Apoptosis In Human Cell Lines Research Articles

Hsa-miR-6165 downregulates insulin-like growth factor-1 receptor (IGF-1R) expression and enhances apoptosis in SW480 cells.

MicroRNAs are small non-coding RNAs that are implicated in various biological processes. Hsa-miR-6165 (miR-6165), located in the p75NTR gene, is known to induce apoptosis in human cell lines, but its mechanism of action is not fully understood yet. Here, we predicted the insulin-like growth factor 1 receptor (IGF-1R) gene as a bona fide target for miR-6165. The overexpression of miR-6165 in SW480 cells resulted in significant downregulation of IGF-1R expression as detected by real time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Also, it resulted in reduced transcript levels of AKT2, AKT3, PI3KR3, PI3KR5, CCND1, c-MYC and P21 genes detected by RT-qPCR analysis. In addition, a direct interaction between miR-6165 and a 3'UTR sequence of the IGF-1R gene was verified through a dual luciferase assay. Furthermore, miR-6165 and IGF-1R showed opposite patterns of expression during the neural differentiation process of NT2 cells. Annexin V analysis and MTT assay showed that miR-6165 overexpression was followed by increased apoptosis and reduced the viability rate of SW480 cells. Moreover, a lower expression level of miR-6165 was detected in high-grade colorectal tumors compared with low-grade tumors. Taken together, the results of our study suggest a tumor suppressive role of miR-6165 in colorectal cancer, which seems to take place by regulating IGF-1R gene expression.

Loss of RASSF4 Expression in Multiple Myeloma Promotes RAS-Driven Malignant Progression.

RAS mutations occur frequently in multiple myeloma (MM), but apart from driving progression, they can also stimulate antitumor effects by activating tumor-suppressive RASSF proteins. Although this family of death effector molecules are often silenced in cancers, functional data about RASSF proteins in MM are lacking. Here, we report that RASSF4 is downregulated during MM progression and correlates with a poor prognosis. Promoter methylation analysis in human cell lines revealed an inverse correlation between RASSF4 mRNA levels and methylation status. Epigenetic modulating agents restored RASSF4 expression. Enforced expression of RASSF4 induced G2-phase cell-cycle arrest and apoptosis in human cell lines, reduced primary MM cell viability, and blocked MM growth in vivo Mechanistic investigations showed that RASSF4 linked RAS to several pro-death pathways, including those regulated by the kinases MST1, JNK, and p38. By activating MST1 and the JNK/c-Jun pathway, RASSF4 sensitized MM cells to bortezomib. Genetic or pharmacological elevation of RASSF4 levels increased the anti-MM effects of the clinical relevant MEK1/2 inhibitor trametinib. Kinome analysis revealed that this effect was mediated by concomitant activation of the JNK/c-Jun pathway along with inactivation of the MEK/ERK and PI3K/mTOR/Akt pathways. Overall, our findings establish RASSF4 as a tumor-suppressive hub in MM and provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib to treat patients with tumors exhibiting low RASSF4 expression.Significance: These findings provide a mechanistic rationale for combining trametinib with HDAC inhibitors or bortezomib in patients with multiple myeloma whose tumors exhibit low RASSF4 expression. Cancer Res; 78(5); 1155-68. ©2017 AACR.

Mechanisms of l-Serine Neuroprotection in vitro Include ER Proteostasis Regulation

β-N-methylamino-L-alanine (L-BMAA) is a neurotoxic non-protein amino acid produced by cyanobacteria. Recently, chronic dietary exposure to L-BMAA was shown to trigger neuropathology in nonhuman primates consistent with Guamanian ALS/PDC, a paralytic disease that afflicts Chamorro villagers who consume traditional food items contaminated with L-BMAA. However, the addition of the naturally occurring amino acidL-serine to the diet of the nonhuman primates resulted in a significant reduction in ALS/PDC neuropathology. L-serine is a dietary amino acid that plays a crucial role in central nervous system development, neuronal signaling, and synaptic plasticity and has been shown to impart neuroprotection from L-BMAA-induced neurotoxicity both in vitro and in vivo. We have previously shown that L-serine prevents the formation of autofluorescent aggregates and death by apoptosis in human cell lines and primary cells. These effects are likely imparted by L-serine blocking incorporation of L-BMAA into proteins hence preventing proteotoxic stress. However, there are likely other mechanisms for L-serine-mediated neuroprotection. Here, we explore the molecular mechanisms of L-serine neuroprotection using a human unfolded protein response real-time PCR array with genes from the ER stress and UPR pathways, and western blotting. We report that L-serine caused the differential expression of many of the same genes as L-BMAA, even though concentrations of L-serine in the culture medium were ten times lower than that of L-BMAA. We propose that L-serine may be functioning as a small proteostasis regulator, in effect altering the cells to quickly respond to a possible oxidative insult, thus favoring a return to homeostasis.

