibit the proliferation of HepG2 cells in a concentration and time dependent manner.2. As2O3 could induce the apoptosis of HepG2 cells associated with MAPK, PI3K/Akt/mTOR, caspase-dependent signaling pathways possibly.3. As2O3 could induce the autophagy in HepG2 cells by the inhibition of PI3K/Akt signaling pathway inducing autophagic death occurred.4. It indicated that As2O3 can induce apoptosis and autophagy, which could exist simultaneously and work cooperation in HepG2 cells. Inhibition of autophagy could resist apoptosis.5. As2O3 showed synergistic action in inhibiting human hepatocarcinoma SMMC-7721 in nude mice.6. As2O3 could induce the apoptosis in SMMC-7721 cells in vivo. |
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