Ciprofloxacin-mediated induction of S-phase cell cycle arrest and apoptosis in COLO829 melanoma cells

Background

Low effectiveness of anti-melanoma therapies makes it necessary to search for new drugs that could improve or replace the standard chemotherapy. Fluoroquinolones are a group of synthetic antibiotics, used in the treatment of wide range of bacterial infections. Moreover, this class of antibiotics has shown promising anti-tumor activity in several cancer cell lines. The aim of this study was to examine the effect of ciprofloxacin on cell viability, apoptosis and cell cycle distribution in COLO829 melanoma cells.

Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest.     

Nitric oxide induces apoptosis and autophagy; autophagy down-regulates NO synthesis in physalin A-treated A375-S2 human melanoma cells

Physalin A is an active withanolide isolated from Physalis alkekengi var. franchetii, a traditional Chinese herbal medicine named Jindenglong, which has been used for the treatment of sore throat, hepatitis, eczema and tumors in China. Our previous study demonstrated that physalin A induced apoptosis and cyto-protective autophagy in A375-S2 human melanoma cells. Induction of reactive oxygen species (ROS) with physalin A triggered apoptosis. In this study, NO generated by physalin A induced apoptosis and autophagy in A375-S2 cells, since physalin A induced the expression of inducible nitric oxide synthase (iNOS) in the cells. Generation of NO partially promoted both apoptosis and autophagy in A375-S2 cells. NO suppressed mTOR expression, which led to autophagy induction. An autophagic inhibitor, 3-methyladenine (3MA) promoted NO production, while acceleration of autophagy with an autophagic agonist rapamycin repressed NO production, suggesting that autophagy and NO production form a negative feedback loop that eventually protects the cells from apoptosis. The results together with the previous study indicate apoptosis and autophagy induced by physalin A in A375-S2 cells; the autophagy, repressing production of reactive nitrogen species (RNS) and ROS, protects the cells from apoptosis.     

In vitro and in vivo anti-melanoma action of metformin

The in vitro and in vivo anti-melanoma effect of antidiabetic drug metformin was investigated using B16 mouse melanoma cell line. Metformin caused a G₂/M cell cycle arrest associated with apoptotic death of melanoma cells, as confirmed by the flow cytometric analysis of cell cycle/DNA fragmentation, phosphatidylserine exposure and caspase activation. Metformin-mediated apoptosis of melanoma cells was preceded by induction of oxidative stress and mitochondrial membrane depolarization, measured by flow cytometry in cells stained with appropriate fluorescent reporter dyes. The expression of tumor suppressor protein p53 was increased, while the mRNA levels of anti-apoptotic Bcl-2 were reduced by metformin, as revealed by cell-based ELISA and real-time RT-PCR, respectively. Treatment with metformin did not stimulate expression of the cycle blocker p21, indicating that p21 was dispensable for the observed cell cycle arrest. The activation of AMP-activated protein kinase (AMPK) was not required for the anti-melanoma action of metformin, as AMPK inhibitor compound C completely failed to restore viability of metformin-treated B16 cells. Metformin induced autophagy in B16 cells, as demonstrated by flow cytometry-detected increase in intracellular acidification and immunoblot-confirmed upregulation of autophagosome-associated LC3-II. Autophagy inhibitors ammonium chloride and wortmannin partly restored the viability of metformin-treated melanoma cells. Finally, oral administration of metformin led to a significant reduction in tumor size in a B16 mouse melanoma model. These data suggest that anti-melanoma effects of metformin are mediated through p21- and AMPK-independent cell cycle arrest, apoptosis and autophagy associated with p53/Bcl-2 modulation, mitochondrial damage and oxidative stress.     

