Induction of differentiation rescues HL-60 cells from Rana catesbeiana ribonuclease-induced cell death

Rana catesbeiana ribonuclease (RC-RNase) exerted strong anti-tumor activity and its cytotoxicity was shown to correlate with differentiation stages of three different hepatoma cell lines. In this study, we demonstrate different RC-RNase cytotoxicity in undifferentiated HL-60 cells and in those that had been induced to differentiate by retinoic acid or dimethylsulfoxide. RC-RNase showed cytotoxicity in undifferentiated HL-60 cells, but not in HL-60 cells undergoing terminal differentiation. Furthermore, the caspase-9/caspase-3 pathway was activated when RC-RNase induced death in undifferentiated HL-60 cells and induction of differentiation led to a reversal of the caspase activation pathway.     

Synergistic cytotoxicity of Rana catesbeiana ribonuclease and IFN-gamma on hepatoma cells

RC-RNase purified from Rana catesbeiana (bullfrog) oocytes is a pyrimidine-guanine sequence-specific ribonuclease. RC-RNase is derived from the RNase superfamily genes exerting distinct ribonucleolytic activity and possesses cytotoxicity to tumor cells, but rarely to primary cells. In this study, we utilized RC-RNase to function with antiproliferative cytokines. The combination with TNF-alpha or TNF-beta would not aggravate cell death. However, the combination with IFN-gamma could induce synergistic cytotoxicity verified by XTT assays toward three hepatoma cell lines bearing different differentiation stages. The distinct cytotoxicity from RC-RNase or RC-RNase/IFN-gamma on different hepatoma cells was correlated with the differentiation extent but not the proliferation rate of the cells. Despite the synergistic cytotoxicity and severe mitochondrial disruptions in the RC-RNase/IFN-gamma-treated cells, we scarcely detected any significant feature of apoptosis or necrosis by FACS analysis on annexin-V/propidium iodide staining. The mechanisms of cell death triggered by RC-RNase or RC-RNase/IFN-gamma require further investigation.     

Withanolide induces apoptosis in HL-60 leukemia cells via mitochondria mediated cytochrome c release and caspase activation

The present study is on the growth inhibitory effect of Withania somnifera methanolic leaf extract and its active component, withanolide on HL-60 promyelocytic leukemia cells. The decrease in survival rate of HL-60 cells was noted to be associated with a time dependent decrease in the Bcl-2/Bax ratio, leading to up regulation of Bax. Both the crude leaf extract and the active component activated the apoptotic cascade through the cytochrome c release from mitochondria. The activation of caspase 9, caspase 8 and caspase 3 revealed that caspase was a key mediator in the apoptotic pathway. DNA fragmentation analysis revealed typical ladders as early as 12h indicative of caspase 3 role in the apoptotic pathway. Flow cytometry data demonstrated an increase of sub-G1 peak upon treatment by 51% at 24h, suggesting the induction of apoptotic cell death in HL-60 cells.     

Involvement of p38 MAP kinase during iron chelator-mediated apoptotic cell death

Iron is an essential element for the neoplastic cell growth, and iron chelators have been tested for their potential anti-proliferative and cytotoxic effects. To determine the mechanism of cell death induced by iron chelators, we explored the pathways of the three structurally related mitogen-activated protein (MAP) kinase subfamilies during apoptosis induced by iron chelators. We report that the chelator deferoxamine (DFO) strongly activates both p38 MAP kinase and extracellular signal-regulated kinase (ERK) at an early stage of incubation, but slightly activates c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) at a late stage of incubation. Among three MAP kinase blockers used, however, the selective p38 MAP kinase inhibitor SB203580 could only protect HL-60 cells from chelator-induced cell death, indicating that p38 MAP kinase serves as a major mediator of apoptosis induced by iron chelator. DFO also caused release of cytochrome c from mitochondria and induced activation of caspase 3 and caspase 8. Interestingly, treatment of HL-60 cells with SB203580 greatly abolished cytochrome c release, and activation of caspase 3 and caspase 8. Collectively, the current study reveals that p38 MAP kinase plays an important role in iron chelator-mediated cell death of HL-60 cells by activating downstream apoptotic cascade that executes cell death pathway.     

Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway

Surfactin has been known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in surfactin-induced apoptosis remain poorly understood. The present study was undertaken to elucidate the underlying network of signaling events in surfactin-induced apoptosis of human breast cancer MCF-7 cells. In this study, surfactin caused reactive oxygen species (ROS) generation and the surfactin-induced cell death was prevented by antioxidants N-acetylcysteine (NAC) and catalase, suggesting involvement of ROS generation in surfactin-induced cell death. Surfactin induced a sustained activation of the phosphorylation of ERK1/2 and JNK, but not p38. Moreover, surfactin-induced cell death was reversed by PD98059 (an inhibitor of ERK1/2) and SP600125 (an inhibitor of JNK), but not by SB203580 (an inhibitor of p38). However, the phosphorylation of JNK rather than ERK1/2 activation by surfactin was blocked by NAC/catalase. These results suggest that the action of surfactin on MCF-7 cells was via ERK1/2 and JNK, but not via p38, and the ERK1/2 and JNK activation induce apoptosis through two independent signaling mechanisms. Surfactin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 expression ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade reaction. The NAC and SP600125 blocked these events induced by surfactin. Moreover, the general caspase inhibitor z-VAD-FMK inhibited the caspase-6 activity and exerted the protective effect against the surfactin-induced cell death. Taken together, these findings suggest that the surfactin induces apoptosis through a ROS/JNK-mediated mitochondrial/caspase pathway.     

Synthetic menthyl α/β-(1→6)-diglucopyranosides-induced cell death in human leukemia cells is dependent on caspases

A series of alkyl α/β-(1→6)-diglucopyranosides 1-12 were synthesized and assessed for cytotoxicity against HL-60, U937, Molt-3 and MCF-7 cancer cell lines. The menthyl derivatives displayed strong cytotoxic properties showing IC(50) values between 6 and 16 μM. Furthermore, we demonstrated that the selected synthetic (+)-menthyl β-(1→6)-diglucopyranoside 5 induces apoptotic cell death in human leukemia cells through a mechanism that involves activation of multiple caspases. Cell death was completely prevented by the non-specific caspase inhibitor z-VAD-fmk and found to be associated with the release of cytochrome c, an increase in the expression of Bax levels and a decrease in the generation of reactive oxygen species.     

Oligonucleosomal DNA Fragmentation in MCF-7 Cells Undergoing Palmitate-Induced Apoptosis

Oligonucleosomal fragmentation of nuclear DNA is the late-stage apoptosis hallmark. In apoptotic mammalian cells the fragmentation is catalyzed by DFF40/CAD DNase primarily activated by caspase 3 through the site-specific proteolytic cleavage of DFF45/ICAD. A deletion in the casp3 gene of human breast adenocarcinoma MCF-7 results in lack of procaspase 3 in these cells. The absence of caspase 3 in MCF-7 leads to disability to activate oligonucleosomal DNA fragmentation in TNF-alpha induced cell death. In this study, sodium palmitate was used as an apoptotic stimulus for MCF-7. It has been shown that palmitate but not TNF-alpha induces both apoptotic changes in nuclei and oligonucleosomal DNA fragmentation in casp3-mutated MCF-7. Activation and accumulation of 40-50 kD DFF40-like DNases in nuclei of palmitate-treated apoptotic MCF-7 were detected by SDS-DNA-PAGE assay. Microsomal fraction of apoptotic MCF-7 does not contain any detectable DNases, but activates 40-50 kD nucleases when incubated with human placental chromatin. Furthermore, microsomes of apoptotic MCF-7 induce oligonucleosomal fragmentation of chromatin in a cell-free system. Both the activation of DNases and chromatin fragmentation are suppressed in the presence of the caspase 3/7 inhibitor Ac-DEVD-CHO. Microsome-associated caspase 7 is suggested to play an essential role in the induction of oligonucleosomal DNA fragmentation in casp3-deficient MCF-7 cells.     

