CLONING AND EXPRESSION OF A GENE ASS0CIATE WITH HL_(60) CELL APOPTOSIS INDUCED BY INHIBITION OF POLYAMINE BIOSYNTHESIS

TosearchwhethsomeblDckedgenes0ftIJmorcellsarerePfOmOtedintheeVentsassociatedwithindUctionofcelldifferenti~andaPOPtsisby~inonofpolytabfotwsis,SCreeninandd0ninofthegenesfrDmffeboconsttuctedtoHhainducedtDdriationandwsisbydeofpolyaIninbiosynthsis,DFMO,andthetwanalsisofcbogenexPresshoandaCthatyofaP0PtsisindUCtiDwereperfOIminthesStUdymromonscenCtheHLoocellswereculturedinPRMIl64()containin1o?Sat37"CandWAn5%CO2.6fnInl/LDFMOwasusedtoinducedifferenthaandwsisofHLtocells.Cellulargro…     

多胺生物合成抑制诱导HL60细胞凋亡相关基因的克隆及表达

目的克隆多胺生物合成抑制剂二氟甲基鸟氨酸（ＤＦＭＯ）诱导ＨＬ６０细胞凋亡的相关基因,分析该基因表达及诱导凋亡的活性。方法采用差示杂交法筛选目的基因克隆;以Ｎｏｒｔｈｅｒｎ杂交、形态观察、流式细胞光度法（ＦＣＭ）分析及ＤＮＡ电泳梯形图,分别证明转染细胞中该基因表达及凋亡诱导。结果成功地克隆了ＤＦＭＯ诱导ＨＬ６０细胞凋亡的相关基因ｄＦ４,并证明了ｄＦ４在转染细胞中的表达及其诱导凋亡的活性。结论提示本研究克隆的ｄＦ４基因可能是与调控ＨＬ６０细胞凋亡有关的基因。     

二氟甲基鸟氨酸对K562细胞生长特性的影响及其与端粒酶活性的相关性

目的研究二氟甲基鸟氨酸(DFMO)对K562细胞生长特性、凋亡及端粒酶活性的影响。方法采用细胞计数、形态观察、FCM分析及DNA电泳分别检测DFMO处理的细胞生长速率、周期分布及细胞凋亡。按TRAP法对细胞端粒酶活性进行检测。结果DFMO处理的细胞生长速率明显减慢;G1期细胞增多而S期细胞减少;细胞表现凋亡诱导,且其端粒酶活性受到抑制。结论DFMO引起的K562细胞生长抑制及凋亡诱导与端粒酶活性抑制有关。端粒酶的失活是抑制多胺生物合成的抗癌分子机制重要事件之一。     

多胺生物合成抑制剂DFMO对人膀胱癌细胞抑制及凋亡的实验研究

 目的 探讨多胺生物合成抑制剂DFMO对人膀胱癌EJ细胞的抑制作用。方法 应用细胞培养、活细胞摄影技术、伊红染色细胞计数和原位DNA片段末端标记，观察DFMO抑制人膀胱癌EJ细胞的增生及诱导其凋亡。结果 活细胞摄像结果显示：与空白对照组相比：4 mmol/L，6 mmol/L，8 mmol/L DFMO给药第5天，细胞生长明显抑制，随DFMO剂量增大，上述变化逐渐明显。伊红染色结果显示：4 mmol/L，6 mmol/L，8 mmol/L的DFMO均可抑制EJ细胞生长，并随剂量的增加，抑制作用逐渐增强。原位DNA片段末端标记结果显示：4 mmol/L，6 mmol/L，8 mmol/L DFMO均可诱导EJ细胞凋亡；6 mmol/L为较适宜剂量；三种检测方法均显示同时加入外源性腐胺后，EJ细胞的凋亡与对照组无明显差别。结论 DFMO可以抑制膀胱癌EJ细胞的增生并诱导其凋亡。     

Decursinol angelate inhibits PGE2-induced survival of the human leukemia HL-60 cell line via regulation of the EP2 receptor and NFκB pathway

