| Background and ObjectiveHepatocellular carcinoma (HCC) is one of the world’s common lethal malignant tumors, which ranked second in the classification of malignant tumor death in China. There are about 700 thousand cases of liver cancer patients each year. At present, the main treatment includes surgery, ablation, liver transplantation, transcatheter arterial chemoembolization, and molecular targeted therapy. There are various combination and improvement of therapy, but the survival rate still cannot be significantly improved. It is mainly because of postoperative recurrence and intrahepatic or extrahepatic metastasis. Therefore the researches for metastasis mechanism of hepatocellular carcinoma become one of the focuses. The metastasis of HCC mainly refers to the migration and invasion of cancer cells. The molecular mechanism is related to the regulation of many important genes and signaling pathways.In recent years, the research on the relationship between Notch signaling pathway and metastasis of HCC has been a hot topic. The research mainly includes screening for abnormal expression of Notch signaling pathway related molecules in HCC cells, biological influences of cancer cells in regulation of Notch pathway molecule (invasion, migration, proliferation and apoptosis), as well as high risk factors of HCC (hepatitis B virus, HBx protein, etc.) which promote the occurrence and development of HCC through the Notch signaling pathway.IL-24 can arrest the tumor cell cycle, inhibit growth, and induce cell apoptosis. Research shows that IL-24 gene has potential ability of antitumor, and can selectively kill tumor cells without influence on normal cells. This phenomenon has been found and reported in the melanoma cells, HCC cells and other tumor cells. And IL-24 is a kind of cytokine which is produced by the human body itself and has no toxicity. It can improve the sensitivity of tumor cells to chemotherapy, and reduce the side effects.In our experiment, HCC cells were treated by Notch pathway blockade agent-GSI or Notch1-siRNA combined with IL-24. Synergistic biological effect and related mechanism of Notch pathway inhibition combined with IL-24 were researched from several aspects:the inhibition of invasion, migration and proliferation, the promotion of apoptosis. This study improves the research of Notch signaling pathway mechanism in HCC invasion, migration, proliferation, and apoptosis. The synergistic effect of GSI combined with IL-24 was deeply researched. It provides a novel therapy of HCC metastasis and recurrence.Part 1 The effect of GSI-I combined with IL-24 on invasion,migration, proliferation and apoptosis of hepatocellular carcinoma MethodsThe HCC cell lines (SMMC7721, HepG2) were treated with gamma secretase inhibitor (GSI-I) and/or IL-24. The cells were divided into three experimental groups (GSI group, IL-24 group, GSI-I/IL-24 group) and control group. The influence on invasion ability of each group was detected by using Transwell assay. The effect on migration ability of each group was detected by using scratch adhesion test. The influence on cell proliferation of each group was detected by using MTT colorimetric assay. The effect on apoptosis of each group was detected by using flow cytometry and Hoechst staining assay. The expression of key molecules related to Notch signaling pathway, epithelial mesenchymal transition (EMT), and apoptosis in each group was detected by using Western bloting.Results1. In HepG2 cells, compared with control group, the invasion cells number in GSI group decreased significantly (323.3±25.1 vs 175.0±23.1, P＜0.01).The invasion cells number in IL-24 group did not change significantly (323.3±25.1 vs 307.3±31.3,P＞0.05). The invasion cells number in IL-24 and GSI combined group was significantly reduced (323.3±25.1 vs 37.7±31.4, P＜0.01). Combination of IL-24 and GSI can significantly inhibit invasion of HepG2 compared with a single treatment group. The invasion cells number in combined group significantly reduced compared with GSI group (37.7±31.4 vs 175.0±23.1, P＜0.01), and also significantly reduced compared with IL-24 group (37.7±31.4 vs 307.3±31.3, P＜0.01). In SMMC7721 cells, compared with control group, the invasion cells number in GSI group