雷帕霉素对黑色素瘤B-16细胞增殖与凋亡的影响

目的观察雷帕霉（Rapamycin,RAPA）对体外培养的黑色素瘤B-16细胞增殖和凋亡的影响。方法应用MTT比色法检测细胞增殖活力;采用ELISA检测细胞凋亡;倒置显微镜观察细胞形态学变化。结果0～40nmol·L^-1 RAPA能使B-16细胞增殖活力降低并使其发生凋亡。且具有时．效和量．效依赖关系;显微形态表现为细胞体积缩小,粘附性能减弱。结论RAPA能抑制黑色素B-16细胞增殖并可诱导其发生凋亡。     

Riccardin D induces cell death by activation of apoptosis and autophagy in osteosarcoma cells

Macrocyclic bisbibenzyls, characteristic components derived from liverworts, have various biological activities. Riccardin D (RD), a liverwort-derived naturally occurring macrocyclic bisbibenzyl, has been found to exert anticancer effects in multiple cancer cell types through apoptosis induction. However, the underlying mechanisms of such effects remain undefined. In addition, whether RD induces other forms of cell death such as autophagy is unknown. In this study, we found that the arrest of RD-caused U2OS (p53 wild) and Saos-2 (p53 null) cells in G1 phase was associated with the induction of p53 and p21^WAF1 in U2OS cells. RD-mediated cell cycle arrest was accompanied with apoptosis promotion as indicated by changes in nuclear morphology and expression of apoptosis-related proteins. Further studies revealed that the antiproliferation of RD was unaffected in the presence of p53 inhibitor but was partially reversed by a pan-inhibitor of caspases, suggesting that p53 was not required in RD-mediated apoptosis and that caspase-independent mechanisms were involved in RD-mediated cell death. Except for apoptosis, RD-induced autophagy occurred as evidenced by the accumulation of microtubule-associated protein-1 light chain-3B-II, formation of AVOs, punctate dots, and increased autophagic flux. Pharmacological blockade of autophagy activation markedly attenuated RD-mediated cell death. RD-induced cell death was significantly restored by the combination of autophagy and caspase inhibitors in osteosarcoma cells. Overall, our study revealed RD-induced caspase-dependent apoptosis and autophagy in cancer cells, as well as highlighted the importance of continued investigation on the use of RD as a potential anticancer candidate.     

A novel aza-naphthoquinone inhibits tumor growth by inducing apoptosis

Natural product menaquinone has been reported to exhibit antitumor activity mediated by inhibiting IDO-1 enzyme, and naphthoquinone compounds like pyranaphthoquinone have attracted extensive research interests since they share the naphthoquinone pharmacophores. In the present study, a novel series of aza-naphthoquinones was synthesized based on 6,7-dichloroquinoline-5,8-dione structure, and their cytotoxicities were screened using lung cancer cells. Among them CY-26-11was identified as the most potent one. CY-26-11 concentration-dependently induced apoptosis in A549 human lung adenocarcinomacells, as indicated by annexin-V staining. Further experimental data show that CY-26-11 down-regulated Bcl-2 while up-regulatedBax and Puma protein levels, and activated caspase-3. Finally, CY-26-11 significantly inhibited the growth of Lewis lung carcinomaxenografts in vivo without significant major toxicities. Taken together, our results demonstrate that CY-26-11 exhibited IDO-1-independent anti-tumor effects both in vitro and in vivo, providing a new anti-tumor mechanism for naphthoquinonecompounds.     

Ochratoxin A at low concentrations inhibits in vitro growth of canine umbilical cord matrix mesenchymal stem cells through oxidative chromatin and DNA damage

Ochratoxin A (OTA) exposure during pregnancy in laboratory animals induces delayed/abnormal embryo development. Foetal adnexa-derived mesenchymal stem cells (MSCs) could help evaluate the developmental risk of exposure to chemicals in advanced gestational age. We tested the effects of OTA at concentrations ranging from 2.5 × 10⁻⁴ to 25 nM on growth parameters of canine umbilical cord matrix (UCM)-derived MSCs. The hypothesis that oxidative chromatin and DNA damage could underlie OTA-mediated cell toxicity was also investigated. After in vitro exposure, OTA significantly decreased cell density and increased doubling time in a passage- and concentration-dependent manner and no exposed cells survived beyond passage 5. Significantly higher rates of cells showed condensed and fragmented chromatin and oxidized DNA, as assessed by OxyDNA assay. These findings showed that in vitro exposure to OTA, at picomolar levels, perturbs UCM-MSC growth parameters through oxidative chromatin and DNA damage, suggesting possible consequences on canine foetal development.     

