The Effects Of Cucurmosin On The Inhibition Of Proliferation And Induction Of Apoptosis In Acute Promyelocytic Leukemia Cell Line NB4

Objective The aim of this study is to investigate the effects of cucurmosin(CUS) on proliferation inhibition and synergistic effects of CUS combined with ATRA and ATO in human Acute Promyelocytic Leukemia cell line NB4, as well as explore the effects and possible mechanisms of induction apoptosis to provide the theoretically rationale and experimental evidence for its potential clinical application for anti-APL.Methods1 Ara-c used as positive control, proliferation inhibition of NB4 cell was detected by MTT assay after CUS treatment.Growth curve of NB4 cells treated with CUS was measured for the effects of growth Using trypan blue exclusion test.2 Results of MTT method test synergistic inhibitory effects of CUS combined with ATRA or ATO in NB4 line.3 Fluorescence microscopic analysis of Hoechst/PI staining and Transmission electron microscope were used to observe morphological chage and study Ultrastructural changes of NB4 cell exposed to CUS.4 Typical DNA ladder was observed through agarose gel electrophoresis in NB4 cell.Propidium iodide (PI) staining associated with flow cytometry was used to study the effect on NB4 cell cycle and apoptosis induced by CUS. Apoptosis rate of NB4 cell was examined by Annexin V/PI double staining analysis.5 Western blot was used to analyze the expressions of apoptosis related proteins including Bax, bcl-2, caspase-3, caspase-8, caspase-9 and downstream effector PARP as well as PML-RARa fusion protein of NB4 cells after CUS treatment.6 RQ-PCR was used to detect the expression of PML-RARa fusion gene mRNA. Results1 The results of MTT assay showed that the rate of proliferation inhibition in a time-dependent and dose-dependent manner increased from 13.3% to 93.7% when NB4 cell exposed to 0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5, 1umol/L CUS for 24, 48 and 72 hours.The 50% inhibitory concentrations(IC50)of CUS for 24, 48 and 72h were 1.235, 0.089 and 0.055 umol/L, respectively.Growth curve through Trypan blue staining test counting viable cell indieated that, compared with control, cell growth of CUS treated group was inhibitied, latent period was prolonged, curve tends to declined gently and cells doubled time extended.2 Inaddition, the inhibitive ratio of CUS combined with ATRA or ATO was higher than CUS, ATRA and ATO alone on NB4 cells, q>0.85, using MTT assay.3 Fluorescence microscopic analysis of Hoechst/PI staining revealed cells of CUS treated group were chromatin condensation and aggregation stained bright blue and nuclear lobes, disintegrate, as well as intact cell completely became fragment up to 1umol/L CUS. Morphological analysis through transmission electron micro- scopy presented, compared with control, the shrinking of cell volume, irregular morphology of nuclear,increase of nuclear heterochromatin and typical morpholo- gic changes of apoptosis such as chromatin condensation, shrunken, fragmentation nucleus as well as"apoptotic body"after NB4 cells exposure to cucurmosin.4 DNA agarose gel electrophoresis revealed a characteristic"ladder"of intemucleo- somal DNA fragmentation and approximately the 180-220bp integral multiple increasing distribution.Flow cytometry was used to evaluate the DNA content present massive hypodiploid cell population(sub-G1) and the percentage of apoptotic cells was 23.1%, 31.9%, 4.2%and 59.8%, respectively after NB4 cell exposure to 0.0156, 0.0312, 0.0625, 0.25umol/L CUS for 72h showing a significant time-dependent manner.The results of annexinV/PI stainin detected cells apoptosis indicated that the rate of apoptosis improved(1.7%, 2.4%, 2.8%, 4.9%, 7.5%and 17.0%)following the augmentation of the CUS concentratio(0, 0.0156, 0.0312, 0.0625, 0.25 and 1umol/L for 72h).5 Western blot analysis showed that bcl-2 and PML-RARa proteins sharply reduced, meanwhile expression of cleaved caspase-3, cleaved caspase-9, 85kD PARP and Bax protein strongly upregulate in a time-dependent and dose-dependent way, but pro-caspase-8 had no obvious changes and cleaved caspase-8 did not express.6 Expression of PML-RARa fusion gene mRNA reduced in dose-dependent maner by using real-time quantitative polymerase chain reaction.Conclusion1 The proliferation of NB4 cell was remarkbly inhibited by CUS in a concentration- and time-dependent manner in vitro. Meanwhile,The combination of CUS and ATRA/ATO could synergistically inhibit NB4 cell growth.2 Cucurmosin can effectively induces apoptosis in a concentration- and time- dependent manner. Mitochondria initiated apoptotic pathway possibly involved in the apoptosis process of NB4 cell induced by CUS.3 It is likely one of antileukemia mechanism in NB4 cell treated with CUS that PML-RARa targeted protein and mRNA was highly down-regulated.