| Beta-escin, a natural triterpenoid saponin, isolated from the seed of Aesculus hippocastannum (Europe), Aesculus turbinata Blume (Japane) and Aesculus wilsonii Rebd (China), is known to generate wide variety of biochemical and pharmacological effects. Specially, it has been shown to be effective as an alternative to medical treatment for chronic venous insufficiency. Beta-escin was found to display significant anti-proliferative activity on sarcoma, lung, and hepatic cell lines. and apoptotic activity on human HT-29 colon cancer cell line recently. The aim of the present work was to determine the anti-proliferative and apoptotic activity of beta-escin in various leukemia cells by in vitro and in vivo methods. Another set of experiments were used in order to investigate the mechanisms involved in beta-escin activity. To our knowledge, this is the first report that about beta-escin's effects on leukemia cells. The results present here should be useful in the search for newpotentialantileukemic agents.Beta-escin was purchased from Shandong Luye Pharmaceutical Co., Ltd., (Shandong, China), which was isolated from the seed of Aesculus wilsonii Rebd (Semen Aesculi). Doxorbicin (DOX) was provided by Zhejiang Haizheng Pharmaceutical Co. Ltd., The human acute myelogenous leukemia cell line HL-60,human chronic mylogenous leukemia cell line K562 and murine lymphoid leukemia cell line P388 and L1210 were purchased from Shanghai Institutes for Biological Sciences (SIBS, Shanghai China). Cleaning inbred strain DBA/2 mouse (18-22g) male/female, were supplied by Shanghai Laboratory Animal Center, Chinese Academy of Sciences (SLACCAS, Shanghai China).The anti-proliferative effects of beta-escin in various leukemia cell lines in vitro were detected by soft agar colony formation assay, MTT assay or/and trypan blue exclusion cell viability assay. In vivo study, mice inoculated with P388 or L1210 murine lymphoid leukemia cells were treated with ten daily doses of beta-escin and each other day of DOX, and were observed for survival. Antileukemic effects were assessed by calculating the increase in lifespan (ILS). The molecular mechnism were expored by the methods of morphology. Annexin V-FITC /PI double staining analysis, DNA fragmentation analysis and flow cytometry DNA content assay using K562 and HL-60 as target cells. The statistic precession was carried out in SPSS 10.0 for windows. Comparison with difference between two groups was performed by student's t-test, the differences between various groups were compared by one-way analysis of variance (ANOVA) and the P value less than 0.05 was considered statistically significant. The IC50 values were obtained by nonlinear regression using the GWBASIC (LOGIT) software.Results showed that after exposure to 30μg/ml of beta-escin for 24, 48 and 72h. the percentage of CFU-K562 colony formation were 76.5±7.6%, 49.5±5.4% and 33.2±4.7% respectively, showed a significant suppression (P<0.05) as compared to the control of 100%. After exposure to 50μg/ml of beta-escin for 24, 48 and 72h, The percentage of CFU-K562 colony formation were 30.5±5.2%, 19.8±4.6% and 11.2±3.6% respectively, showed a significant suppression (P<0.01) as compared to the control of 100%. After exposure to 70μg/ml of beta-escin for 24, 48 and 72h. a significant suppression (P<0.001) of CFU-K562 were showed by percentage of 93.5%. 100% and 100% respectively. In cells exposed to 10, 30 and 50μg/ml of beta-escin for 24, 48 and 72h, there were no significant decrease in cell viability. However, 70μg/ml of beta-aescin showed obvious cytotoxic effect in K562 cells, cell's viability decreased to less than 50% after an exposure for 24, 48 and 72h.After exposure to 30μg/ml of beta-escin for 24, 48 and 72h, the percentage of CFU-HL-60 colony formation were 68.5±5.4%, 45.2±4.9% and 33.2±5.7% respectively, showed a significant suppression (P<0.05) as compared to the control of 100%. After exposure to 50μg/ml of beta-escin for 24, 48 and 72h, The percentage of CFU-HL-60 colony formation were 35.5±4.1%, 27.8±3.2% and 16.2±2.9% respectively, showed a significant suppression (P<0.01) as compared to the control of 100%. After exposure to 70μg/ml of beta-escin for 24, 48 and 72h, a significant suppression (P<0.001) of CFU-HL-60 were showed by percentage of 92%, 100% and 100% respectively In cells exposed to 10, 30 and 50μg/ml of beta-escin for 24. 