Expression and Function of hsa-miR-6165 in Human Cell Lines and During the NT2 Cell Neural Differentiation Process.

MicroRNAs are small non-coding RNAs that posttranscriptionally regulate mRNA expression. hsa-miR-6165 which was previously discovered in our group is located in the forth intron of p75NTR gene and its function is still under investigation. As P75NTR has diverse cellular functions, some of the complexity of its function could be attributed to the internally located microRNA. Our analysis revealed that treatment of HCT116 cells with 5-azacytidine promoted differential expression of hsa-miR-6165 from its host gene which is consistent with the bioinformatic prediction of an independent promoter for hsa-miR-6165. In addition, hsa-miR-6165 promoter is capable of driving GFP reporter gene in HeLa cells. The putative target gene expression level which was detected using RT-qPCR is inversely proportional to the expression level of hsa-miR-6165 during NT2 cell neural differentiation. Furthermore, hsa-miR-6165 overexpression resulted in significant downregulation of ABLIM-1, PVRL1, and PDK1 target genes, while it attenuates NT2 neural differentiation. Hsa-miR-6165 overexpression in SW480 cells also resulted in significant downregulation of PKD1, DAGLA, and PLXNA2 putative target genes, while it increases the sub-G1 cell population of SW480 and HEK293T cells as detected by flow cytometry. Overall, in this study, we report an independent promoter for hsa-miR-6165 which is active in HeLa cells. Additionally, hsa-miR-6165 targets ABLIM-1, PVRL1, PKD1, PLXNA2, and PDK1 genes, and unlike in HEK293T and SW480 cells, hsa-miR-6165 overexpression does not affect HeLa cells while its downregulation reduces sub-G1 cell population. Our results validate that hsa-miR-6165 affects the cell cycle progression and could increase apoptosis in human cell lines.

Human SUV3 helicase regulates growth rate of the HeLa cells and can localize in the nucleoli.

The human SUV3 helicase (SUV3, hSUV3, SUPV3L1) is a DNA/RNA unwinding enzyme belonging to the class of DexH-box helicases. It localizes predominantly in the mitochondria, where it forms an RNA-degrading complex called mitochondrial degradosome with exonuclease PNP (polynucleotide phosphorylase). Association of this complex with the polyA polymerase can modulate mitochondrial polyA tails. Silencing of the SUV3 gene was shown to inhibit the cell cycle and to induce apoptosis in human cell lines. However, since small amounts of the SUV3 helicase were found in the cell nuclei, it was not clear whether the observed phenotypes of SUV3 depletion were of mitochondrial or nuclear origin. In order to answer this question we have designed gene constructs able to inhibit the SUV3 activity exclusively in the cell nuclei. The results indicate that the observed growth rate impairment upon SUV3 depletion is due to its nuclear function(s). Unexpectedly, overexpression of the nuclear-targeted wild-type copies of the SUV3 gene resulted in a higher growth rate. In addition, we demonstrate that the SUV3 helicase can be found in the HeLa cell nucleoli, but it is not detectable in the DNA-repair foci. Our results indicate that the nucleolar-associated human SUV3 protein is an important factor in regulation of the cell cycle.