Anticancer properties of 10-hydroxycamptothecin in a murine melanoma pulmonary metastasis model in vitro and in vivo

Lung cancer, including lung metastatic cancer, remains one of the most difficult types of cancer to treat. Therefore, the search for new agents for its treatment is very important. 10-Hydroxycamptothecin (HCPT) was proved to have ideal anticancer activity in curing series cancer cells. In this study, the anticancer effect of HCPT on melanoma lung metastasis cancer was investigated by several administration routes, and whether the effect may be attributed to the induction of tumor cells apoptosis was determined. MTT assay results showed that HCPT exhibited selective cytotoxic activity against B16-F10 cells in a concentration- and time-dependent manner. Hoechst 33258 staining and transmission electron microscopy showed typical apoptotic morphology such as condensed chromatin, irregular nuclei, and apoptotic body formation. Flow cytometry analysis indicated a growth on apoptotic cells and a cell-cycle arrest in S phase after treatment with HCPT. In vivo melanoma pulmonary metastases were inhibited by treatment with HCPT. A more significant inhibition was observed if HCPT was administered by aerosol inhalation than that given by i.v. or i.p. administration. Thus, HCPT exhibited potential anticancer activity against B16-F10 cells in vitro and in vivo. However, the possible mechanisms involved still need to be investigated to explain this behavior.     

Zearalenone induces apoptosis and cytoprotective autophagy in primary Leydig cells

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. ZEA causes reproductive disorders, genotoxicity, and testicular toxicity in animals. However, little is known about the functions of apoptosis and autophagy after exposure to ZEA in Leydig cells. This study investigated the effects of ZEA on rat Leydig cells. Results showed that ZEA at different doses significantly inhibited the growth of Leydig cells by inducing apoptosis. ZEA treatment upregulated Bax expression, promoted cytochrome c release into the cytosol, and triggered mitochondria-mediated apoptosis. Consequently, caspase-9 and downstream effector caspase-3 were activated, followed by the cleavage of poly(ADP-ribose) polymerase (PARP), resulting in Leydig cell apoptosis. ZEA treatment also upregulated LC3-II and Beclin-1 expression, suggesting that ZEA induced a high level of autophagy. Pretreatment with chloroquine (an autophagy inhibitor) and rapamycin (an autophagy inducer) increased and decreased the rate of apoptosis, respectively, in contrast to other ZEA-treated groups. Autophagy delayed apoptosis in the ZEA-treated Leydig cells. Therefore, autophagy may prevent cells from undergoing apoptosis by reducing ZEA-induced cytotoxicity.     

Induction of apoptosis and autophagy by sodium selenite in A549 human lung carcinoma cells through generation of reactive oxygen species

Selenium in the form of sodium selenite has been reported to exert anti-tumor effects in several cancer cell types by inducing autophagic cell death and apoptosis mediated by reactive oxygen species (ROS). However, the exact molecular pathways underlying these effects have not been fully established. The present study used A549 human lung carcinoma cells for further investigation of the anti-cancer mechanism of sodium selenite. We showed that sodium selenite modulated both the extrinsic and intrinsic apoptotic pathways, which were interconnected by Bid truncation. We used z-VAD-fmk, a pan-caspase inhibitor, to demonstrate that sodium selenite-induced apoptosis was dependent on the activation of caspases. Sodium selenite also increased autophagy, as indicated by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3II, and elevation of autophagic flux. Pretreatment with bafilomycin A1 enhanced sodium selenite-induced apoptosis, indicating that sodium selenite-induced autophagy functioned as a survival mechanism. Sodium selenite treatment also resulted in generation of ROS, which abrogated mitochondrial membrane potential (MMP) and regulated both apoptosis and autophagy. Phospho-nuclear factor erythroid 2-related factor 2 (p-Nrf2) showed a ROS-dependent translocation to the nucleus, which suggested that Nrf2 might increase cell survival by suppressing ROS accumulation and apoptosis mediated by oxidative stress. Sodium selenite treatment of A549 cells therefore appeared to trigger both apoptosis and cytoprotective autophagy, which were both mediated by ROS. The data suggest that regulation of ROS generation and autophagy can be a potential strategy for treating lung cancer that is resistant to pro-apoptotic therapeutics.     

