Journal of Experimental Hematology * AA005. AA005 > 200 nmol /L AA005 NB4 U937 K h R R A

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1 Journal of Experimental Hematology Article ID * NB4 CCK-8 NB4 Western Blot caspase-3 PARP-1 < 200 nmol /L > 200 nmol /L NB4 U937 K h NB4 caspase-3 PARP-1 caspase Z-VAD-fmk < 200 nmol /L G 2 /M G 2 /M R R A Effect of Annonaceous Acetogenin Mimic on Proliferative Inhibition of Leukemia Cells in vitro and Its Possible Mechanisms HAN Bing YAO Zhu-Jun 1 WANG Li-Shun * Biomedical Research Institute Shanghai Jiaotong University School of Medicine Ruijin Hospital Shanghai China 1 Shanghai Institute of Organic Chemistry Chinese Academy of Sciences Shanghai China * Corresponding Author WANG Li-Shun Senior Scientist. Tel Jywangls@ shsmu. edu. cn Abstract This study was aimed to investigate the biological behavior of annonaceous acetogenin mimic in various kinds of leukemia cells and further elaborated its possible mechanisms in acute promyelocytic leukemia APL cell line NB4. The proliferative inhibition of leukemia cells was measured by CCK-8 method. Cell death was determined by trypan blue. Cell morphological features of NB4 treated with were examined by microscopy after Wright' s staining. The form of cell death was measured by flow cytometry. Proteins PARP-1 and caspase-3 were detected by Western blot. Flow cytometry was used to detect the cell cycle arrest induced by of low concentration. The results showed that > 200 nmol /L significantly inhibited proliferation of all tested leukemia cell lines in a concentration-dependent manner. The vast majority of cells went to die after leukemia cell lines of NB4 U937 and K562 were treated with different concentration of for 48 h. Typical morphologic changes significantly appeared in NB4 cells after treatment. almost simultaneously induced early apoptosis and late apoptosis. The little cleavage of PARP-1 and activation of caspase-3 happened in -induced cell death and caspase-3 inhibitor Z-VAD-fmk could not block the cell death. The non-toxic concentrations of < 200 nmol /L caused NB4 cells G 2 /M-phase arrest. It is concluded that annonaceous acetogenin mimic induces significant proliferative inhibition of various leukemia cell lines in a concentration-dependent manner which may be associated with cell death and G 2 /M-phase arrest induced by. Key words annonaceous acetogenin mimic acute promyelocytic leukemia cell proliferation cell death cell cycle J Exp Hematol NSFC QH * Jywangls@ shsmu. edu. cn

2 550 J Exp Hematol SDS-PAGE Bio-Rad / 1 acute promyelocytic leukemia NB4 U937 APL t PM L-RARα Kasumi-1 K562 PML RARα 2 HL-60 T Jurkat 10% 100 U /ml μg /ml 2 mmol /L RPMI 1640 DMEM 37 5% CO all-trans retinoic acid ATRA CCK-8 APL 4 CCK-8 APL ATRA ATRA 10 μl CCK h 450 nm 5 annonaceous acetogenin mimic bullatacin 0. 4% min ApoAlert Annexin-Ⅴ kit PBS μl μl Annexin-Ⅴ 5 μl propidium iodine PI 15 min 300 μl 1 Caspase 3 Western blot Gibco RPMI mmol /L Tris Sigma NSC % SDS 0. 2% 20% 5% β caspase- 3 PARP Santa Cruz β-actin Oncogene HRP DAKO HRP- Pierce ApoAlert Annexin-Ⅴ kit BD Biosciences caspase-3 Z-VAD-fmk Sigma min 8% - 15% SDS-PAGE 5% TBS 1 h 2 h 4 TBS HRP 1 htbs 3 HRP- X BD FACSCalibur CO 2 Thermo PBS 1 Thermo Nikon 75% - 20 PBS

3 551 2 PBS100 μl PBS 100 mg /ml RNase A ph 7. 4 Tris-HCl 30 min 25 μg /ml PI 20 min 400 μl PBS DNA t- P < NB4 U937 K562 Kasumi-1 HL-60 JurkatCCK-8 Figure 2. Death in NB4 U937 K562 leukemia cells μmol /L induced by for 48 h. A NB4 cells. B U937 cells. C K562 cells. 48 h * P < ** P < caspase NB4 1 NSC A NB4 48 h Annexin-Ⅴ PI 3B μmol /L Annexin- Ⅴ + / PI % 3. 83% Figure 1. Inhibitory effect of 48 h on different leukemia cell lines detected by CCK-8 method. CCK-8 U937 NB4 K562 CCK-8 2 Figure 3. Death of NB4 cells induced by. A NB4 cell morphological features of NB4 examined by microscopy after Wright' s staining. B cell death measured by flow NB4 cytometry. C proteins PARP-1 and caspase-3 detected by 1 μmol /L 20 μmol /L NSC Western blot.

4 552 J Exp Hematol % % Annexin-Ⅴ + / PI % 5. 91% % % Caspase -3 Western blot caspase-3 2 G 1 S 3C 1 μmol /L NB4 24 h G 2 /M 6 caspase-3 NB4 G 2 /M PARP-1 caspase h caspase-3 48 h 60% NSC % caspase-3 caspase-3 Z-VADfmk NB4 NSC Z-VAD-fmk NSC nmol /L NB4 2 d Figure 5. Proliferative inhibition and viability of NB4 cells after treatment with in low concentration. A the proliferative inhibition of NB4 cells measured by CCK-8 assay. B the cell viability measured by trypan blue assay. Figure 4. Cell death measured by flow cytometry after and NSC treatment without or with Z-VAD-fmk. Figure 6. Changes of cell cycle in NB4 cells treated with in non-toxic concentrations. 200 nmol /L U937 NB4 K % 61% 33% 13% 34% 11% NADH- - Ⅰ nmol /L NB4 9 CCK-8 5A 5B 2

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