decreased significantly (266.7±17.8 vs 157.7±14.0, P ＜0.01). The invasion cells number in IL-24 group did not change significantly (266.7±17.8 vs 245.67±21.5,P＞0.05).The invasion cells number in IL-24 and GSI combined group was significantly reduced (266.7±17.8 vs 76.0±22.3,P＜0.01). Combination of IL-24 and GSI can significantly inhibit invasion of SMMC7721 compared with a single treatment group. The invasion cells number in combined group significantly reduced compared with GSI group (76.0±22.3 vs 157.7±14.0, P ＜0.01), and also significantly reduced compared with IL-24 group (76.0±22.3 vs 245.67±21.5,P＜0.01).2. In HepG2 cells, compared with control group, the wound healing rate in GSI group decreased (83.6±3.9 vs 66.5±4.9, P<0.01).The wound healing rate in IL-24 group also decreased (83.6±3.9 vs 52.6±3.4, P<0.01). The wound healing rate in IL-24 and GSI combined group reduced significantly (83.6±3.9 vs 6.8±1.9, P ＜0.001). Combination of IL-24 and GSI can significantly inhibit migration of HepG2 compared with a single treatment group. The wound healing rate in combined group reduced significantly compared with GSI group (6.8±1.9 vs 66.5±4.9, P＜0.001), and also significantly reduced compared with IL-24 group (6.8±1.9 vs52.6±3.4, P ＜0.001). In SMMC7721 cells, compared with control group, the wound healing rate in GSI group did not change significantly(56.3±6.8 vs 48.4±7.2, P＞0.05). The wound healing rate in IL-24 group also did not change significantly (56.3±6.8 vs 50.4±5.7, P>0.05).The wound healing rate in IL-24 and GSI combined group was significantly reduced (56.3±6.8 vs 25.4±4.0, P＜0.001). Combination of IL-24 and GSI can significantly inhibit migration of SMMC7721 compared with a single treatment group. The wound healing rate in combined group significantly reduced compared with GSI group (25.4±4.0 vs 48.4±7.2, P＜0.01), and also significantly reduced compared with IL-24 group (25.4±4.0 vs 50.4±5.7, P＜0.01)..3. In the HepG2 cells treated with low concentration of IL-24, the expression of SNAIL1, SNAIL2 reduced slightly, but the expression of E-cadherin protein did not changed significantly. GSI-I inhibited the expression of Notchl, SNAIL1, SNAIL2, SNAIL, and upregulated the expression of E-cadherin. The Combination of IL-24 and GSI significantly inhibited the expression of Notchl protein, and also inhibited the expression of EMT related protein SNAIL1, SNAIL2. The upregulation of E-cadherin protein was accompanied by downregulation of the snail protein.4. With the GSI concentration of 2.5 μM, the average proliferation rate of SMMC7721 reduced 3.5 compared with the control (GSI-I concentration OμM), while the average proliferation rate of HepG2 cells decreased more significantly (reduced 65.1). The proliferation rate in combination group of IL-24 and GSI-I showed a downward trend with the increase of GSI-I concentration. In SMMC7721 cells, the mean cell proliferation rate decreased by 35.1 with the in under the concentration of IL-24 50ng/ml and GSI-I 2.5 μM. While in HepG2 cells, the inhibiting effect of IL-24 and GSI-I combination on the growth of tumor cells was more obvious than those in SMMC7721 cell. GSI-I in the 2.5 M concentration, the mean cell proliferation rate (less than 20%) decreased by 83.4 compared with control group.5. In HepG2 cells, there was no significant apoptosis in GSI-I group, compared with the control. Some of HepG2 cells appeared apoptosis in IL-24 group, and most of the apoptotic cells were in early stage. The apoptotic rate of IL-24 and GSI-I combined in HepG2 cells was 14.81±1.33, which is significant different from the IL-24 or GSI-I group and the control one (P＜0.01). The apoptosis rate of SMMC7721 cells treated with GSI-I alone was not significantly different from control group (P>0.05). In HepG2 cells treated with IL-24 alone, the apoptosis rate was higher than that of the control group (P＜0.05). Compared with the control group and GSI-I group, the apoptosis rate of combined group were significantly increased (P＜0.01). The apoptosis rate of combined group was higher (P＜0.05) than that of IL-24 group.Conclusion1. Although IL-24 in low concentration only has weak ability to inhibit migration, and no ability to inhibit invasion, but IL-24 can significantly