Bisphenol A affects cell viability involved in autophagy and apoptosis in goat testis sertoli cell

Bisphenol A (BPA) is shown to be the endocrine disruptor that induces reproductive dysfunction in male animals. In this study, we aim to probe the effects of BPA exposure on induction of autophagy in goat Sertoli Cells (gSCs), as well as the relationship between autophagy and apoptosis. Results indicated that exposure to BPA (100, 200, 300, 400, 500 and 600 μM) decreased the cell viability in a concentration-dependent manner. Exposure of gSCs to 500 μM BPA for 12 h resulted in in vitro triggered loss of mitochondrial membrane potential (ΔΨm) and increased reactive oxygen species (ROS) production. Apoptosis with an increase in Bax:Bcl-2 ratio and higher rates of autophagy, such as autophagosome formation and increased expression of autophagy-related markers were also induced in gSCs exposed to 500 μM BPA. Furthermore, treatment with 350 nM Rapamycin (Rap, autophagy activator) alleviated a decrease in cell viability, intracellular ROS production, and reduction of ΔΨm, as well as decreasing apoptosis. Collectively, our results indicated that gSCs viability was disrupted after BPA treatment through affecting ROS production, mitochondrial membrane potential and inducing autophagy/apoptosis.     

Mulberry fruits extracts induce apoptosis and autophagy of liver cancer cell and prevent hepatocarcinogenesis in vivo

Hepatocellular carcinoma (HCC) is one of the most common malignancies in Taiwan. Many risks factors induce liver chronic inflammation, fibrosis, cirrhosis, and hepatocellular carcinoma. Mulberry fruits containing polyphenols to remove free radicals and mitigate inflammation has been reported to not only against gastric cancer, melanoma and leukemia but also prevent liver injury induced by alcohol or CCl₄ in previous researches. The aim of this study is to examine whether Mulberry could inhibit hepatocarcinogenesis. In animal experiment, diethylnitrosamine (DEN) was used to induce hepatic tumorgenesis. After injecting DEN, the rats treated with mulberry water extracts (MWE) had less and smaller tumor than others without MWE. Moreover, MWE reduced the serum ALT and AST, HCC marker, cleavage caspases, Ser-15-p53 and Ser46-p53 induced by DEN. Further, we observed that mulberry polyphenol extracts (MPE) inhibited the cell growth of HepG2 cell and Hep3B cell. By using flow cytometry and western blotting methods, MPE induced HepG2 cell apoptosis by increase subG1 cells and the elevated expression of caspase-3/8/9. Instead of apoptosis, MPE caused Hep3B cells autophagy by inhibiting Akt and mTOR phosphorylation. Comprehensively, mulberry extracts has a potential to be a health supplement to prevent hepatocarcinogenesis in the future.     

Cytotoxic activity of Apomine is due to a novel membrane-mediated cytolytic mechanism independent of apoptosis in the A375 human melanoma cell line

Summary Apomine, a novel bisphosphonate ester, has demonstrated anticancer activity in a variety of cancer cell lines; however, its mechanism of cytotoxicity is not well understood. Previous work has demonstrated that Apomine induces cell death by activation of caspase-3 in several cancer cell types. However, we have demonstrated that Apomine induces cell death in the A375 human melanoma cell line through a novel membrane-mediated mechanism that is independent of caspase-3 activation. This mechanism of membrane lysis may apply to other bisphosphonates and may be an important mechanism for overcoming resistance to apoptosis. Interestingly, Apomine-mediated cell death in the A375 and UACC 3093 human melanoma cell lines is also independent of N-Ras farnesylation, which was a previously described mechanism of action for Apomine in other cancer cell types. These data suggest that Apomine induces cell death through a novel plasma membrane-mediated cytolytic pathway, independent of caspase-3 activation and N-Ras farnesylation.     