Toxicity assessment of carbon black waste: A by-product from oil refineries

In Singapore, approximately 30t/day of carbon-based solid waste are produced from petrochemical processes. This carbon black waste has been shown to possess physical properties that are characteristic of a good adsorbent such as high external surface area. Therefore, there is a growing interest to reutilize and process this carbon black waste into secondary materials such as adsorbents. However, the carbon black waste obtained from petrochemical industries may contain heavy metals that are hazardous to human health and the environment, hence restricting its full potential for re-utilization. Therefore, it is important to examine the possible toxicity effects and toxicity mechanism of carbon black waste on human health.In this study, inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis showed that the heavy metals, vanadium (V), molybdenum (Mo) and nickel (Ni), were present in the carbon black waste in high concentrations. Three human cell lines (HepG2 cells, MRC-5 cells and MDA-MB-231 cells) were used to investigate the toxicity of carbon black waste extract in a variety of in vitro assays. Results from MTS assays indicated that carbon black waste extract decreased the viability of all three cell lines in a dose and time-dependent manner. Observations from confocal microscopy further confirmed this phenomenon. Flow cytometry assay also showed that carbon black waste extract induced apoptosis of human cell lines, and the level of apoptosis increased with increasing waste concentration. Results from reactive oxygen species (ROS) assay indicated that carbon black waste extract induced oxidative stress by increasing intracellular ROS generation in these three human cell lines. Moreover, induction of oxidative damage in these cells was also observed through the alteration of glutathione (GSH) and superoxide dismutase (SOD) activities. Last but not least, by treating the cells with V-spiked solution of concentration equivalent to that found in the carbon black waste extract, V was identified as the main culprit for the high toxicity of carbon black waste extract. These findings could potentially provide insight into the hazards of carbon black waste extract and its toxicity mechanism on human cell lines.

Inactivation of DNA–Dependent Protein Kinase Promotes Heat–Induced Apoptosis Independently of Heat–Shock Protein Induction in Human Cancer Cell Lines

The inhibition of DNA damage response pathway seems to be an attractive strategy for cancer therapy. It was previously reported that in rodent cells exposed to heat stress, cell growth was promoted by the activity of DNA-dependent protein kinase (DNA-PK), an enzyme involved in DNA non-homologous end joining (NHEJ) required for double-strand break repair. The absence of a functioning DNA-PK was associated with down regulation of heat shock protein 70 (HSP70). The objective of this study is thus to investigate the role of DNA-PK inhibition in heat-induced apoptosis in human cell lines. The inhibitors of phosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) at Ser2056, such as NU7026 and NU7441, were utilized. Furthermore, knock down of DNA-PKcs was carried out using small interfering RNA (siDNA-PKcs). For heat exposure, cells were placed in water bath at 44°C for 60 min. Apoptosis was evaluated after 24 h incubation flow cytometrically. Proteins were extracted after 24 h and analyzed for HSP70 and HSP40 expression by Western blotting. Total RNA was extracted 6 h after treatment and analyzed using a GeneChip® microarray system to identify and select the up-regulated genes (≥1.5 fold). The results showed an enhancement in heat-induced apoptosis in absence of functioning DNA-PKcs. Interestingly, the expression levels of HSP70 and HSP40 were elevated in the absence of DNA-PKcs under heat stress. The results of genetic network analysis showed that HSPs and JUN genes were up-regulated independently of DNA-PKcs in exposed parent and knock out cells. In the presence of functioning DNA-PKcs, there was an observed up-regulation of anti-apoptotic genes, such as NR1D1, whereas in the absence of DNA-PKcs the pro-apoptotic genes, such as EGR2, were preferentially up-regulated. From these findings, we concluded that in human cells, the inactivation of DNA-PKcs can promote heat-induced apoptosis independently of heat-shock proteins.

A novel Cu(ii)–mal–picoline complex induces mitotic catastrophe mediated by deacetylation of histones and α-tubulin leading to apoptosis in human cell lines