Evidence for the involvement of carbon-centered radicals in the induction of apoptotic cell death by artemisinin compounds

Artemisinin and its derivatives are currently recommended as first-line antimalarials in regions where Plasmodium falciparum is resistant to traditional drugs. The cytotoxic activity of these endoperoxides toward rapidly dividing human carcinoma cells and cell lines has been reported, and it is hypothesized that activation of the endoperoxide bridge by an iron(II) species, to form C-centered radicals, is essential for cytotoxicity. The studies described here have utilized artemisinin derivatives, dihydroartemisinin, 10beta-(p-bromophenoxy)dihydroartemisinin, and 10beta-(p-fluorophenoxy)dihydroartemisinin, to determine the chemistry of endoperoxide bridge activation to reactive intermediates responsible for initiating cell death and to elucidate the molecular mechanism of cell death. These studies have demonstrated the selective cytotoxic activity of the endoperoxides toward leukemia cell lines (HL-60 and Jurkat) over quiescent peripheral blood mononuclear cells. Deoxy-10beta-(p-fluorophenoxy)dihydroartemisinin, which lacks the endoperoxide bridge, was 50- and 130-fold less active in HL-60 and Jurkat cells, respectively, confirming the importance of this functional group for cytotoxicity. We have shown that chemical activation is responsible for cytotoxicity by using liquid chromatography-mass spectrometry analysis to monitor endoperoxide activation by measurement of a stable rearrangement product of endoperoxide-derived radicals, which was formed in sensitive HL-60 cells but not in insensitive peripheral blood mononuclear cells. In HL-60 cells the endoperoxides induce caspase-dependent apoptotic cell death characterized by concentration- and time-dependent mitochondrial membrane depolarization, activation of caspases-3 and -7, sub-G(0)/G(1) DNA formation, and attenuation by benzyloxycarbonyl-VAD-fluoromethyl ketone, a caspase inhibitor. Overall, these results indicate that endoperoxide-induced cell death is a consequence of activation of the endoperoxide bridge to radical species, which triggers caspase-dependent apoptosis.     

Involvement of Akt in mitochondria-dependent apoptosis induced by a cdc25 phosphatase inhibitor naphthoquinone analog

Vitamin K-related analogs induce growth inhibition via a cell cycle arrest through cdc25A phosphatase inhibition in various cancer cell lines. We report that 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (DDN), a naphthoquinone analog, induces mitochondria-dependent apoptosis in human promyelocytic leukemia HL-60 cells. DDN induced cytochrome c release, Bax translocation, cleavage of Bid and Bad, and activation of caspase-3, -8, -9 upon the induction of apoptosis. Cleavage of Bid, the caspase-8 substrate, was inhibited by the broad caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), whereas cytochrome c release was not affected, suggesting that activation of caspase-8 and subsequent Bid cleavage occur downstream of cytochrome c release. DDN inhibited the activation of Akt detected by decreasing level of phosphorylation. Overexpression of constitutively active Akt protected cells from DDN-induced apoptosis, while dominant negative Akt moderately enhanced cell death. Furthermore, Akt prevented release of cytochrome c and cleavage of Bad in DDN-treated HL-60 cells. Taken together, DDN-induced apoptosis is associated with mitochondrial signaling which involves cytochrome c release via a mechanism inhibited by Akt.     

