| Hepatocellular carcinoma (HCC) is a clinical common disease, and leading cause of death for urban and rural residents in china. Most patients, who diagnosed as advanced stage, usually lost the timing of surgery but only select non-surgical treatment. However, the pathogenesis of HCC has remained unclear so far. Being not sensitive to conventional chemotherapy medicines and prone to drug resistance, HCC has poor prognosis with high recurrence rate, and thus seriously endangers the public health. In recent years, how to figure out the nature of autophagy and particular the relationship between autophagy and HCC emerge as an important but unresolved proposition, which injected novel research content for the pathogenesis, diagnosis and treatment of HCC. In this study, different techniques involving molecular biology, cell biology and experimental zoology have been used to explore the role of autophagy in HCC in response to EGCG with a pharmacological respect to this medicine.Part I:The characteristics of autophagy in hepatoma cells and associated impact on chemotherapeutic drugs in HCC Objecctive To study morphology and molecular characteristics of autophagy in HCC cells and explore the process and significance of autophagy involved in chemotherapeutic drugs in HCC.Methods HCC cell lines Hep3B, HepG2 were maintained in high glucose DMEM containing 10% fetal bovine serum. According to different experimental protocols, cells were incubated with different concentrations of doxorubicin. Autophagosomes formation was observed by transmission electron microscopy after Hep3B cells were treated with DOX. LC3 protein was detected by immunofluorescence staining.The expression of Beclinl and Atg5 were detected by Western blot. The cell proliferation inhibition rates of Hep3B and HepG2 by different concentrations of DOX were calculated using MTT assay. After Hep3B and HepG2 were pretreated with autophagy inhibitor 3-MA, the cells death rate was measured by trypan blue staining.Results (1) Autophagosomes and autophagic lysosomal were found by transmission electron microscopy in Hep3B. When Hep3B cells were treated with doxorubicin, more poisoning vacuoles, typical autophagosomes were observed in cytoplasm. (2) DOX induced protein expression of LC3 by fluorescence microscope in HCC cells. (3)Treatment with doxorubicin dose-dependently induced Atg5, Beclinl protein expression by Western blot analysis. (4) DOX can dose-dependently inhibit proliferation in Hep3B and HepG2 by MTT assay. Based on MTT assas of cultured cells, the IC50 of DOX in Hep3B was 4.06μM and HepG2 was 0.60μM. (5) Trypan blue staining showed that cell death rate of Hep3B was (9.76±1.3)% after 2.5μM DOX treatment for 48h. In contrast, cell death rate increased to (14.15±4.64)%(P <0.01) when Hep3B was pretreated with 3MA. For HepG2, cell death rate was (8.91±2.87)% when 0.5μM DOX treatment for 48h. Using 3MA pretreatment, the DOX-induced cell death rate increased to (18.00±4.57)%(P<0.01).Conclusion:Doxorubicin inhibit proliferation of HCC cells and induce the enhancement of autophagy signaling at the same time.Autophagy induced by doxorubicin in HCC cells may be self-protection mechanism against chemotherapy.Part II:Effect of EGCG on the Autophagy in Hepatoma Hep3B CellsObjective:To investigate the effects of epigallocat-echin-3-gallate (EGCG) on autophagy and proliferation in hepatocellular carcinoma (HCC) Hep3B cells, and explore the associated relevance of such intracellular machinery in this cell line.Methods:Hep3B cells were routinely cultured in Dulbecco's modified Eagle's medium in the presence of EGCG with different concentration and different incubated time. First, transmission electron microscopy technique was used to record the change in autophagosomes in Hep3B with or without EGCG. And then real-time PCR and western blot were respectively used to detect the autophagy-related genes and protein expression level. Later on, MTT assay was performed to determine the proliferation of Hep3B as well as the IC50 of EGCG.Results:Compared with control, the number of autophagosomes as well as expression level of Beclinl and Atg5 in Hep3B was found to be substantially reduced after EGCG treatment. It was confirmed that the inhibition of Hep3B cells by EGCG was significantly and positively related to its concentration and working time course (the correlation coefficient for 24h, 48h and 72h were 0.873,0.96,0.978, respectively, P<0.05). Accordingly, experimental IC50 of EGCG were (174.77±60.42)μg/mL. (64.72±10.36)μg/mL and (37.49±13.96)μg/mL. The decreasement of mRNA transcription of autophagy-related genes, including Beclinl and Atg5, was found to be negative correlation with EGCG concentration (r=-0.923 and -0.848, P<0.05) and positive correlation with cell viability(r=0.999 and 0.990, P<0.01). EGCG induced change in protein translation of Beclinl and Atg5 was similar to that of mRNA level in this hepatoma cell.Conclusion:EGCG can down-regulate the autophagic level and this may contribute to proliferative inhibition in Hep3B cells.Part III Study of the autophagy mechanism involved in EGCG combined with DOX therapy for HCC in vitro.Objective:Study the synergistic effect of EGCG combined with DOX therapy for HCC in vitro; evaluate whether the autophagy mechanism contribute to the combined EGCG/DOX treatment for HCC.Methods:Hepatocellular carcinoma cell line Hep3B was used, MTT assay and the coefficient of drug interaction (CDI) was performed to examine the inhibitory effects of combined EGCG/DOX on the proliferation of Hep3B; Analysis of cell apoptosis induced by EGCG/DOX combination by Flow-cytometry. Cell viability was assessed by the Trypan blue staining assay after these two drugs combination, then further evaluation of synergistic effect