Decursinol angelate (DA), an active pyranocoumarin compound from the roots of Angelica gigas, has been reported to possess anti-inflammatory and anti-cancer activities. In a previous study, we demonstrated that prostaglandin E₂ (PGE₂) plays a survival role in HL-60 cells by protecting them from the induction of apoptosis via oxidative stress. Flow cytometry and Hoechst staining revealed that PGE₂ suppresses menadione-induced apoptosis, cell shrinkage, and chromatin condensation, by blocking the generation of reactive oxygen species. Treatment of DA was found to reverse the survival effect of PGE₂ as well as restoring the menadione-mediated cleavage of caspase-3, lamin B, and PARP. DA blocked PGE₂-induced activation of the EP2 receptor signaling pathway, including the activation of PKA and the phosphorylation of CREB. DA also inhibited PGE₂-induced expression of cyclooxygenase-2 and the activation of the Ras/Raf/ Erk pathway, which activates downstream targets for cell survival. Finally, DA greatly reduced the PGE₂-induced activation of NF-κB p50 and p65 subunits. These results elucidate a novel mechanism for the regulation of cell survival and apoptosis, and open a gateway for further development and combinatory treatments that can inhibit PGE₂ in cancer cells.     

Tetra-arsenic tetra-sulfide (As4S4) promotes apoptosis in retinoid acid -resistant human acute promyelocytic leukemic NB4-R1 cells through downregulation of SET protein

Tetra-arsenic tetra-sulfide (As₄S₄) is an arsenic compound with antitumor activity, especially in acute promyelocytic leukemia (APL) that are resistant to retinoic acid (RA). Although recent studies have revealed that the therapeutic action of As₄S₄ is closely associated with the induction of cellular apoptosis, the exact molecular mechanism underlying this action in RA-resistant APL remains to be clarified. In this study, we found that As₄S₄-induced apoptosis was accompanied by reduced mRNA and protein expression of SET gene in RA-resistant NB4-R1 cells. Moreover, RNAi knockdown of SET gene further promoted As₄S₄-induced apoptosis, while SET overexpression recovered the cell viability, suggesting that As₄S₄ induces apoptosis through the reduction of SET protein in NB4-R1 cells. We also observed that the knockdown of SET gene resulted in the upregulation of protein phosphatase 2 (PP2A) expression and the downregulation of promyelocytic leukemia and retinoic acid receptor α fusion gene (PML-RARα) expression, which were enhanced by As₄S₄ treatments. By contrast, overexpression of SET gene resulted in PP2A downregulation and PML-RARα upregulation, which were abolished by As₄S₄ pretreatment. Since PP2A is a proapoptotic factor and PML-RARα is an antiapoptotic factor, our results suggest that As₄S₄-induced apoptosis in RA-resistant NB4-R1 cells is through the downregulation of SET protein expression, which, in turn, increases PP2A and reduces PML-RARα expressions to lead to cell apoptosis.     

二氟甲基鸟氨酸及反义bcl-2协同诱导HL60细胞凋亡

目的　观察二氟甲基鸟氨酸 (DFMO)及反义bcl 2调控bcl 2基因表达对HL6 0细胞凋亡的诱导作用。方法　构建反义bcl 2逆转录病毒重组体 ,建立产病毒细胞系 ,转染HL6 0细胞 ,用形态观察、生长曲线、FCM分析、集落形成、DNA电泳图谱、分子杂交及免疫组化等方法研究细胞生长特性及细胞凋亡诱导。结果　成功地构建了反义bcl 2逆转录病毒重组体及产病毒细胞系。以此转染HL6 0细胞 ,引起了bcl 2mRNA及蛋白表达下降 ,但生长速率、细胞周期分布及鸟氨酸脱羧酶 (ODC)基因表达水平与亲本细胞比较差别不大 ;无明显的细胞凋亡 ,仅集落形成能力有所减弱。用小剂量DFMO处理反义bcl 2转染的细胞 ,不仅生长受到明显抑制 ,而且可诱导细胞凋亡。结论　DFMO与反义bcl 2对HL6 0细胞增殖抑制及凋亡诱导有协同作用。抑制bcl 2表达能提高肿瘤细胞对DFMO作用的敏感性。     

Induction of apoptosis by SeO<Subscript>2</Subscript> in human lung carcinoma cell line GLC-82

Objective:To investigate the effect of selenium dioxide(SeO2) on human pulmonary adenocarcinoma GLC-82 cell lines to reveal its probable mechanism and the relationship between apoptosis and SeO2. Methods: Methyl thiazolyl tetrazolium (MTT) method, flow cytometry (FCM), DNA agarose gel electrophoresis, light and electron microscope were used to study cell apoptosis in human pulmonary adenocarcinoma cell line GLC-82 treated by SeO2 at different concentrations (3, 10, 30μmol/L) and for different times (24, 48, and 72 h). Results: SeO2 significantly inhibited the proliferation and induced the cell apoptosis of GLC-82 at the different concentrations after treatment of 48h and 72 h. Conclusion: Selenium dioxide could inhibit the growth of lung cancer GLC-82 cells through inducing apoptosis. The effect of inhibition is dose-dependant and time-dependant.     

