Research On The Mechanism Of P38MAPK Regulate The Apoptosis And Autophagy Induced By Huaier In Hepatocellular Carcinoma Cells

Trametes robiniophila murris(Huaier) is a type of fungus found in C hina, has been approved by the Chinese Food and Drug Administration to be applied in the clinical treatment of patients with malignant tumor, chemical analyses revealed that Huaier consists mainly of proteoglycans. Rece nt studies have found that Huaier exerts an apoptotic effect on the cells of a variety of human cancers, such as breast cancer, hepatocarcinoma(HCC), lung adenocarcinoma, and ovarian cancer. In addition, Huaier suppresses cancer cell metastasis and motility, and exhibits an anti-angiogenic activity and enhances host immune system function. Together, these data indicate that Huaier shows promising results against cancer in the pre-clinical trials. Although several studies indicated that Huaier induces apoptosis in HCC cells via different signaling pathways, the detailed mechanism remains to be explored.Autophagy is a series of biochemical processes in eukaryotic cells by degradation of cytoplasm and organelles of its implementation of "self digestion". Autophagy plays a promoting or inhibitory effect in the development of liver cancer, in different periods of liver cancer development, the role of autophagy is not consistent. There is mutual relationship between apoptosis and autophagy. In some cases, autophagy is the survival pathways in cancer cells by inhibition of apoptosis, but the autophagy itself will also induced cell death, or autophagy and apoptosis influence each other. Another possible, autophagy can induce death in cancer cells under the apoptosis defects. The two pathways are interrelated, mutual control.Mitogen activated protein kinase(MAPK) is a serine/threonine protein kinase, including extracellular signal regulated protein kinase(ERK), C-Jun N terminal kinase(JNK), and p38 kinase(p38MAPK). MAPK pathway can regulate gene expression in the period of before transcription, post transcription and translation, it regulates nearly all cellular processes, including gene expression, cell cycle regulation, cell survival and death, cell movement, through the regulation of Bcl-2 family proteins to the downstream genes. MAPK signaling pathway is also involved in regulation of autophagy, p38 MAPK has a dual effect on autophagy, promote or inhibit; JNK and ERK can be stored in autophagy.The Bcl-2 family proteins are key regulate factors in regulation of mitochondria apoptosis. Bax/Bcl-2 ratio increased intracellular can induce the release of cytochrome c from mitochondrial to form apoptotic bodies, and then activate Caspase 9, and Caspase 3, eventually leading to cell apoptosis. Bcl-2 has also been shown can be involved in autophagy regulation, Bcl-2 combined with Beclin 1 to inhibit the autophagy. In addition, Bcl-2 phosphorylation is also involve in the process of regulation of autophagy and apoptosis. In the early stage, based on the rapid onset of phosphorylation of Bcl-2, autophagy induced by the separation of Bcl-2-Beclin-1 complex, the organelle damage can be repaired; with the prolongation of time, self-help behavior cannot be reguarantee which induced by autophagy in cell survival, Bcl-2 phosphorylation interfere the formation of Bcl-2-Bax complex, and cause the activation of apoptotic pathway dependent on Caspase 3.The methods of this study include flow cytometry, MTT, application of apoptos is and autophagy related inhibitors, immunofluorescence and Western blot, et al. We plan to observe the changes of proliferation and apoptosis in Hep G2 and Huh7 cells after Huaier treatment; detect the expression of some proteins including Caspase 3, PARP, Survivin, Bcl-2 family proteins, MAPK signal pathway proteins, AKT/mTOR signal pathway proteins and the expression of autophagy proteins after Huaier treatment; observe the changes of proliferation and apoptosis in HCC cells by using inhibitors of apoptosis, autophagy induction or inhibition drugs, at the same time, we detect the expression of Caspase 3, PARP, Survivin, Bcl-2 family proteins and the expression of autophagy related proteins.The purpose of this study is to observe the effects of Huaier on apoptosis and autophagy and their influence on HCC cell proliferation, and explore the mechanism of its induction and regulation on the apoptosis and autophagy in hepatocellular carcinoma cell, provide theoretical and experimental basis of Huaier for the treatment of hepatocellular carcinoma. The study results are as follows: 1. Huaier can inhibit proliferation and induce apoptosis of hepatocellular carcinoma cells in vitroTo investigate the effect of Huaier on HCC cells, MTT