Amplification loop cascade for increasing caspase activity induced by docetaxel

The hierarchy of events accompanying induction of apoptosis by the microtubule inhibitor docetaxel was investigated in HL-60 human leukemia cells. Treatment of HL-60 cells with docetaxel resulted in the production of reactive oxygen species (ROS), activation of caspase-3 (-like) protease, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation, bcl-2 phosphorylation and apoptosis. Docetaxel elicited ROS production from NADPH oxidase as demonstrated by specific oxidase inhibitor diphenylene iodonium (DPI). ROS mediated the caspase-3 activation and apoptosis in HL-60 cells. The caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) effectively inhibited JNK/SAPK activation, bcl-2 phosphorylation and partially attenuated the ROS production induced by docetaxel. Docetaxel-induced bcl-2 phosphorylation was completely blocked by expression of dominant negative JNK or the JNK/SAPK inhibitor SP600125. Overexpression of bcl-2 partially prevented docetaxel-mediated ROS production and subsequent caspase-3 activation, thereby inhibiting apoptotic cell death. It is thus conferred that such sequent events as ROS production, caspase activation, JNK/SAPK activation, bcl-2 phosphorylation and the further generation of ROS should be parts of an amplification loop to increase caspase activity, thereby facilitating the apoptotic cell death process.     

Essential requirement of cytochrome c release for caspase activation by procaspase-activating compound defined by cellular models

Mitochondrial cytochrome c (cyt. c) release and caspase activation are often impaired in tumors with Bcl-2 overexpression or Bax and Bak-defective status. Direct triggering of cell death downstream of Bax and Bak is an attractive strategy to kill such cancers. Small molecule compounds capable of direct caspase activation appear to be the best mode for killing such tumors. However, there is no precise model to screen such compounds. The currently employed cell-free systems possess the inherent drawback of lacking cellular contents and organelles that operate in integrating cell death signaling. We have developed highly refined cell-based approaches to validate direct caspase activation in cancer cells. Using this approach, we show that PAC-1 (first procaspase-activating compound), the first direct activator of procaspases identified in a cell-free system, in fact requires mitochondrial cyt. c release for triggering caspase activation similar to other antitumor agents. It can induce significant caspase activation and cell death in the absence of Bax and Bak, and in cells overexpressing Bcl-2 and Bcl-xL. This study for the first time defines precise criteria for the validation of direct caspase-activating compounds using specialized cellular models that is expected to accelerate the discovery of potential direct caspase activators.     

p38 mitogen-activated protein kinase is a key regulator of 5-phenylselenyl- and 5-methylselenyl-methyl-2'-deoxyuridine-induced apoptosis in human HL-60 cells

Two novel, modified thymidine nucleosides, 5-phenylselenyl-methyl-2'-deoxyuridine (PhSe-T) and 5-methylselenyl-methyl-2'-deoxyuridine (MeSe-T), trigger reactive oxygen species (ROS) generation and DNA damage and thereby induce caspase-mediated apoptosis in human HL-60 cells; however, the mechanism leading to caspase activation and apoptotic cell death remains unclear. Therefore, we investigated the signaling molecules involved in nucleoside derivative-induced caspase activation and apoptosis in HL-60 cells. PhSe-T/MeSe-T treatment activated two mitogen-activated protein kinases (MAPKs), extracellular-receptor kinase (ERK) and p38, and induced the phosphorylation of two downstream targets of p38, ATF-2 and MAPKAPK2. In addition, the selective p38 inhibitor SB203580 suppressed PhSe-T/MeSe-T-induced apoptosis and activation of caspase-3, -9, -8, and -2, whereas the jun amino-terminal kinase (JNK) inhibitor SP600125 and the ERK inhibitor PD98059 had no effect. SB203580 and an ROS scavenger, tiron, inhibited PhSe-T/MeSe-T-induced histone H2AX phosphorylation, which is a DNA damage marker. Moreover, tiron inhibited PhSe-T/MeSe-T-induced phosphorylation of p38 and enhanced p38 MAP kinase activity, indicating a role for ROS in PhSe-T/MeSe-T-induced p38 activation. Taken together, our results suggest that PhSe-T/MeSe-T-induced apoptosis is mediated by the p38 pathway and that p38 serves as a link between ROS generation and DNA damage/caspase activation in HL-60 cells.     