The induction of apoptosis is a common feature of the cytotoxic action of ether-linked glycerophospholipids in human leukemic cells

The ability of 2 recent ether-lipid derivatives, aza-phospholipids BN52205 and BN522II, to induce apoptosis in different leukemia cell lines was investigated using I-octadecyl-2-methly-rac-glycero-3-phosphocholine (ET-18-OCH₃) as a positive control. HL60, K562, Molt-4 and U937 cells were exposed for 24 hr to 20 μM of drug. The 2 aza-derivatives were as cytotoxic as ET-18-OCH₃: BN52205 and BN522II selectively induced apoptotic death in HL60, Molt-4 and U937 cells, but not in the KS62-resistant cell line. Around 50% of DNA was fragmented in HL60 cells after exposure to the aza-derivatives, and 34% and 20% of DNA was fragmented in Molt-4 and U937 cells respectively. Similar results were obtained when cells were exposed to ET-18-OCH₃. Our data confirm that ether lipids induce apoptosis in a variety of human leukemic cells, providing a possible explanation for their selectivity and mechanism of action.     

Reverse resistance to radiation in KYSE-150R esophageal carcinoma cell after epidermal growth factor receptor signal pathway inhibition by cetuximab

Background and purpose

The purpose of our study is to examine the capacity of cetuximab to reverse radiation resistance and investigate molecular mechanisms in human radiation-resistant esophageal carcinoma cell line KYSE-150R.

Materials and methods

The radioresistant cell line KYSE-150R was established by using fractionated irradiation (FIR). The KYSE-150R cell line was exposed to radiation, treatment with cetuximab, and combined treatment. Cell cycle distribution and apoptosis were analyzed using flow cytometry. Radiation survival was analyzed using clonogenic assays. RT² profiler™ PCR array was performed to analyze EGF/PDGF signaling pathway genes.

Results

The established esophageal carcinoma cell line KYSE-150R showed higher radioresistance than parental cell line. Cetuximab could reverse the radiation resistance of KYSE-150R cells. Cell cycle analysis showed that combination with radiation and cetuximab resulted in the accumulation of cells in G1 and G2/M phases, with the reduction of cells within the S phase. Cetuximab enhanced the apoptosis induced by radiation. RT² profiler™ array showed that some intracellular signaling genes deriving from EGF/PDGF signaling pathway regulated by cetuximab.

Conclusions

Irradiation combined with EGFR blocked by cetuximab may reverse the resistance to radiation in radioresistant esophageal carcinoma cell. The mechanisms may include cell cycle perturbation and enhancement of radiation-induced apoptosis. Further studies are needed to evaluate the role of cetuximab in combination with radiotherapy in the management of esophageal carcinoma.     

G2/M cell cycle arrest and apoptosis induced by COH-203 in human promyelocytic leukemia HL-60 cells

The combretastatin A-4/oltipraz hybrid (COH), 5-(3-amino-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-3H−1,2-dithiole-3-one (COH-203) is one of the COH compounds synthesized by our previous study, which has been reported to affect a number of cancer cell lines, such as SGC-7901, KB, HT-1080, HepG2, SMMC-7721 and BEL-7402. The sensitivity of human acute leukemia cell lines to COH-203, and the mechanism underlying its anti-proliferative effects remain unknown, which was investigated in the present study. In the present study, it was demonstrated that COH-203 had notable time- and dose-dependent antiproliferative effects on the human acute promyelocytic leukemia HL-60 cell line. Furthermore, COH-203 treatment resulted in cell cycle arrest at G₂/M phase in a dose-dependent manner, and subsequently induced apoptosis. Western blot analysis revealed that upregulation of cyclin B was associated with G₂/M arrest. In addition, treatment with COH-203 resulted in downregulated expression of Bcl-2. This result revealed that COH-203-induced apoptosis in HL-60 cells may occur via the mitochondrial pathway in a caspase-dependent manner.     