assay showed that Huaier can significantly inhibite the proliferation of HepG2 and Huh7 cells in a time and concentration dependent manner. Cell survival rate decreased with prolonging drug effects and increasing drug concentration. The single cell clone formation assay showed cell clone formation rate was significantly lower than the control group after Huaier affected, and the higher of the concentration of Huaier, the lower clonal ability of hepatocellular carcinoma cell. With the increase of Huaier concentration, cell cycle arrest became more obvious, cycle distribution showed a concentration dependent manner. Results of Flow cytometry and Hoechst 333258 staining showed that cells apoptosis rate increased significantly both in Hep G2 and Huh7 cells, compared with the control group after Huaier treatment. With the extension of Huaier action time, the apoptosis rate of HepG2 and Huh7 cells increased significantly, Huaier induced apoptosis of HCC cells with a time dependent manner.To explore the way of apoptosis and inhibition in proliferation, we detected apoptosis rate and some apoptosis related proteins with or without apoptosis inhibitors by flow cytometry, MTT and WB. Results showed that Huaier inhibited the proliferation of HCC cells through the blocking of cell cycle in G1 phase. Expression of C yclin cycle associated protein D1 and C DK2 significantly decreased after the effect of Huaier.Expression of pro-apoptotic proteins of Bcl-2 family increased, but decreased in anti-apoptosis proteins after Huaier treatment. In HepG2 and Huh7 cells, expression of anti-apoptosis proteins Bcl-2, Bcl- xL and Mcl-1 were significantly reduced, on the contrary the expression of pro-apoptotic protein Bim, Bax were significantly increased, while the tumor suppressor protein P53 expression was significantly increased.In Hep G2 cells, PARP, Caspase 3, 8, 9 cracking, and the expression level was significantly increased, but the expression had no significant change with the time prolonging; in Huh7 cells, after 4 hours of Huaier treatment, PARP and Caspase protein cleavage, and the expression level increased gradually with the time prolonging. Flow cytometry and MTT detection showed that after the application of Z-VAD-FMK, the inhibition of Huaier on hepatocellular carcinoma cells proliferation decreased, cell proliferation significantly rebound, and apoptosis decreased significantly. While expression of Caspase 3 and PARP was significantly decreased compared with Huaier after Z-VAD-FMK affected. 2. p38 MAPK signal pathway regulate the apoptosis of Huaier induced by Bcl-2 family proteins and Survivin in HCC cellsTo explore the mechanism of apoptosis induced by Huaier, we detected MAPK signal pathway proteins and observed the apoptosis rate and some apoptosis related proteins with or without MAPK inhibitors by flow cytometry, MTT and WB.The MAPK signal pathway was activated, four hours after the Huaier treatment, the phosphorylation levels of ERK1/2, JNK, and p38 MAPK in Hep G2 and Huh-7 cells were markedly increased. Intriguingly, p38 MAPK was robustly activated in the time interval from 4 to 24 h in both cell lines.The pharmacological inhibition of p38 MAPK attenuates the Huaier- induced apoptosis in both Hep G2 and Huh-7 cells. Results of flow cytometry displayed that, JNK and p38 MAPK inhibitors made the cell apoptosis rate decreased significantly, ERK inhibitors had no significant effect on apoptosis rate of hepatocellular carcinoma cell; while in Huh7 cell, p38 MAPK inhibitors also made the cell apoptosis rate decreased significantly, JNK and ERK inhibitors had no significant effect on HCC cells after Huaier treatment. The results of MTT and clone formation showed that p38 MAPK inhibitor can significantly improve the proliferation ability of HCC cell except ERK and JNK inhibitors.In these two cell types, p38 MAPK inhibitor can make the expression of C leaved Caspase 3 and PARP decreased, JNK inhibitor did not change the proteins expression significantly, ERK inhibitor significantly increased the expression of C leaved Caspase 3 and PARP after Huaier affected. p38 MAPK regulate the apoptosis of hepatocellular carcinoma cells by Bcl-2 family proteins and Survivin. SB203580 treatment significantly increased the expression of Bcl-2 and Survivin in the Huaier-treated Hep G2 cells compared to the treatment with Huaier alo ne, whereas the expression of Bax was substantially downregulated by the combination treatment. Similar results were obtained in the Huh-7 cells. However, the expression levels of Bax, Bcl-2, and Survivin were all enhanced in the HepG2 and Huh-7 cells following the combination treatment with SP600125 and Huaier compared