Nec-1对铝致神经细胞凋亡和自噬的作用

目的观察和探讨在铝致神经细胞死亡中,坏死抑制剂1(Nec-1)对凋亡和自噬的作用。方法用体外原代培养小鼠神经细胞的方法,制造铝损伤神经细胞模型,然后用细胞活力检测、荧光定量PCR(qRT-PCR)、蛋白印迹(Western-Blot)以及流式细胞术等方法从多角度对Nec-1的作用进行研究。结果①流式结果显示,在Al3+(2mmol/L)作用于神经细胞后,随着Nec-1剂量的增加,细胞的凋亡率呈下降趋势,且差异有统计学意义(P0.05);②qRT-PCR结果显示,以Al3+(2mmol/L)为对照,Nec-1(60和90μmol/L)可使神经细胞的凋亡和自噬相关基因的表达呈显著性下降(P0.05);③Western-Blot结果显示,同Al3+(2mmol/L)组比,Nec-1(60和90μmol/L)组凋亡相关蛋白caspase-3的表达下降(P0.01),Nec-1(30和60μmol/L)可使自噬相关蛋白LC3-Ⅱ表达下降(P0.05)。结论在本试验条件下,Nec-1可减少铝导致的神经细胞凋亡和自噬,起到保护神经细胞的作用。     

异丙安替比林通过抑制蛋白激酶B通路诱导人肾癌细胞凋亡和自噬

目的 探究异丙安替比林(Propyphenazone)对人肾癌细胞Caki-1增殖抑制和凋亡的影响及其分子机制。方法 使用MTT(四甲基偶氮唑盐)法和克隆形成抑制实验检测异丙安替比林在不同时间和浓度下对Caki-1细胞增殖能力的影响;Annexin V-FITC/PI双染色检测对Caki-1细胞凋亡的影响;Hoechst 33342染色法检测Caki-1细胞染色质固缩状态;蛋白质印迹法(Western Blot)检测Caki-1细胞的丝氨酸/苏氨酸蛋白激酶AKT(蛋白激酶B)、磷酸化的丝氨酸/苏氨酸蛋白激酶p-AKT以及DNA修复酶PARP蛋白表达变化;激光共聚焦显微镜检测LC3蛋白在细胞中的点状聚集。结果 MTT实验结果表明异丙安替比林可抑制人肾癌细胞Caki-1的增殖(P<0.05),同时,异丙安替比林分别处理24 h、48 h、72 h后,所对应的半抑制浓度(IC₅₀)分别为105 μM、83 μM、56 μM;同时,克隆形成抑制实验表明20 μM和40 μM的异丙安替比林处理Caki-1细胞后,克隆形成率分别降为38%(P<0.05)和20%(P<0.01);流式结果表明,当使用0 μM、40 μM、80 μM和100 μM 的异丙安替林处理Caki-1细胞后,细胞凋亡率分别为7.4%、13.5%、34.5%和50.9%;Western Blot结果表明,随着异丙安替比林浓度的增加,p-AKT蛋白表达降低,并伴随DNA修复酶PARP失活。免疫荧光实验结果表明异丙安替比林可能诱导细胞发生自噬。结论异丙安替比林显著抑制人肾癌Caki-1细胞增殖,并通过抑制AKT通路诱导肾癌细胞发生凋亡和自噬。     

Cordycepin-induced apoptosis and autophagy in breast cancer cells are independent of the estrogen receptor

Cordycepin (3-deoxyadenosine), found in Cordyceps spp., has been known to have many therapeutic effects including immunomodulatory, anti-inflammatory, antimicrobial, and anti-aging effects. Moreover, anti-tumor and anti-metastatic effects of cordycepin have been reported, but the mechanism causing cancer cell death is poorly characterized. The present study was designed to investigate whether the mechanisms of cordycepin-induced cell death were associated with estrogen receptor in breast cancer cells. Exposure of both MDA-MB-231 and MCF-7 human breast cancer cells to cordycepin resulted in dose-responsive inhibition of cell growth and reduction in cell viability. The cordycepin-induced cell death in MDA-MB-231 cells was associated with several specific features of the mitochondria-mediated apoptotic pathway, which was confirmed by DNA fragmentation, TUNEL, and biochemical assays. Cordycepin also caused a dose-dependent increase in mitochondrial translocation of Bax, triggering cytosolic release of cytochrome c and activation of caspases-9 and -3. Interestingly, MCF-7 cells showed autophagy-associated cell death, as observed by the detection of an autophagosome-specific protein and large membranous vacuole ultrastructure morphology in the cytoplasm. Cordycepin-induced autophagic cell death has applications in treating MCF-7 cells with apoptotic defects, irrespective of the ER response. Although autophagy has a survival function in tumorigenesis of some cancer cells, autophagy may be important for cordycepin-induced MCF-7 cell death. In conclusion, the results of our study demonstrate that cordycepin effectively kills MDA-MB-231 and MCF-7 human breast cancer cell lines in culture. Hence, further studies should be conducted to determine whether cordycepin will be a clinically useful, ER-independent, chemotherapeutic agent for human breast cancer.     