Low dose cytosine-arabinoside has only minimal differentiation inducing capacity in HL60 cells

Cytosine-arabinoside (ARA-C) in low doses induces complete remissions in myelodysplastic syndromes and acute leukemia. Evidence is accumulating that these remissions are not reached by differentiation induction but through cytotoxicity. In HL60 cells differentiation was measured by a comprehensive panel of quantitative and qualitative markers of maturation. After exposure to ARA-C (10⁻⁷ M) for 4 days HL60 cells did not mature morphologically. Cell volume increased. The increase in esterase activity was small and did not reach the amount measured in normal monocytes. There was no significant difference in latex phagocytosis and NBT reduction between cultures with and without ARA-C. HL60 cells were arrested in S-phase and clonogenic capacity persisted. The observed changes after exposure to ARA-C seem to be caused by impeded cell division while synthesis of protein continues. We conclude that ARA-C in low dose exerts its effect by halting proliferation through cytotoxic effects and not by differentiation induction.     

转染外源CC10对肺腺癌细胞系A549的CyclinD1蛋白及mRNA的影响

Objective： To investigate the effects of exogenous CC10 gene transfection on cell cycle and the expression of cyclinD1 protein and mRNA in A549 cells. Methods： A549 cells in all test groups （group A to E） and control group （group F） were transfected with exogenous CC10 gene by liposome for 8, 16, 24, 36, 48 and 0 h respectively. CC10 protein expression was detected in A549 cells by Western blot. The growth inhibitory rate was detected by MTT method. Flow cyometry analysis （FCS） and AnnexinV-PI staining were used to determine the changes of cell cycle progression and apoptosis rate in all groups. CyclinD1 protein and mRNA expression in A549 cells was detected by the methods of immunocytochemistry and RT-PCR. Results： Exogenous CC10 gene could inhibit the growth of A549 cells, and the growth inhibitory rates in all test groups （from group A to E） were 24.7%, 33.1%, 44.3%, 61.7% and 74.2% respectively, and that in group F was 6.24%. CC10 blocked the cell cycle progression at G0/G1 and induced apoptosis gradually. In A549 cells of test groups, the expression of cyclinD1 protein and mRNA was significantly decreased. Conclusion： The inhibitory effects of the transfection of exogenous CC10 gene on G0/G1 cycle of lung cancer cells might be related with the down-regulation of cyclinD1 gene.     

Induction of apoptosis by heat and γ-radiation in a human lymphoid cell line; role of mitochondrial changes and caspase activation

Purpose: The aim of the study was to investigate the molecular mechanisms involved in apoptosis of human promyelocytic cells (HL60) induced by hyperthermia and to compare this to radiation-induced apoptosis as a reference model.

Materials and methods: Apoptosis of HL60 cells was induced by heat-treatment (43°C during 1?h) or by γ-radiation (8?Gy) and followed at increasing time periods after treatment with Annexin V binding to phosphatidylserine (PS). The transition of the mitochondrial membrane potential (Δψ_m) was estimated by the extent of mitochondrial JC-1 uptake. Bcl-2 and Bax protein expression levels were monitored using fluorescent-labelled antibodies. Caspase activation was studied using a fluorochrome-labelled pan-caspase inhibitor (FLICA), which also allowed one to study the kinetics of the apoptotic cascade.

Results: After heat-treatment or irradiation of HL60 cells, a decreased Δψ_m as well as PS membrane expression were detectable after 8?h. Bcl-2 and Bax protein expression levels were decreased and increased, respectively, 1?h after heat-treatment or irradiation. The apoptotic rate of HL60 cells, as measured by the FLICA binding, was faster with heat-treatment as compared to γ-irradiation. Addition of a pan-caspase inhibitor prevented PS externalization after heat-treatment but not after irradiation. The presence of a pan-caspase inhibitor did not influence the decrease of Δψ_m both after heat-treatment and γ-irradiation. However, the addition of the specific caspase-2 inhibitor zVDVAD-fmk prevented the mitochondrial breakdown after heat-treatment. Inhibition of caspase-2 had no effect on the γ-irradiation induced apoptosis.

Conclusion: These results suggest that the commitment to apoptosis in HL60 cells after heat-treatment is started by mitochondrial membrane transition involving the Bcl-2 family members and is mainly executed in a caspase-dependent pathway. The results suggest that caspase-2 plays a key role in the heat-induced apoptosis.     