In this study, we investigated mitotic catastrophe followed by apoptosis induced in human cell lines [HeLa, HepG2 and THP1] by a novel Cu(II) complex having malonate as the primary ligand and protonated 2-amino-4-picoline as the counter ion; whose in vitro DNA binding ability was demonstrated previously (B. Saha et al., J. Phys. Chem. B, 2010, 114(17), 5851–5861). Using the auto-fluorescence property of the complex, it was observed that the complex entered into the cells within 15 min after the exposure and was able to kill cells as determined by clonogenic survivability and MTT assay in a dose and time dependent manner. While dissecting the cell killing mechanisms, it was found that initially the complex induced multinucleated cells by inhibiting acetylation of a histone acetyl transferase (HAT) domain of CBP/p300, although histone deacetylase 6 (HDAC6) expression did not change much. As a result, histone proteins, H3 and H2AX, along with a non-histone protein, α-tubulin, were mostly deacetylated after 48 h of the treatment. This eventually led to mitotic catastrophe (MC), as histone acetylation–deacetylation dynamics is essential for the successful mitosis. DNA damage-induced γH2AX and 53BP1 foci in the treated cells were also observed after 72 h of treatment, as abnormal mitosis with decondensed chromosomes are prone to nucleolytic attack. These molecular phenomena ultimately rendered apoptosis. Taken together, our results provided evidence that the said complex perturbed the signaling events associated with mitosis and consequently induced cell death.

PDCD2, a protein whose expression is repressed by BCL6, induces apoptosis in human cells by activation of the caspase cascade

We have previously reported that the human programmed cell death-2 gene ( PDCD2), a target of BCL6 repression, is likely to be important in the pathogenesis of certain human lymphomas. We now demonstrate that transfection of a construct expressing PDCD2 induces apoptosis in human cell lines, that this occurs, at least in part, through activation of the caspase cascade, and, furthermore, that caspase inhibitors block this effect. Immunohistochemical studies in human benign lymphoid and lymphoma tissues support these findings. In addition, transfection of a VP16-BCL6 zinc fingers fusion protein, which competes with the binding of endogenous BCL6 in a Burkitt lymphoma cell line, increases PDCD2 protein expression and apoptosis, and knockdown of the PDCD2 protein in this cell line by PDCD2-specific small interfering RNA duplexes inhibits apoptosis. These studies indicate that one function of PDCD2 is to promote apoptosis in several human and mammalian cell lines and tissues, including lymphoma. Although the pathways involved in lymphomagenesis are likely to be multiple and complex, it is plausible that repression of PDCD2 expression by BCL6, which, in turn, leads to downregulation of apoptosis, is one mechanism involved in BCL6-associated lymphomatous transformation. The usefulness of increasing PDCD2 expression in the treatment of certain lymphomas merits further investigation.

Epstein-Barr virus-encoded LMP1 promotes cisplatin-induced caspase activation through JNK and NF-κB signaling pathways

Our previous studies have shown that Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) potentiates chemotherapeutic agent-induced apoptosis in human cell lines of epithelial origin: cervical carcinoma-derived HeLa cells and nasopharyngeal carcinoma-derived TW03 cells. LMP1 acted upstream of caspase-dependent mitochondrial perturbation, and the effect was mapped to the C-terminal signaling domain of LMP1, designated CTAR2. CTAR2 is known to engage the c-Jun N-terminal kinase (JNK) and NF-κB pathways, and we show here that SP600125, a selective JNK inhibitor, suppresses LMP1 potentiation of cisplatin-induced mitochondrial damage and caspase activation in HeLa cells. Moreover, the potentiation of cisplatin-triggered caspase activation was blocked by Bay11-7082, a potent inhibitor of NF-κB. Similar results were obtained when a dominant negative form of IκB, a specific repressor of NF-κB, was co-expressed with LMP1. The current data support the notion that LMP1 modifies stress-induced apoptosis in epithelial cells through molecular interactions downstream of its C-terminal signaling domain.

Features that determine telomere homolog oligonucleotide‐induced therapeutic DNA damage‐like responses in cancer cells

Cancer is the second leading cause of death in the USA, with metastatic disease proving a particular management challenge. Treatment modalities for patients with metastatic disease are limited, and survival beyond 5 years is uncommon. We have reported that an 11-base DNA oligonucleotide 100% homologous to the telomere 3' overhang can induce apoptosis, senescence and/or differentiation of several types of malignant cells in vitro and in vivo, while having minimal effect on normal cells. We now report that 22 oligonucleotides, 9-20 bases in length, with or without a 5' phosphate group and with varying homology (40-100%) to the 3' overhang, inhibit growth and induce apoptosis of human cell lines derived from breast cancers, pancreatic and ovarian carcinomas, and malignant melanoma, lines that lack p53 and/or p16 and harbor a variety of other abnormalities in key regulatory signaling pathways. Cytosine (C) content adversely affected oligonucleotide efficacy, decreasing their effect on cellular apoptosis by > or =80%. These data confirm and expand our earlier work suggesting that such telomere homolog oligonucleotides (T-oligos) target an innate anti-cancer defense system in human cells and may provide an effective treatment for cancers of multiple different cellular origins and genetic profile.