48 and 72h, there were no significant decrease in cell viability. However, 70μg/ml of beta-escin showed obvious cytotoxic effect in HL-60 cells, cell's viability decreased to less than 50% after an exposure for 24, 48 and 72h. Beta-escin was also found to be able to inhibit the proliferation of P388 murine leukemia cells by MTT assay. The effects were in dose- and time- dependent manner. The IC50 value of beta-escin in P388 cells after 72h was 23.15±4.61μg/ml.In vivo study showed that after treating the mice inoculated with P388 cells with higher, middle and low dosage (4.5, 3.5 and 2.5 mg/kg) of beta-escin for ten consecutive days, The increase in lifespan (ILS) were 23.5% (p<0.01), 29.4% (p<0.01) and 17.6% respectively. Moreover, we observed 47.1% of ILS when mice were treated with a low dose of beta-escin in combination with DOX (2mg/kg). which is significantly increased compared with 29.4% of ILS when mice were treated with single DOX (P<0.01). After treating the mice inoculated with L1210 cells with higher, middle and low dosage (4.5, 3.5 and 2.5 mg/kg) of beta-escin for ten consecutive days, The increase in lifespan (ILS) were 25.8% (p<0.01), 32.3% (p<0.01) and 12.9% respectively. Moreover, we observed 45.2% of ILS when mice were treated with a low dose of beta-escin in combination with DOX (2mg/kg), which is significantly increased compared with 29.0% of ILS when mice were treated with single DOX (P<0.01).The molecular mechnism results showed that morphological evidence of apoptosis, including reduction in cell volume, chromatin condensation and vacuolization were observed in K562 cells treated with 30 and 50μg/ml of beta-escin for 24h. Significant increase of population of annexin V+ and PI- cells (early apoptotic cells) of the total cells were observed in cells treated with beta-escin (30-50μg/ml) for 24h. Typical DNA ladder, DNA with a unit length of about 180bp, were detecteded in cells treated with beta-escin (30-50μg/ml) for 48 and 72h by agarose gel electrophoresis. Flow cytometry cell cycle analysis revealed that beta-escin induced G1-S arrest and led to an accumulation of sub G1 population in K562 cells.Morphological evidence of apoptosis, including vacuolization, nuclear fragmentation and formation of apoptotic body, were observed in HL-60 cells treated with 30μg/ml of beta-escin for 24, 48 and 72h respectively. Significant increase of population of annexin V+ and PI- cells (early apoptotic cells) of the total cells were observed in cells treated with beta-escin (20-50μg/ml) for 24h. Typical DNA ladder, DNA with a unit length of about 180bp, were detecteded in cells treated with beta-escin (20-50μg/ml) for 48h by agarose gel electrophoresis. Flow cytometry cell cycle analysis revealed that beta-escin induced G1-S arrest and led to an accumulation of sub G1 population in HL-60 cells.In summary, beta-escin, a natural triterpenoid saponin from the seed of Aesculus wilsonii Rebd (Semen Aesculi), can effectively inhibit cell proliferation in human acute mylogenous leukemia HL-60 cells, human chronic mylogenous leukemia K562 cells and murine lymphoid leukemia P388 cells in vitro, that were in a dose- and time- dependent manner. Beta-escin can also significant increase the lifespan in mice inoculated with P388 or L1210 murine lymphoid leukemia cells, and low dose of showes coordination effects in combination with DOX by increasing DOX's antileukemic efficacy. Moreover, beta-escin can induce apoptosis and led to an accumulation of sub G1 population in K562 and HL-60 leukemia cells, which indicate that the molecular mechanism of beta-escin's antiproliferate activity is related to its apoptitic ability. The data presented here indicate that beta-escin is a potent natural inhibitor of proliferation and inducer of apoptosis in various leukemia cells and beta-escin has the potential to be a promising candidate for cancer treatment.
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