| BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors and threatens seriously to the health and life of humans, and about half of the patients with liver cancer focused in China, where about 110,000 people die from liver cancer each year, ranking the second in malignant tumor mortality. Presently, there still existed some problems in clinical diagnosis and treatment of liver cancer, such as a difficult early diagnosis to make, high rate of recurrence and metastasis, and lack of relevance for treatment. Chemotherapy is one of the important ways in the comprehensive treatment for liver cancer, while the tumor multidrug resistance (MDR) is one of the main causes for the failure of chemotherapy-based treatment modalities of malignant tumors.MDR refers to the drug resistance of tumor cells to a kind of antitumor drug, simultaneously, cross resistance also occurs to other antitumor drugs with different structure and mechanism. It is not only the defense mechanism of tumour cell to antineoplastic agents but also the major cause of the failure of chemotherapy-based treatment modalities of malignant tumors.In recent years, the people focused gradually on the role of ceramide in MDR. Ceramide acts as a second messenger in the mediation of cellular apoptosis. Glucosyl- ceramide synthase (GCS) is one kind of glucose transferring enzyme which participates in the metabolism of ceramide. It can convert ceramide to glucosylceramide (GlcCer), prevent cells from apoptosis induced by ceramide and generate MDR. GlcCer, a simple glycosylated form of ceramide, is prosoma substance for synthesizing other glycosphingolipids. It is correlative with biological behaviour of tumor cells, maintaining the normal structure and functions of cell as well as participating in cell proliferation, differentiation, immunological recognition and signal transduction, and resist the cellular apoptosis induced by drug.At present, the research on the role of GCS in the tumor multidrug MDR focus on breast cancer, leukemia, gastric carcinoma, oophoroma, melanoma and epidermoid carcinoma, but the study of GCS on multidrug resistance in hepatocarcinoma has been reported and the mechanism is unclear.Some medicine reversing MDR has been discovered in recent years but was limited in clinical application due to low specificity and grave adverse effects. The gene therapy of reversing MDR also has some defects such as low specificity and efficacy, difficulty in clinical application. Therefore, it is important to find an efficient and harmfulless drug to reverse MDR.ObjectiveThis study was to construct a recombinant plasmid carrying human GCS gene and transfected into HepG2, investigate the effect of the overexpression of GCS gene on drug resistance in hepatocarcinoma cell line HepG2. The expression of the multidrug resistance gene (MDR1) was assayed in the transgenic cell line HepG2/GCS, and to study the relevance of GCS and MDR1 on multidrug resistance in hepatocarcinoma. To explore the role of AstragalosideⅣin reversing multidrug resistance of drug-resistant hepatocellular carcinoma cell line HepG2/GCS and its possible mechanisms.Methods:1. The GCS gene eukaryon expression vector pEGFP-GCS was constructed and transfected into HepG2 by lipid-media transfection. After screening with G418, the stable transgenic cell line HepG2/GCS were set up. The HepG2 cells transfected by the blank vector pEGFP was as control.2. The reverse transcription polymerase chain reaction (RT-PCR) and Western blot test were used to analyze the expression level of GCS mRNA and protein in HepG2 be transfected by pEGFP-GCS.3. The flow cytometry was used to detect the apoptosis and tolerance of HepG2/GCS cells affected by 5-Fu.4. The expression level of MDR1 mRNA and P-gp in HepG2/GCS cells was analyzed by RT-PCR and Western blot test.5. The MTT colorimetric method was used to detect the 50% inhibition concentration (IC50) of doxorubicin (ADM) on HepG2 and HepG2/GCS cells.6. The MTT colorimetric method was used to detect the cytotoxicity of AstragalosideⅣand the drug susceptibility of the HepG2/GCS cells be treated by AstragalosideⅣto ADM.7. The Hoechst 33258 dye was used to detect the apoptosis of HepG2/GCS cells affected by ADM combined with AstragalosideⅣ.8. The Western blot was used to analyze the expression level of GCS protein and P-gp in HepG2/GCS cells treated by AstragalosideⅣand the expression level of Caspase3 in HepG2/GCS cells treated by AstragalosideⅣand ADM.9. The SPSS13.0 statistical package program was used to make data statistics. The data were expressed with x±s. The t-test was used to compare these data, P<0.05 was treated as statistical significance.Results:1. The GCS gene eukaryon expression vector pEGFP-GCS was successfully constructed and transfected into HepG2 by lipid-media transfection. HepG2/GCS cells selected successfully by being cultured with the antibiotic G418.2. It is shown with the results of RT-PCR and WesternBlot that the relative expression amount of GCS mRNA were 2.42±0.13 in HepG2/GCS, significantly higher than in HepG2/pEGFP (0.34±0.02) and HepG2(0.41±0.03) (P<0.05). The expression levels of GCS protein in HepG2/GCS was 4.8 times compared with HepG2, the difference had statistical significance (P<0.05).3. The flow cytometry showed that the apoptosis rates of HepG2/GCS cells induced by 200μg/ml 5-Fu were decreased significantly.4. The expression of MDR1 mRNA in HepG2/GCS were enhanced significantly than HepG2/pEGFP and HepG2 (P<0.05), and the expression levels of P-gp was 1.55 times compared with HepG2, the difference had statistical significance (P<0.05).5. The IC50 of ADM on HepG2 and HepG2/GCS cells were respectively (11.6±1.05)μg/ml and (6.2±0.35)μg/ml, the difference had statistical significance (P<0.05), and the resistant factor was 1.87.6. The result of MTT test showed that AstragalosideⅣhas no cytotoxicity effect on HepG2/GCS. After treated by AstragalosideⅣin finally density at 40μg/ml, HepG2/GCS cells are more sensitive to adriamycin. The IC50 was 7.7±0.24, greatly decreased being compared with that of untouched HepG2/GCS cells (P<0.05). The reversed effect was 1.50.7. The result of Hoechst 33258 dye showed that HepG2/GCS cells were more sensitive to adriamycin after treated by AstragalosideⅣ, the apoptosis and necrosis were observed in HepG2/GCS cells. Many cells separated nestling up to the wall and cannot be observed, while most of the other cells present the typical formation of apoptosis.8. The result of Western blot test showed that, the expression of GCS protein in HepG2/GCS cells was significantly down-regulated after be treated by AstragalosideⅣ. The expression level was 1.05±0.09, while it was 1.71±0.2 before treated. The expression of P-gp had not changed.9. The result of Western blot test showed that, after using AstragalosideⅣcooperated with adriamycin, the content of Caspase-3 in HepG2/GCS cells increased to 1.98±0.13, presenting a significant difference compared with that of single-treated-with-adriamycin group which was 1.17±0.15 (P< 0.05).Conclusion:1. Overexpressed GCS may decrease the sensitivity of HepG2/GCS cells to 5-Fu and adriamycin, suggesting that GCS was involved in mediating the multidrug resistance of HepG2/GCS cells.2. The expression of multidrug resistance related genes (MDR1) in HepG2/GCS cells was up-regulation, suggesting that GCS genes and MDR1 genes have positive relation in the process of multidrug resistance, which may be one of mechanism of GCS on multidrug resistance.3. AstragalosideⅣmay increase the sensitivity of HepG2/GCS cells to adriamycin, the possible mechanism may be to down-regulate the expression of GCS protein in HepG2/GCS cells, and the effect of cell apoptosis induced by adriamycin is enhanced through activating caspases cascading passage. It is indicated through the experiment that, AstragalosideⅣis a low-poisonous and effective reversal agent of drug resistance, and is expected to being applied in clinical research.
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