Induction of apoptosis in human cells by RNAi-mediated knockdown of hARD1 and NATH, components of the protein N-α-acetyltransferase complex

Protein N-epsilon-acetylation is recognized as an important modification influencing many biological processes, and protein deacetylase inhibitors leading to N-epsilon-hyperacetylation of histones are being clinically tested for their potential as anticancer drugs. In contrast to N-epsilon-acetyltransferases, the N-alpha-acetyltransferases transferring acetyl groups to the alpha-amino groups of protein N-termini have only been briefly described in mammalians. Human arrest defective 1 (hARD1), the only described human enzyme in this class, complexes with N-acetyltransferase human (NATH) and cotranslationally transfers acetyl groups to the N-termini of nascent polypeptides. Here, we demonstrate that knockdown of NATH and/or hARD1 triggers apoptosis in human cell lines. Knockdown of hARD1 also sensitized cells to daunorubicin-induced apoptosis, potentially pointing at the NATH-hARD1 acetyltransferase complex as a novel target for chemotherapy. Our results argue for an essential role of the NATH-hARD1 complex in cell survival and underscore the importance of protein N-alpha-acetylation in mammalian cells.

ATRA can enhance apoptosis that is induced by Flt3 tyrosine kinase inhibition in Flt3-ITD positive cells

Among activating Flt3 mutations that have been shown in 25–30% of acute myeloid leukaemia (AML) Flt3-internal tandem duplication (ITD) mutations are predominant. We investigated the influence of all- trans-retinoic acid (ATRA) and granulocyte colony stimulating factor (G-CSF) for their effects on differentiation and apoptosis in human cell lines with different Flt3 variants (THP-1 versus MV4-11 and MOLM13) dependent on the inhibition of Flt3 tyrosine kinase by the bis(lH-2-indolyl)methanone D-65476. While myeloid differentiation was not observed in both Flt3-ITD cell lines (MV4-11 and MOLM13), we demonstrate an enhanced proapoptotic effect of D-65476 in the presence of ATRA that was restricted to the Flt3-ITD expressing cells. The combined treatment with ATRA and D-65476 also led to a pronounced down-regulation of surviv in on mRNA and protein level in Flt3-ITD but not in Flt3 wildtype expressing cells (THP-1). Surprisingly, there was no differential expression of important proteins like Bcl-X L, Bcl-2 or Bax that might explain enhanced apoptosis. Furthermore, Akt phosphorylation after stimulation with Flt3 ligand dependent on D-65476 was not affected by pretreatment with ATRA. We suggest that regulation of inhibitors of apoptosis might play a crucial role how ATRA can increase the proapoptotic effect of Flt3 inhibitors in myeloid leukemia cells expressing Flt3-ITD. This effect can potentially be exploited for the treatment of Flt3-ITD positive acute myeloid leukemia.

Apoptosis Induced in Human Cell Lines by a Butanol Extract from Chlorophytum comosum Roots

The antiproliferative effects of a n-butanol extract from Chlorophytum comosum (C. comosum) (spider plant) roots was tested in vitro against four human cell lines: HeLa, CCRF-HSB-2, HL-60 and U937 cells. The extract was found to have cell antiproliferative effects against HeLa, CCRF-HSB-2, HL-60 and U937 cell lines, with an IC50 value of 6.6, 5.4, 8.7 and 10.2 μg/ml, respectively. The dying cells showed characteristics of apoptosis, such as DNA fragmentation, as assessed by DNA electrophoresis analysis and the Terminal deoxynucleotidyl transferase (TdT)-mediated biotin dUTP Nick End-Labeling (TUNEL) method. These results indicate that extract from C. comosum roots can induce apoptosis in human cell lines.