Gaseous signal molecule SO2 regulates autophagy through PI3K/AKT pathway inhibits cardiomyocyte apoptosis and improves myocardial fibrosis in rats with type II diabetes

Myocardial fibrosis is a key link in the occurrence and development of diabetic cardiomyopathy. Its etiology is complex, and the effect of drugs is not good. Cardiomyocyte apoptosis is an important cause of myocardial fibrosis. The purpose of this study was to investigate the effect of gaseous signal molecule sulfur dioxide (SO₂) on diabetic myocardial fibrosis and its internal regulatory mechanism. Masson and TUNEL staining, Western-blot, transmission electron microscopy, RT-qPCR, immunofluorescence staining, and flow cytometry were used in the study, and the interstitial collagen deposition, autophagy, apoptosis, and changes in phosphatidylinositol 3-kinase (PI3K)/AKT pathways were evaluated from in vivo and in vitro experiments. The results showed that diabetic myocardial fibrosis was accompanied by cardiomyocyte apoptosis and down-regulation of endogenous SO₂-producing enzyme aspartate aminotransferase (AAT)_1/2. However, exogenous SO₂ donors could up-regulate AAT_1/2, reduce apoptosis of cardiomyocytes induced by diabetic rats or high glucose, inhibit phosphorylation of PI3K/AKT protein, up-regulate autophagy, and reduce interstitial collagen deposition. In conclusion, the results of this study suggest that the gaseous signal molecule SO₂ can inhibit the PI3K/AKT pathway to promote cytoprotective autophagy and inhibit cardiomyocyte apoptosis to improve myocardial fibrosis in diabetic rats. The results of this study are expected to provide new targets and intervention strategies for the prevention and treatment of diabetic cardiomyopathy.     

Novel derivatives of spirohydantoin induce growth inhibition followed by apoptosis in leukemia cells

Hydantoin derivatives possess a variety of biochemical and pharmacological properties and consequently are used to treat many human diseases. However, there are only few studies focusing on their potential as cancer therapeutic agents. In the present study, we have examined anticancer properties of two novel spirohydantoin compounds, 8-(3,4-difluorobenzyl)-1′-(pent-4-enyl)-8-azaspiro[bicyclo[3.2.1] octane-3,4′-imidazolidine]-2′,5′-dione (DFH) and 8-(3,4-dichlorobenzyl)-1′-(pent-4-enyl)-8-azaspiro[bicyclo[3.2.1]octane-3,4′-imidazolidine]-2′,5′-dione (DCH). Both the compounds exhibited dose- and time-dependent cytotoxic effect on human leukemic cell lines, K562, Reh, CEM and 8E5. Incorporation of tritiated thymidine ([³H] thymidine) in conjunction with cell cycle analysis suggested that DFH and DCH inhibited the growth of leukemic cells. Downregulation of PCNA and p-histone H3 further confirm that the growth inhibition could be at the level of DNA replication. Flow cytometric analysis indicated the accumulation of cells at subG1 phase suggesting induction of apoptosis, which was further confirmed and quantified both by fluorescence-activated cell sorting (FACS) and confocal microscopy following annexin V-FITC/propidium iodide (PI) staining. Mechanistically, our data support the induction of apoptosis by activation of the mitochondrial pathway. Results supporting such a model include, elevated levels of p53, and BAD, decreased level of BCL2, activation and cleavage of caspase 9, activation of procaspase 3, poly (ADP-ribosyl) polymerase (PARP) cleavage, downregulation of Ku70, Ku80 and DNA fragmentation. Based on these results we discuss the mechanism of apoptosis induced by DFH and its implications in leukemia therapy.