The Effects And Mechanism Of Aminosteroid Z1 On MEG-01 Leukemia Cell

Objective To explore the proliferation-inhibiting and differentiation-inducing effects and the potential mechanisms of aminosteroid Zl on human chronic myelogenous leukemia MEG-01 cells.Methods Proliferation was detected by cell count, colony count and MTT assay. Differentiation was measured by morphology, AS-D NAE stain and flow cytometry. The changes of intracellular [Ca2+]i were verified by fluorescence spectrophotometer. The changes of calcium channel protein mRNA and bcr/abl oncogene mRNA were testified by RT-PCR.Results1.The effects of aminosteroid Zl on proliferation of normal peripheral blood cells and MEG-01 cells.The growth of MEG-01 cells was inhibited after the treatment with 10-8-10-4mol/L aminosteroid Zl for 5 days. MTT assay showed that after the treatment with 10-6mol/L aminosteroid Zl for 3days, the data of the normal peripheral blood cells (MNC) was 0.035±0.003,there was no difference between the treatment group and the control group (0.036±0.002,p>0.05).The inhibition rate for MEG-01 cells in MTT assay was (50.87±11.67)% after treatment with 10-8 mol/L aminosteroid Zl for 5 days and it increased with the drug concentrations. The colony culture assay was also demonstrated that aminosteroid Zl inhibited the proliferation of MEG-01 cells in a dose-dependent manner, but no obvious effect was shown on normal peripheral blood cells.2.The effects of aminosteroid Zl on differentiation of MEG-01 cells.The morphology showed the differentiation tendency after the treatment with aminosteroid Zl for 5 days. The increased A value was detected after treatment with aminosteroid Zl in AS-D NAE stain assay. Data from flow cytometry showed that aminosteroid Zl induced MEG-01 cells toward megakaryocytic differentiation at 10-6mol/L treatment after cultured for 5 days, in which the surface-marker CD41 expression was 76%.3.The mechanisms of the proliferation-inhibiting and differentiation-inducing effects of aminosteroid Zl on MEG-01 cells.(1) The effects of aminosteroid Zl on intracellular [Ca2+]i in MEG-01 cells.MEG-01 cells preloaded with Fura-2/AM were treated with 10-6mol/L aminosteroid Zl for 1 hour. Data from fluorospectrophotometer showed that A value decreased in the treatment group compared with the control group.(2) The TRPV6 calcium channel gene mRNA expression in MEG-01 cell after treatment with 10-6mol/L aminosteroid Zl by Real-time PCR (RT-PCR).The relative quantity of MEG-01 cell calcium channel trpv6 gene mRNA from treatment group and control group were (2.36±0.18)×10-3 and (6.65±0.40)×10-3 respectively. Calcium channel gene mRNA was down-regulated by aminosteroid Zl.(3) The bcr/abl fusion gene mRNA expression in MEG-01 cell after treatment with 10-6mol/L aminosteroid Zl by RT-PCR.The relative quantity of MEG-01 cell bcr/abl fusion gene mRNA from treatment group and control group were (2.22±0.18)×10-2 and (6.2±0.22)×10-2 respectively. It was showed that aminosteroid Zl down-regulated the bcr/abl fusion gene mRNA at 10-6mol/L doConclusion1. Aminosteroid Zl inhibited the proliferation of MEG-01 cells in a dose-dependent manner, but no obvious effect was shown on normal peripheral blood cells.2. Aminosteroid Zl induced MEG-01 cells toward megakaryocytic differentiation.3. Aminosteroid Zl decreased intracellular [Ca2+]i in MEG-01 cells, and this may be caused by the blocking effects on calcium channel.4. Aminosteroid Zl down-regulated the TRPV6 calcium channel gene mRNA in MEG-01 cells, and this may acount for the intracelullar calcium decrease and be related with the aminosteroid Zl effects on the proliferation inhibition and differentiation induction.5. Aminosteroid Zl down-regulated the bcr/abl fusion gene mRNA in MEG-01 cells, and this may be associated with the mechanism of the aminosteroid Zl underlying its effects on the leukemia cells such as inhibiting proliferation and inducing differentiation.