Effects And Mechanisms Of Bufalin-induced Apoptosis In Human Hepatocarcinoma Multidrug Resistance Cell Line BEL-7402/5-FU

BACKGROUND: Intrinsic and acquired resistance to chemotherapeutic agents is a significant issue in the treatment of patients with hepatocelluar carcinoma (HCC) which attributes to the failure of chemotherapy. However, there is no effective medicine used in the chemotherapy for HCC up to now and thus the exploitation in chemotherapy medicine will still be an important point in the treatment of HCC. Venenum bufonis, which is a traditional medicine in China, has a long history in the treatment of HCC. In the previous studies, we found that bufalin, an effective component in Venenum bufonis, showed potent effects in anti-hepatocarcinoma and would be a potential medicine in overcoming multidrug resistance and treatment of advanced stage HCC. In the present study, we investigated the effects and mechanisms of bufalin on inducing apoptosis in human Hepatocellular carcinoma multidrug resistance cell line BEL-7402/5-FU in vitro and in vivo.OBJECTIVE: To investigate the effects of bufalin on inducing apoptosis and clarify its mechanisms in HCC multidrug resistance cell BEL-7402/5-FU. To evaluate the effects of bufalin in multidrug resistance Hepatocellular carcinoma nude mice.METHODS: 1. The inhibition effects of Fluorouracil, Vincristine, Adriamycin, Oxaliplatin, Cytoxan and Bufalin in HCC multidrug resistance cell BEL-7402/5-FU, the time- and dose-dependent on inhibiting parental and drugresistant cell proliferation were observed by MTT assay. 2. Cell morphology and fluorescence staining on cell membrane and nucleus in BEL-7402/5-FU cell were observed by inverted phase contrast microscope and laser scanning confocal microscope (LSCM) respectively after 24 h treatment with indicated concentration of bufalin (1, 0.1, 0.01μM). 3. Cell cycle and apoptosis were detected by flow cytometry after 24 h treatment with different concentrations of bufalin (I, 0.1, 0.01μM). 4. The effects of bufalin on Bcl-XL and Bax expression in BEL-7402/5-FU cell were observed by immunocytochemical and Western blot methods. The time-effects relationship of bufalin on mitochondrial membrane potential was measured with flow cytometry. The effects of bufalin on Caspase-3,Caspase-8,Caspase-9 and time- and dose- dependent on Caspase-9 were analyzed by flow cytometry. Moreover, the effect of Caspase-9 special inhibitor on bufalin induced cell apoptosis was studied. 5. An orthotopic transplantation tumour model of human hepatocellular carcinoma in nude mice was established. Twelve days later, the nude mice with hepatocellular carcinoma were randomly divided into 4 groups according to the tumor size as follows: normal saline (NS) group, 5-Fluorouracil treatment group (20 mg/kg·d, one time every other day), bufalin treatment group (1 mg/kg·d, one time every other day) and 5-Fluorouracil ad bufalin treatment group (5-Fluorouracil 20 mg/kg·d, bufalin 1 mg/kg·d, one time every other day). The nude mice were sacrificed and the effect of bufalin in nude mice was evaluated after 14 days treatment. The volumes of tumor were calculated and the biochemistry parameters of blood were detected. Tumor tissues, myocardium, liver and kidney were sectioned, stained with hematoxylin and eosin, and then viewed by microscope. The apoptosis rates of tumor were observed by the TUNEL labeling.RESULTS: 1. The drug resistance indexes of HCC BEL-7402/5-FU cell to Fluorouracil, Vincristine, Adriamycin, Oxaliplatin and Cytoxan were 18.66±2.85, 10.22±2.85, 2.12±0.20, 5.91±1.08 and 8.92±2.46 respectively. There was no obvious drug resistance to bufalin and the proliferation inhibition of bufalin in HCC BEL-7402/5-FU cell line showed time- and dose-dependent relationship. 2. Marked morphological changes, characterizations of shrinkage of cells and losts of the originally confluent monolayer, could be seen after 24 h, which indicated cell apoptosis. 3. The Cell cycle analysis demonstrated an accumulation of cells arrested in G0/G1 and G2/M stages (%) decrease with the concentration increase of bufalin, 25.58±3.70, 48.09±2.49, 55.26±1.97 and 39.78±1.55, 32.1±1.14, 19.42±1.04 respectively, and the differences were significant between bufalin groups and control (P<0.01). The apoptosis rates (%) of the three different concentrations of Bufalin (1, 0.1, 0.01μM) were 42.58±2.11, 23.27±1.02, 18.65±1.36, the differences between bufalin groups and control were significant (P<0.01). 4. The apoptosis rates in 1μM group were higher than 0.1μM group (P<0.01) and the apoptosis rates in 0.1μM group were higher than 0.01μM group (P<0.01). 5. Western blot indicated that bufalin could down-regulate the Bcl-XL expression, and up-regulate the Bax expression. 6. Bufalin reduced the mitochondrial membrane potential. 7. Bufalin could increase the activity of Caspase-3 and Caspase-9 (P<0.01), and the effects could be inhibited by Z-VAD-FMK, a pan-caspase inhibitor. But the activity of Caspase-8 showed little changes (P > 0.05). The release of Caspase-9 could be increased by bufalin and showed time-concentration dependent relationship within 24 h. 8. The apoptosis rates of HCC BEL-7402/5-FU induced by bufalin could be inhibited by Caspase-9 inhibitor. 9. The expression of thymidylate synthase (TS) in orthotopic transplantation tumour model was higher than parental cells. There were significant differences of tumor volume between bufalin group and NS group(P<0.01) . There was significant difference between bufalin group and bufalin ad 5-fluorouracil group (P<0.01). There was no significant difference between NS group and 5-fluorouracil group (P>0.05). Tumor apoptosis rates were detected by Tunel method, significant differences had been noted among bufalin group, bufalin ad 5-fluorouracil group and NS group (P<0.01), but there was nonsignificant difference between the fluorouracil and the NS (P>0.05). There was no significant difference in liver function, renal function and biochemical parameter between NS and bufalin treatment groups (P>0.05). The AST, CL~- and K~+ levels had significant difference between NS and 5-fluorouracil treatment (P<0.01).CONCLUSIONS: 1. Bufalin has no cross-multidrug resistance and has proliferation inhibition effects on HCC multidrug resistance cell BEL-7402/5-FU in vitro and in vivo. It has no significant toxicity on liver, heart and kidney at the indicated dosage. 2. Bufalin may induce apoptosis in HCC multidrug resistance cell BEL-7402/5-FU. 3. Up-regulating the Bax expression, down-regulating the Bcl-XL expression, promoting the mitochondria membrane potential dissipation and then activating Caspase-9 may be one of the mechanisms of bufalin in inducing apoptosis in HCC multidrug resistance cell BEL-7402/5-FU.