| Objective Acute myeloid leukemia(AML) is typically a disease of stem progenitor cell origin. Although the development of better chemotherapy regimens has improved induction remission and overall survival, relapse and side-effects of the chemotherapeutic agents remain the serious problems. The development of new anti-leukemic compounds from natural plants with higher bioactivity and less side effects has become one of the focuses in pharma ceutical research. Emodin is one of the bioactive components extracted from the root of Rheum palmatum.Extensive studies have reported that emodin possesses a number of biological properties. E35, one of emodin derivatives, has exhibited stronger anticancer activity when compared to emodin.This study was focused on the investigation of the effects of E35 on human acute myeloid leukemia cell lines HL-60 and Kasumi.The roles of E35-induced apoptosis and autophagy were highlighted in this study. Methods 1. Cell proliferation inhibition was accessed by MTT assay.Human acute myeloid leukemia(AML) cell lines HL-60 and Kasumi were included in this study. 2. Apoptotic cells were measured by flow cytometry after Annexin V PE/7-AAD double staining. 3. The morphology changes of cell apoptosis and autophagy were observed by fluorescence microscope after AO/EB and MDC staining.4. Changes of Beclin-1 expression level were detected by quantitative PCR. 5. Expression levels of apoptosis and autophagy related proteins, PARP, Caspase-3, Caspase-9, LC3-I/II and Beclin-1were analyzed by western blot. 6. Time-dependent changes for expression levels of apoptosis and autophagy related proteins, PARP, LC3-I/II were analyzed by western blot. 7. The effects of E35-induced cell apoptosis and autophagy were also investigated after HL-60 cells were treated with E35 alone, or E35 in combination with autophagy inhibitor(3-MA), autophagy inducer(rapamycin) and apoptosis inhibitor(z VAD-fmk). Result1. E35 dose- and time- dependently inhibited HL-60 and Kasumi cell proliferation with an average IC50 values of 4.76±0.35?mol/L and 6.06±0.29?mol/L, respectively. 2. E35 induced HL-60 and Kasumi cell apoptosis in a dose-dependent manner. The percentages of apoptotic cell in E35-conditioned groups were significantly higher than those without E35 treatment. 3. AO/EB and MDC staining results showed that cell cytoplasm appeared large sizes of apoptotic bodies and autophagic vacuoles in E35- cocultured groups, which presented in a E35 dose-dependent way. 4. The Beclin-1 m RNA expression levels in both cell lines were enhanced as higher concentrations of E35 were added in the culture system. 5. Western blot results showed that the apoptosis-related proteins, cleaved Caspase-9 and PARP, were induced when increasing doses of E35 were set up. The similar induction effects were found in LC3 and Beclin-1, the marker proteins of autophagy. 6. Autophagic protein LC3-II was induced in E35-treated HL-60 cells approximately 6 hours, and the maximum amount of LC3-II occurred at 12 h after treatment,the expression decreased at 24 h after. Apoptosis protein was presented at 24 h after HL-60 cells responsed to E35 stimuli. The presence of E35 stimulation first activated autophagy, and then induced apoptosis in HL-60 cells.7. Higher percentages of apoptosis cells were induced when E35 was combined with 3-MA treatment. While it didn't increase the percentages of apoptotic cells when HL-60 cells conditioned either E35 plus rapamycin or E35 plus z VAD-fmk treatment. Conclusion 1. Emodin derivative E35 could efficiently inhibit proliferation in AML cell lines including HL-60 and Kasumi cells, and it also induced cell apoptosis and autophagy at the same time. 2. E35-induced autophagy might play an adaptive response role in AML cells, and protected leukemia cells from existence pressured and maintained leukemic cell survival. 3. E35 in combination with autophagy inhibitor was able to further induce apoptosis in HL-60 cells, which indicated that would be a helpful strategy to further improve the anti-leukemic activity of E35. |
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