EFFECTS OF SeO2 ON REGULATORY REGIONS p250 OF c—fos GENE

Objective: This study was designed to determine whether regulatory regions p250 of c-fos gene were responsive to SeO₂ and to seek the possible mechanisms of regulation. Methods: HeLa cells were transfected with plasmids p250-tk CAT containing upstream regulating regions of c-fos gene. Cells were treated by SeO₂ for 20 min. CAT expression in transfected cells was observed by thin layered chromatography. Results: In transfected HeLa cells CAT expression showed obvious increase after exposure to SeO₂, especially in 10 μmol/L and 30 μmol/L group (P<0.05). Conclusion: Through affecting regulatory regions p250 of c-fos gene, SeO₂ exerted biological effect on tumor cells. SeO₂ possibly had anti-tumor effects. Biography: YU Hai-jian (1963–), male, doctor of medicine, associate professor, Peking Union Medical College Hospital, majors in research on the respiratory diseases.     

Estradiol control of ornithine decarboxylase mRNA,enzyme activity,and polyamine levels in MCF-7 breast cancer cells: therapeutic implications

Previous studies have shown that natural polyamines - putrescine, spermidine, and spermine - play a key role in the mechanism of action of estrogens in breast cancer. Ornithine decarboxylase (ODC) is the first enzyme of the polyamine biosynthetic pathway. To examine estrogenic regulation of polyamine biosynthesis in breast cancer, we measured ODC mRNA, ODC activity, and polyamine levels in G₁ synchronized MCF-7 cells. ODC mRNA and activity increased four-fold over that of cells in G₁ phase between 8 to 16 h after the addition of estradiol. Polyamine levels showed a sharp increase by 8 h after the addition of estradiol and decreased by 12 h. We further examined whether synthetic homologs of putrescine or spermidine could replace natural polyamines in supporting MCF-7 cell growth. Treatment of MCF-7 cells with 1 mM difluoromethylornithine (DFMO), an inhibitor of ODC, suppressed putrescine, spermidine, and spermine levels by 74, 78, and 10%, respectively, within 48 h. Cells treated with DFMO for 48 h were supplemented with either putrescine or its homologs or spermidine or its homologs. Diaminopropane, diaminobutane (putrescine), and diaminopentane were capable of fully or partially reversing the growth inhibitory effects of DFMO, whereas diaminoethane had no significant effect. Among a series of triamines, H₂N(CH₂)_nNH(CH₂)₃NH₂ (where n = 2 to 8; abbreviated as APn n = 4 for spermidine, or AP4), spermidine was most effective in reversing the effects of DFMO, whereas compounds with shorter or longer methylene bridging regions were less effective. AP8 was ineffective in reversing the growth inhibitory effects of DFMO. At 10 µM concentration, AP8 also inhibited DNA synthesis by 66%, as measured by [³H]-thymidine incorporation. These data show that MCF-7 cells have a strong requirement for polyamines for their growth and that estradiol stimulates the polyamine cascade by inducing the ODC mRNA level. Our results also suggest that polyamine homologs such as AP8 might be potentially useful in breast cancer therapy.     

DFMO通过Fas/FasL通路诱导人肺癌细胞凋亡的研究

背景与目的:Fas/FasL系统是细胞凋亡的重要通路。为了解Fas/FasL系统在肿瘤多胺生物合成抑制导致恶性表型逆转中的作用,本研究拟探讨多胺生物合成抑制剂α-二氟甲基鸟氨酸(α-difluoromethylornithine,DFMO)对人肺癌A549细胞生长、凋亡的影响及其与人肺癌相关抗原、rasP21蛋白及Fas/FasL表达的相关性。方法:用MTT法观察细胞生长,流式细胞仪分析细胞周期,DNAladder电泳法观察细胞凋亡,SP免疫组化法及RT-PCR法检测细胞蛋白及基因表达。结果:DFMO可抑制A549细胞生长并诱导凋亡,使G1期细胞增多(61.0±2.08)%,S期细胞减少(21.2±0.88)%,出现DNAladder;同时下调A549细胞人肺癌相关抗原及rasP21蛋白表达,上调Fas基因mRNA及蛋白表达。结论:DFMO通过Fas/FasL通路诱导肺癌A549细胞的凋亡,并可能与人肺癌相关抗原及rasP21蛋白表达调控有关。     

in vivo effects of 4-amidinoindan-l-one 2′-amidinohydrazone (CGP 48664A) and α-difluoromethylornithine (DFMO) on L1210 growth,cell-cycle phase distribution and polyamine contents