The histone deacetylase inhibitor sodium butyrate interacts synergistically with phorbol myristate acetate (PMA) to induce mitochondrial damage and apoptosis in human myeloid leukemia cells through a tumor necrosis factor-alpha-mediated process

Interactions between the histone deacetylase inhibitor sodium butyrate (SB) and phorbol 12-myristate 13-acetate (PMA) were examined in human myeloid leukemia cells (U937 and HL-60). Exposure of U937 cells to 1 mM SB and 1 nM PMA (24 h) markedly induced caspase activation and apoptosis, events accompanied by impaired differentiation induction (e.g., reduced plastic adherence and diminished expression of CD11b) as well as reduced clonogenic survival. The PKC inhibitor GF109203X blocked SB-/PMA-mediated apoptosis. Comparable results were obtained in HL-60 cells. Apoptosis was associated with early procaspase 8 activation and Bid cleavage, accompanied by pronounced mitochondrial damage (e.g., loss of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release). Neutralization of endogenous TNFalpha by a human soluble TNF receptor substantially blocked SB-/PMA-induced cytochrome c release and apoptosis. Consistent with this, ectopic expression of a mutant dominant-negative caspase 8 or CrmA resulted in a significant decrease in SB-/PMA-induced apoptosis, whereas Bcl-2 overexpression did not. SB/PMA treatment also triggered a decline in the S and G(2)M populations, and dephosphorylation of p34(cdc2). These results indicate that SB interacts with low concentrations of PMA to induce apoptosis in human leukemia cells and that this process proceeds through a PKC-/TNFalpha-dependent pathway in which procaspase 8 and Bid activation play key roles.     

The analysis of biochemical markers of MCF-7 cells apoptosis induced by recombinant analog of lactaptin

Earlier we isolated and characterized human milk pro-apoptotic peptide - lactaptin and generated its engineered analog - RL2. It was shown that both lactaptin and RL2 are capable to induce apoptotic death of MCF-7 cells. In this report we have analyzed biochemical markers of RL2 induced MCF-7 apoptosis. The activation of initiator and effector caspases as well as mitochondrial membrane potential and cytoplasm membrane changes were analyzed using flow cytometry and Western-blot methods. We have found that RL2 induced apoptotic death of MCF-7 cells was accompanied by PS exposure on the plasma membrane surface. It also was shown that RL2 has induced dissipation of mitochondrial membrane potential and resulted in activation of initiator caspases 8, 9 and effector caspase 7.     

Disparity in the induction of glutathione depletion, ROS formation, poly(ADP-ribose) polymerase-1 activation, and apoptosis by quinonoid derivatives of naphthalene in human cultured cells

