Essential Role Of JAK/STAT/SOCS Signal Pathways In Selective Bystander Effect Induced By RhIL-24/MDA-7

Apoptosis-inducing effects on hepatocellular carcinoma in nude mice by replication-incompetent adenovirus vector-mediated mda-7/IL-24Objective:To study the apoptosis-inducing effects after the treatment of replication-incompetent adenovirus vector-mediated mda-7/IL-24 on hepatocellular carcinoma in nude mice.Methods:The recombinant replication-incompetent adenovirus vector carrying melanoma differentiation-associated gene-7 (Ad.mda-7) was constructed. The xenograft tumors were established by subcutaneous (sc) injection of 1×105 HCC cells suspended in serum-free saline into the hind legs. When the diameter of the tumors reached 5-6 mm (day 0), animals were randomized into three treatment groups as follows:blank control (saline injection; n= 8), negative control (3×1010 vp in three fractions on days 1,3, and 5; n=8), Admda7 (3×1010 vp in three fractions on days 1,3, and 5; n=8). Their antitumor effects on the average volume of the tumor and the survival rate of the mice were observed in each group and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining was performed after administration of different substances in every group in vivo.Results:Recombinant replication-incompetent adenovirus vector carrying mda-7/IL-24 was constructed successfully. The mice treated with Ad.mda-7 had significantly longer survival time (61.4±1.67d) and greater tumor inhibition compare to other two groups (P<0.01). In the group treated with Ad.mda-7, apoptosis index was 25.7%±3.1%. There is significant difference between Ad.mda-7 group and the other two control group (P<0.05).Conclusion:Ad.mda-7 has stronger antitumor potency on metastastic hepatocellular carcinoma mouse model. The mechanism of this antitumor effect is related to tumor cells apoptosis-inducing.The specific antitumor activity of recombinant human IL-24 against hepatocellular carcinoma cells in vitroObjective:To investigate the biological effect of MDA-7/IL-24 protein on the hepatocellular carcinoma HepG2 cells in vitro.Methods:Treated normal liver L-02 cells, human hepatocellular carcinoma HepG2 cells and human non-small-cell lung carcinoma A549 cells with the recombinant human mda-7/IL-24 cytokines. The mRNA expression of IL-24 receptor complex (IL-20R1/IL-20R2 and IL-22R/IL-20R2) in three kinds of cells was confirmed by RT-PCR. MTT assay and CFSE assay were used to study tumor cell proliferation in vitro. Annexin-V/PI staining was studied to indicate the apoptosis effect. And the flow cytometry was used to assess the cell cycle.Results:(1) RT-PCR analysis showed that normal liver cell L-02 membrane surface IL-20R1 (+) IL-20R2 (+) IL-22R1 (+), hepatocellular carcinoma cell line HepG2 membrane surface IL-20R1 (-) IL-20R2 (+) IL-22R1 (+); (2) 160nmol/L rhIL-24 could promote normal liver L-02 cell proliferation (P<0.05), and with the concentration of rhIL-24, rate of the cell proliferation also increased (P<0.05). However, it can inhibit hepatocellular carcinoma cell growth. There were significant difference (P<0.01); (3) cell proliferation was blocked after treated hepatocellular carcinoma cells HepG2 with 160noml/L rhIL-24 which was detected by CFSE, while the cell proliferation was promoted after treatment in normal liver cells, and there were significant difference compared with the corresponding control group (P<0.05); (4) normal liver cells and hepatocellular carcinoma cells were arrested in G0/G1 phase after treated with high concentrations of rhIL-24. there was significantly decrease in the proportion of cells in S phase; (5) Apoptosis rate increased significantly after treated with 160nmol/L rhIL-24 for 48h later in HepG2 cells group compared with the L-02 cells group determined by flow cytometry (P<0.01); while the L-02 cell apoptosis rate did not change significantly (P>0.05). Conclusions:Our studies suggested that human MDA-7/IL-24 protein exerts a significant bystander effect, and cell apoptosis can be induced by high concentrations of rhIL-24 in hepatocellular carcinoma cells.Essential role of JAK/STAT/SOCS3 signal pathways in selective anti-tumor effect induced by rhIL-24/MDA-7Objective:To investigate the mechanisms of the selective bystander effect of recombinant human MDA-7/IL-24 cytokines (rhIL-24) on hepatocellular carcinoma cell line HepG2.Methods:Normal liver cell line L02 and hepatocellular carcinoma cell line HepG2 were treated with high concentrations of recombinant human MDA-7/IL-24 cytokines (rhIL-24). Western-blot was used to detected the protein expressions of total STAT1, total STAT3, phosphorylated STAT1, phosphorylated STAT3 and the JAK/STAT signaling pathway downstream signal molecule Bax, caspase-3, P53 after the intervention to normal liver cells and hepatocellular carcinoma cells with high concentrations of rhIL-24 at different time points. SOCS family mRNA expressions were determined by RT-PCR method after the treatment with rhIL-24 in defferent cell lines. After that, the appropriate chemically modified siRNA to SOCS family which was expressed highly after interventions of rhIL-24 was designed, and then Annexin-V and PI staining by flow cytometry was used to study the cell apoptosis after siRNA intervention and/or rhIL-24 on normal liver cells and hepatocellular carcinoma cells.Results:(1) the expressions of the total STAT1 and total STAT3 were no significant difference after the rhIL-24 intervention in normal liver cells and hepatocellular carcinoma cells at different time points (P>0.05), pSTAT1 is activated in normal liver cells or hepatocellular carcinoma cells, and there were the lower contents than the contents of pSTAT3. But the difference was not statistically significant (P>0.05); (2) the expressions of Bax and caspase-3 proteins were decreased with the time extension after treated with high concentrations of rhIL-24 in normal liver cells (P<0.05), but the expression of P53 protein increased (P<0.05); however, the expression of Bax anc caspase-3 protein were increased after intervention in liver cancer cells at different time point (P<0.05), but the expression of P53 protein increased at the same upward trend (P<0.05); (3) there were almost no SOCS1, SOCS3, CIS mRNA expression in hepatocellular carcinoma cells HepG2 after intervention with 160nmol/L rhIL-24. However, small amounts of SOCS1 mRNA expression and significant SOCS3 mRNA expression could be detected in normal liver cells L-02 after the intervention; (4) When intervention with siRNA-SOCS3 8h later, not only HepG2 apoptotic cells was significantly increased (P<0.01), more importantly, cell apoptosis occurred in a large number of normal liver cells L-02. The total percentage of apoptosis increased from 4.35% to 35.26%, and there were significant difference (P<0.01).Conclusion:SOCS3 may inhibit the activated JAK/STAT signaling pathway, thereby protecting normal liver cells from cell apoptosis induced by high concentrations of rhIL-24, thus making rhIL-24 has significant selective bystander anti-tumor effects.