enhance the inhibitory effects of GSI on invasion and migration of HCC cell.2. It was by significantly upregulating E-cadherin and downregulating MMP-2 that the IL-24 and GSI-I combination inhibit the invasion and migration of HCC cells.3. The combination of IL-24 and GSI-I could significantly inhibit the proliferation of HCC cells, as well as downregulating of XIAP, and obviously induced apoptosis in HCC cells.4. The combination of IL-24 and GSI-I may be a new method for the treatment of HCC.Part 2 The efficacy of IL-24 combined with siNotchl on the invasion and apoptosis of hepatocellular carcinomaMethodsFor further confirmation of the Notch pathway that IL-24 and GSI act through, we choose Notch1 as the target gene, and designed the corresponding small interference RNA (siRNA). Then the Notch signaling pathway was downregulated with the siRNA of Notch1 from the gene level, and the invasion, apoptosis, and the expression of EMT associated protein were detected again. Meanwhile, the synergistic inhibitory effect of IL-24 was also detected. The cells were divided into three experimental groups (siNotch1 group, IL-24 group, siNotch1/IL-24 group) and the control group. The invasion and apoptosis of HepG2 cells in each group were detected. And the expression of key molecules related to Notch signaling pathway, EMT, and apoptosis in each group was detected by using Western bloting. The apoptosis was detected by PARP and Caspase3/7 assay.Results1.In HepG2 cells, compared with control group, the invasion cells number in GSI group decreased significantly(508.7±14.6 vs 196.7±13.9,P＜0.01).The invasion cells number in IL-24 group did not change significantly (508.7±14.6 vs 496.0±15.4, P＞0.05).The invasion cells number in IL-24 and GSI combined group was significantly reduced (508.7±14.6 vs 27.7±7.6,P＜0.01). Combination of IL-24 and GSI can significantly inhibit invasion of HepG2 compared with a single treatment group. The invasion cells number in combined group significantly reduced compared with GSI group (27.7±7.6 vs 196.7±13.9, P＜0.01), and also significantly reduced compared with IL-24 group (27.7±7.6 vs496.0±15.4,P＜0.01)2. In the HepG2 cells treated with low concentration of IL-24, the expression of SNAIL 1, SNAIL2 reduced slightly, but the expression of E-cadherin protein did not changed significantly. The siNotchl inhibited the expression of Notch1, SNAIL1, SNAIL2, SNAIL, and upregulated the expression of E-cadherin. The Combination of IL-24 and siNotchl significantly inhibited the expression of Notchl protein, and also inhibited the expression of EMT related protein SNAIL1, SNAIL2. The upregulation of E-cadherin protein was accompanied by downregulation of the SNAIL protein. Meanwhile, further study showed that compared with the siNotchl group, the SNAIL1 levels did not significantly changed, but the E-cadherin expression increased significantly. The combination of IL-24 and siNotchl inhibited the occurrence of EMT by inhibiting the Notch signaling pathway (Notchl downregulation), thereby inhibited the invasion and migration of HCC cells. The changes of the pathway and signal molecules in the process are similar with the effect of IL-24 and GSI-I combination.3. The enhancement of the Caspase-3/7 activity was detected in IL-24 or siNotchl group. The caspase-3/7 activity in IL-24 and siNotchl combination enhanced significantly compared with the control group and the IL-24 or siNotch1 group. The unit of enzyme activity in combination group is about 5 times of the control group, and about 2-3 times of IL-24 or siNotchl group. IL-24 or siNotchl could induce a certain degree of early apoptosis alone, but the apoptosis in the combination group was more significant.Conclusion1. Although IL-24 in low concentration only has weak ability to inhibit migration, and no ability to inhibit invasion, but IL-24 can significantly enhance the inhibitory effects of siNotchl on invasion and migration of HCC cell.2. The combination of IL-24 and siNotchl could significantly induce apoptosis in HCC cells.3. It was verified from the gene level that IL-24 and GSI-I inhibited the invasion and induce apoptosis through inhibiting the Notch signaling pathway. |
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