The purpose of this study is to examine the differences in the induction of cytotoxic effects and poly(ADP-ribose) polymerase-1 activation in human MCF-7 breast cancer cells by quinonoid derivatives of naphthalene, including 1,2-naphthalenediol (NCAT), 1,4-naphthalenediol (NHQ), 1,2-naphthoquinone (1,2-NQ), and 1,4-naphthoquinone (1,4-NQ). Results from the cytotoxic response analyses in cells indicated that all naphthalene quinonoids induced cell death in MCF-7 cells at concentrations ranging from 0.1 to 100microM where NHQ and 1,4-NQ were more efficient than NCAT and 1,2-NQ in the induction of cell death. Results from Western blot analyses confirmed that treatment of cells with NCAT and NHQ resulted in up-regulation of p53 protein expression and a significant shift in bax/bcl2 ratio, suggesting the induction of p53-dependent apoptosis in MCF-7 cells. Additionally, we observed that all naphthalene quinonoids induced increases in reactive oxygen species (ROS) formation and glutathione (GSH) depletion in MCF-7 cells. The induction of ROS formation and GSH depletion in cells by naphthalene quinonoids decreases in the rank order 1,4-NQ>NHQ>1,2-NQ approximately equal to NCAT. Further investigation indicated that least-squares estimates of the overall rates of elimination (k(e)) of naphthalene quinonoids in MCF-7 cells decreased in the rank order 1,4-NQ>1,2-NQ>NHQ>NCAT. Values of k(e) were estimated to be between 0.280h(-1)(T(1/2)=151min) and 13.8h(-1)(T(1/2)=3.05min). These results provide evidence that the para-isomeric form of naphthalene quinonoids tend to induce acute production of ROS and alterations in intracellular redox status in cells, leading to the subsequent cell death. Further, all naphthalene quinonoids induced decreases in intracellular NAD(P)H and NAD(+) in MCF-7 cells at non-cytotoxic concentrations. The reduction of intracellular NAD(P)H in cells exposed to NCAT and 1,2-NQ was blocked by two types of poly(ADP-ribose) polymerase (PARP) inhibitors whereas PARP inhibitors did not prevent the reduction of NAD(P)H in cells exposed to NHQ and 1,4-NQ. Further investigation confirmed that increases in the number of DNA single-strand breaks were detected in MCF-7 cells exposed to NCAT and 1,2-NQ as measured by the single-cell gel electrophoresis (Comet) assay whereas NHQ and 1,4-NQ did not induce increases in the number of single-strand breaks in MCF-7 cells. Overall, results from our investigation suggest that while NHQ and 1,4-NQ are more efficient in the induction of cell death, NCAT and 1,2-NQ are prone to induce depletion of NAD(P)H and NAD(+) mediated by PARP-1 activation through formation of DNA single-strand breaks in human cultured cells.     

Cardiotoxin III induces c-jun N-terminal kinase-dependent apoptosis in HL-60 human leukaemia cells

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. The molecular effects of CTX III on HL-60 cells were dissected in the present study. We found that the antiproliferative action of CTX III on HL-60 cells was mediated through apoptosis, as characterized by an increase of sub G1 population, DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. Upregulation of Bax, downregulation of Bcl-2, the release of mitochondrial cytochrome c to cytosol and the activations of capase-9 and -3 were noted, while CTX III had no appreciable effect on the levels of Bcl-X(L) and Bad proteins. Moreover, c-Jun N-terminal kinase (JNK) was activated shortly after CTX III treatment in HL-60 cells. Consistently, the SP600125 compound, an anthrapyrazolone inhibitor of JNK, suppressed apoptosis induced by CTX III. As expected, this JNK inhibitor also attenuated the modulation of Bax and Bcl-2, as well as the cytosolic appearance of cytochrome c and the activation of caspase-3 and caspase-9 that induced by CTX III. These findings suggest that CTX III can induce apoptosis in HL-60 cells via the mitochondrial caspase cascade and the activation of JNK is critical for the initiation of the apoptotic death of HL-60 cells.     

Induction of lactoferrin gene expression in myeloid or mammary gland cells by human T-cell leukemia virus type 1 (HTLV-1) tax: implications for milk-borne transmission of HTLV-1

Human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia, is transmitted vertically via breastfeeding. We have previously demonstrated that lactoferrin, a major milk protein, enhances HTLV-1 replication, at least in part by upregulating the HTLV-1 long terminal repeat promoter. We now report that HTLV-1 infection can induce lactoferrin gene expression. Coculture with HTLV-1-infected MT-2 cells increased the levels of lactoferrin mRNA in myeloid-differentiated HL-60 cells, as well as MCF-7 cells, models of two probable sources (neutrophils and mammary epithelium) of lactoferrin in breast milk. MT-2 cell coculture could be replaced with cell-free culture supernatants of MT-2 cells to exert the same effect. Furthermore, extracellularly administered Tax protein also induced lactoferrin gene expression at physiologically relevant concentrations. In transient-expression assays, Tax transactivated the lactoferrin gene promoter in HL-60 or MCF-7 cells. Experiments with Tax mutants, as well as site-directed mutants of the lactoferrin promoter reporters, indicated that the NF-kappaB transactivation pathway is critical for Tax induction of the lactoferrin gene promoter activity in myeloid-differentiated HL-60 cells, but not in MCF-7 cells. These results suggest that HTLV-1 infection may be able to induce expression of lactoferrin in a paracrine manner in the lactic compartment. Our findings, in conjunction with our previous study, implicate that mutual interaction between HTLV-1 and lactoferrin would benefit milk-borne transmission of this virus.     