to application of Huaier alone. Interestingly, the treatment with PD98059 considerably augmented the expression of Bax in the Huaier-treated Hep G2 cells in comparison to its rate when only Huaier was utilized, while the expression degree of Survivin was decreased, and that of Bcl-2 remained unchanged. In the Huaier-treated Huh-7 cells, the expression of Survivin was significantly reduced in the presence of PD98059, accompanied by a decline in that of Bcl-2 and an unchanged level of the exhibited by Bax. The application fo Survivin plasmid transfection and SB203580 inhibitor showed that Survivin could promote the cancer cell proliferation and reduce apoptosis, while the effect was enhanced by combination with SB203580. 3. p38 MAPK signal pathway is involved in the regulation of HCC cell apoptosis and autophagy induced by HuaierTo observe the effect of Huaier on autophagy and investigate the mechanism of regulation between apoptosis and autophagy, we detected some autophagy proteins and AK T/mTOR signal pathway proteins and observed the apoptosis rate and some apoptosis related proteins with or without autophagy and MAPK inhibitors by flow cytometry, MTT, and WB. Results showed that autophagy wa s induced by Huaier in HCC cells, and the AK T/mTOR signal pathway was inhibited. The expression level of p62 decreased significantly with the Huaier affection, the expression of Beclin 1 and LC3 II/LC3 I ratios increased in a time dependent manner. Express ion of AKT, mTO R and p70S6 K in HepG2 and Huh7 cel s were significantly decreased.Autophagy significantly influenced the apoptosis of hepatocellular carcinoma cells. In Hep G2 and Huh7 cells, compared with Huaier group, rapamycin can significantly reduce the apoptosis induced by Huaier in HCC cells, while the apoptosis increased slightly after CQ treatment, but showed no significant change compared with the Huaier group. MTT assay showed that the treatment of rapamycin in HCC cells improve the proliferation of cell compared with Huaier group, while CQ decreased the cell proliferation rate significantly.In HepG2 cells, after rapamycin treatment, C leaved Caspase3 expression decreased obviously compared with Huaier group, and PARP decreased with no significant differences; while the expression of C leaved Caspase3 increased significantly after CQ affected, there was no significant difference in PARP expression compared with Huaier group. In Huh7 cells, compared with Huaier group, expression of C leaved Caspase3 a nd PARP was significantly reduced after treatment with rapamycin, while the expression level of C leaved Caspase3 increased significantly after CQ affected, there was no significant difference in PARP expression compared with Huaier group.p38MAPK involved in the regulation of Huaier induced autophagy in hepatocellular carcinoma cells. In HepG2 cells, the p38 MAPK inhibitor significantly enhanced autophagy, the expression of Beclin 1 and LC3 II/LC3 I ratios significantly increased, and p62 expression decreased significantly; while expression of Beclin 1 and LC3 and II/LC3 I ratio decreased with JNK inhibitors, the expression of p62 increased; ERK inhibitors had no significant effect on autophagy, the expression of Beclin 1, p62 and LC3 did not change obviously. In Huh7 cells, autophagy enhanced by the inhibition of p38 MAPK, expression of LC3 and LC3 II/LC3 I ratio increased significantly, but there was no significant changes in the expression of Beclin 1 except p62; JNK inhibitors reduced the expression of Beclin 1 and LC3 and II/LC3 I ratio, the expression of p62 increased; ERK inhibitor has no significant effect on autophagy, the expression of Beclin 1, p62 and LC3 did not change obviously.Immunofluorescence results showed that in HepG2 and Huh7 cells, the p38 MAPK inhibitor make LC3 increased significantly, while the JNK inhibitor reduced LC3 significantly, ERK inhibitors had no significant effect on LC3. Conclusion 1. Huaier could inhibit proliferation of HCC cells through blocking the cell cycle in G1 phase, and induces apoptosis by activating endogenous and exogenous pathway of apoptosis in HCC cells, thereby inhibiting the proliferation of HCC cells. 2. MAPK signaling pathway activate and involve in the apoptosis induced by Huaier, and the regulation of apoptosis is mainly depended on p38 MAPK signaling pathway by the expression of Bcl-2 family proteins and Survivin. Survivin may be a target protein of p38 MAPK. 3. Huaier can induce autophagy in HCC, autophagy plays a role of inhibitor on apoptosis of HCC cells induced by Huaier, and p38 MAPK signaling pathway partially involve in the regulation of autophagy through Bcl-2 family proteins in hepatocellular carcinoma cel s.