We studied the in vivo effects of 4-amidinoindan-l-one 2′-amidinohydrazone (CGP 48664A), α-difluoromethylornithine (DFMO) and a combination of CGP 48664A-DFMO on tumor growth, cell-cycle phase distribution and polyamine contents. DBA-2 mice were inoculated i.p. with 10⁵ L1210 cells on day 0, treated i.p. on days 1–4 and killed on day 5. As compared to controls, CGP 48664A, DFMO and the CGP 48664A-DFMO combination reduced L1210 cell numbers by 33, 43 and 85%, respectively. CGP 48664A did not affect cell-cycle phase distribution. DFMO and the CGP 48664A-DFMO combination caused a moderate and a heavy accumulation in G₀/G₁- and G₂/M-phases, respectively. Compared with controls, the CGP 48664A-DFMO combination reduced putrescine, spermidine and total polyamines, but did not affect spermine. Compared with CGP 48664A, the CGP 48664A-DFMO combination caused lower putrescine and total polyamines, higher spermine, but no change in spermidine. Compared with DFMO, the CGP 48664A-DFMO combination caused higher putrescine and spermidine, lower spermine, but no change in total polyamine levels. We conclude that CGP 48664A potentiates the cystostatic effect of DFMO in vivo. The resulting growth inhibition is accompanied by an accumulation in G₀/G₁- and G₂/M-phases and a reduction of putrescine and spermidine. The data suggest that perturbed polyamine composition rather than reduced spermidine or total polyamine pool size causes a profound growth inhibition. © 1995 Wiley-Liss, Inc.     

Bax overexpression enhances apoptosis induced by adriamycin in HCC-9204 cells

Objective:To investigate the effect of overexpression of Bax to the sensitivity of human HCC-9204 cells to adriamycin (ADR). Methods:Human cultured hepatocellular carcinoma cell line HCC-9204 was exposed in vitro to adriamycin for various time. An inducible vector containing Bax gene,with ZnSO4 as external inducer was constructed. Cell apoptosis was ascertained by morphological criteria,detection of apoptotic DNA fragmentation by TUNEL assay and flow cytometry. Tetrazolium blue (MTT) assay was used to evaluate the differences in drug sensitivity of HCC-9204 cells after Bax-transfection. Results:HCC-9204 cells treated with adriamycin at 20μmol/L showed extensive cell death. TUNEL assay showed nucleus fragmentation. And apoptotic peak was also shown by flow cytometry. FACS analyses showed a significant sub-G1 peak and apoptosis in 31% cells at 24h after treatment. Furthermore, the time-course of cell viability following exposure of HCC-9204/Bax cells to adriamycin showed that Bax was able to significantly decrease cell survival following exposure to adriamycin.Conclusion: These results indicate that apoptosis can be induced effectively by adriamycin and overexpression of Bax can sensitize HCC-9204 cells to apoptosis induced by adriamycin.     

M2-A induces apoptosis and G2–M arrest via inhibiting PI3 K/Akt pathway in HL60 cells

Amonafide, a naphthalimide derivative, although selected for exploratory clinical trials for its potent anticancer activity, has long been challenged by its unpredictable side effects. In the present study, a novel amonafide analogue, M₂-A 2-(2-(dimethylamino)ethyl)-6-(thiophene-2-ylmethylamino)-1H-benzo[de]isoquinoline-1,3(2H)-dione was ascribed to its potent effects on topoisomerase IIα. Moreover, our investigation indicates that M₂-A induces G₂/M phase growth arrest through inhibiting PI3 K/Akt pathway. M₂-A inhibits proliferation of HeLa, HL60, HCT-8, A375, MCF-7 and MRC-5 cells, especially inhibits proliferation of HL60 with an IC₅₀ value of 18.86 μM. M₂-A can not only induce DNA fragmentation, but also enhance Annexin V-FITC binding of the cells. On the one hand the expression levels of protein Cyclin B1, Cdk1 changed in response to M₂-A treatment in HL60 cells. On the other hand we observed the inhibition of NF-κB nuclear translocation, up-regulation of Bax and down-regulation of Bcl-2, the caspase -3, -9 activity increase in HL60 cells after treated with M₂-A, which indicated that the mitochondrial pathway was involved in the apoptosis signal pathway. Our results showed that the phosphorylation of p85/PI3 K and Akt decreased following M₂-A treatment. In summary, M₂-A displayed a significant anti-tumor effect through cell cycle arrest and apoptotic induction in HL60 cells, which suggested that M₂-A might have therapeutic potential against leukaemia.