of EGCG combined with DOX therapy for HCC in vitro. LC3 was monitored as a marker of autophagy, LC3 transiently overexpressed in Hep3B by transfected pEGFP-LC3 plasmid; parallel group design was employed to observe the autophagosome formation treated with EGCG/DOX combination in Hep3B. Also, Beclinl and Atg5 as essential effectors of autophagy pathways were examined using Western blot analysis. Rapamycin induced autophagy and that inhibition of autophagy by small interfering RNA (siRNA) were employed to analyze whether autophage is involved in EGCG/DOX combination anticancer effect.Results:(1) MTT assay demonstrated that the growth inhibition rates in Hep3B by different concentration of EGCG/DOX combination were significantly higher than treated with EGCG or DOX alone. Within a certain range, CDI were less than 1 in Hep3B in the prescence of EGCG combined with DOX. (2) Trypan blue exclusion assay showed that cell death rate is (10.90±1.0)%(P<0.01) when Hep3B treated with DOX alone; significantly increased cell death rates (17.67±1.28% and 27.87±1.57%, P<0.01) were obtained when combined EGCG (10μg/mL and 20μg/mL) and DOX (2.5μM) treatment. (3) Flow-cytometry assay showed that apoptosis rate of Hep3B is 9.69±0.98% when cell treated with 2.5μM DOX, the apoptosis rates were increased significantly when combined two drug treatment (17.34±0.92%, P<0.01). (4) Using fluorescence microscope to observe the autophagosome formation, we found that EGCG decreased the autophagosome formation induced by DOX. (5) Western blot showed that EGCG inhibited the expression of beclinl and Atg5, and DOX increased the expression of Beclinl and Atg5; EGCG/DOX treatment decreased Beclinl and Atg5 expression compare to treat with DOX alone. (6) After rapamycin induced autophagy, cell death rate decreased from 10.24±1.42% to 6.91±1.07%(P<0.01) when treated with DOX alone, however, cell death rate decreased from 17.46±1.08% to 12.41±0.87%(P<0.01) when treated with EGCG/DOX combination. (7) Inhibitory effect of Beclinl-siRNA and Atg5-siRNA showed that cell death rate increased significantly in combined two drug treatment group compared to the control group (increased from 14.85±1.17% to 22.63±1.14%), indicated that EGCG/DOX combination had the synergistic anticancer effect.Conclusion:EGCG/DOX combination has the synergistic anticancer effect. Autophagy mechanism involved in the synergistic effect. Autophagy mediates the chemotherapeutics resistance induced by DOX in HCC therapy.Part IV:Investigate on the synergistic effect of EGCG and doxorubicin on nude mice with liver cancerObjective:To determine whether EGCG and doxorubicin play synergistic therapeutic roles in the nude mice with hepatocellular carcinoma, and to further explore associated autophagic mechanism.Methods:Human hepatoma Hep3B cells were subcutaneously injected into nude mice to establish xenografts model. After the modeling, the Hep3B xenografts mice were respectively administrated with EGCG or doxorubicin or combination of these medicines for 15 days. Next, general pathological observation as well as H&E staining assay were performed to evaluate the therapeutic effects of EGCG and doxorubicin on HCC. Meanwhile, immunohistochemistry and Western blot technique were used to qualitatively and quantitatively analyze the expression of autophagic signaling molecules in hepatoma tissues. And then, TUNEL assay were applied to detect such drugs-induced apoptotic levels in hepatic cancer cells from those tissue sections. Finally, by statistical correlation analysis, it was attempted to explore the relationship between the synergistic anticancer effects and change in autophagy signaling by EGCG and doxorubicin.Results:The inhibitory effect of EGCG or doxorubicin alone was not obvious, with a tumor inhibition rate of 10.94% and 15.73% respectively. Moreover, it was showed dramatic enhanced inhibitory effect on hepatoma by together usage of EGCG and doxorubicin, with an inhibition rate of 36.62%(P<0.05). By light microscope, H&E staining has shown that the necrotic area in HCC in response to two-drug administration was larger than in solo. A larger number of lymphocytes, mononuclear cells infiltration was also found in synergistic presence of EGCG and doxorubicin. By Immunohistochemistry and Western blot analysis, it was showed that doxorubicin stimulated significant incensement in autophagic molecules such as Beclinl, Atg5 and LC3II in the liver cancer tissue. Interestingly, as previous findings in vitro, EGCG was found to significantly attenuate the doxorubicin induced autophagy signaling in HCC. Furthermore, the TUNEL assay was showed that the apoptotic rate in doxorubicin alone and combination with EGCG was (5.27±1.29)% and (10.47±2.26)%, respectively (P<0.05). The induced apoptosis by combination of EGCG and doxorubicin was closely related to the suppression expression of autophagy by this combination therapy. The combination treatment was found to be strongly positive correlation with apoptotic rates (OR=2.836,95% CI=1.026-7.839) but negative correlation with autophagy levels in those HCC (OR=0.874,95%CI=0.762-1.003) (P=0.036<0.05)Conclusions:(1) Doxorubicin exerts an anti-cancer effect and it also induces the expression of autophagy in HCC. EGCG can suppress HCC growth, which down-regulate the level of autophagy Combination of these two drugs can modify the autophagic expression pattern in tumor and promote tumor suppressor and anti-cancer properties. (2) All data indicated that EGCG-induced downregulation in autophagy level can enhance the anti-cancer effect of doxorubicin in HCC, which give rise to a novel pharmacological basis for combined application of EGCG and doxorubicin to treat HCC in the future.
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