Effect of thymol on peripheral blood mononuclear cell PBMC and acute promyelotic cancer cell line HL-60

Thymol, a naturally occurring phenolic compound, has been known for its antioxidant, anti microbial, and anti inflammatory activity. Thymol has also been reported as anti-cancer agent, but its anti-cancer mechanism has not yet been fully elucidated. Thus, we aimed to investigate anticancer activity of thymol on HL-60 (acute promyelotic leukemia) cells. In our study, thymol demonstrated dose dependent cytotoxic effects on HL-60 cells after 24 h of exposure. However, thymol did not show any cytotoxic effect in normal human PBMC. The cytotoxic effect of thymol on HL-60 cells appears to be associated with induction of cell cycle arrest at sub G0/G1 phase, and apoptotic cell death based on genomic DNA fragmentation pattern. Thymol also showed significant increase in production of reactive oxygen species (ROS) activity, increase in mitochondrial H₂O₂ production and depolarization of mitochondrial membrane potential. On performing Western Blot analysis, thymol showed increase in Bax protein level with a concomitant decrease in Bcl2 protein expression in a dose dependent manner. Our study also showed activation of caspase -9, -8 and -3 and concomitant PARP cleavage, which is the hallmark of caspase-dependent apoptosis. Moreover, to rule out the involvement of other mechanisms in apoptosis induction by thymol, we also studied its effect on apoptosis inducing factor (AIF). Thymol induced AIF translocation from mitochondria to cytosol and to nucleus, thus indicating its ability to induce caspase independent apoptosis. We conclude that, thymol-induced apoptosis in HL-60 cells involves both caspase dependent and caspase independent pathways.     

Mechanism of LIGHT/interferon-gamma-induced cell death in HT-29 cells

LIGHT is a member of tumor necrosis factor (TNF) superfamily, and previous studies have indicated that in the presence of interferon-gamma (IFN-gamma), LIGHT through LTbetaR signaling can induce cell death with features unlike classic apoptosis. In present study, we investigated the mechanism of LIGHT/IFN-gamma-induced cell death in HT-29 cells, where the cell death was profoundly induced when sub-toxic concentrations of LIGHT and IFN-gamma were co-treated. LIGHT/IFN-gamma-induced cell death was accompanied by DNA fragmentation and slight LDH release. This effect was not affected by caspase, JNK nor cathepsin B inhibitors, but was partially prevented by p38 mitogen-activated protein kinase (MAPK) and poly (ADP-ribose) polymerase (PARP) inhibitors, and abolished by aurintricarboxylic acid (ATA), which is an inhibitor of endonuclease and STATs signaling of IFN-gamma. Immunobloting reveals that LIGHT/IFN-gamma could induce p38 MAPK activity, Bak and Fas expression, but down-regulate Mcl-1. Besides, LIGHT/IFN-gamma could not activate caspase-3 and -9, but decreased mitochondrial membrane potential. Although LIGHT could not affect IFN-gamma-induced STAT1 phosphorylation and transactivation activity, which was required for the sensitization of cell death, survival NF-kappaB signaling of LIGHT was inhibited by IFN-gamma. These data suggest that co-presence of LIGHT and IFN-gamma can induce an integrated interaction in signaling pathways, which lead to mitochondrial dysfunction and mix-type cell death, not involving caspase activation.