Casein Kinase I Attenuates Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis by Regulating the Recruitment of Fas-Associated Death Domain and Procaspase-8 to the Death-Inducing Signaling Complex

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide variety of malignant cell lines, in contrast to normal cells, but with considerable heterogeneity in response. Death receptor-mediated apoptosis may be attenuated by a variety of different mechanisms, including phosphorylation-based signaling pathways. We have demonstrated that casein kinase I can attenuate TRAIL-induced apoptosis in human cell lines derived from colon adenocarcinoma (HT29 and HCT8) and pediatric rhabdomyosarcoma (JR1). Inhibition of casein kinase I (CKI) phosphorylation events in HT29, HCT8, and JR1 cells by CKI-7 dramatically increased apoptosis after exposure to TRAIL, in the absence of apoptosis induced by TRAIL treatment alone. CKI inhibition enhanced the recruitment of Fas-associated death domain and procaspase-8 to the death-inducing signaling complex after TRAIL treatment and enhanced cleavage of procaspase-8 at the death-inducing signaling complex. In HT29 cells studied further, rapid cleavage of caspase-8, caspase-3, Bid, and the caspase substrate poly(ADP-ribose) polymerase occurred when CKI-7 and TRAIL were combined. Overexpression of Bcl-2, Bcl-xL, or mutant DN-Fas-associated death domain protected HT29 cells from TRAIL-induced apoptosis in the presence of the CKI inhibitor. In addition, TRAIL combined with CKI-7 promoted the release of cytochrome c, Smac/DIABLO, HtrA2/Omi, and AIF from the mitochondria and down-regulated the expression of XIAP and c-IAP1. Small hairpin RNAs directed against CKI revealed that the CKIalpha isoform contributed significantly to the inhibition of TRAIL-induced apoptosis. These findings suggest that CKIalpha plays an antiapoptotic role through the generation of phosphorylated sites at the level of the death-inducing signaling complex, thereby conferring resistance to caspase cleavage mediated by TRAIL.

Dimerization of two novel apoptosisinducing proteins and its function in regulating cell apoptosis

Asy (apoptosis/saibousi Yutsudo) is a novel apoptosis-inducing gene found in 1999 by Yutsudo group in Japan. In 2000, Qi Bing et al. cloned another novel gene, named hap (homologue of ASY protein), which encoded the ASY interacting protein, from human lung cell line (WI-38) cDNA library by using yeast two-hybrid system. It has been proved that ASY formed homodimer in yeast and human cell line, ASY and HAP formed heterodimer in yeast cells, and both induced cell apoptosis in human tumor cell lines Sao2 and CGL4. This paper showed that HAP could form homodimer in yeast cells by yeast two-hybrid system; HAP and ASY could produce heterodimer in human cell line by cross-immunoprecipitation test; by using apoptosis-testing technologies such as AnnexinV, TUNEL, DNA ladder and Flow Cytometry, the cell apoptosis in human normal or tumor cell lines transfected with hap or asy individually or cotransfected by the both was qualified or quantified. It was firstly demonstrated that ASY or HAP induced cell apoptosis not only in human tumor cell lines, but also in human normal cell lines. Moreover, we proved that the heterodimer between ASY and HAP decreased apoptosis-inducing activity from the homodimer of ASY or HAP. It revealed that by choosing to form heterodimer or homodimer between ASY and / or HAP is an important mechanism of regulating apoptosis in human cell lines.

Mechanism of Apoptosis Induced by a New Topoisomerase Inhibitor through the Generation of Hydrogen Peroxide

TAS-103, a new anticancer drug, induces DNA cleavage by inhibiting the activities of topoisomerases I and II. We investigated the mechanism of TAS-103-induced apoptosis in human cell lines. Pulsed field gel electrophoresis revealed that in the leukemia cell line HL-60 and the H(2)O(2)-resistant subclone, HP100, TAS-103 induced DNA cleavage to form 1-2-Mb fragments at 1 h to a similar extent, indicating that the DNA cleavage was induced independently of H(2)O(2). TAS-103-induced DNA ladder formation in HP100 cells was delayed compared with that seen at 4 h in HL-60 cells, suggesting the involvement of H(2)O(2)-mediated pathways in apoptosis. Flow cytometry revealed that H(2)O(2) formation preceded increases in mitochondrial membrane potential (DeltaPsim) and caspase-3 activation. Inhibitors of poly(ADP-ribose) polymerase (PARP) prevented both TAS-103-induced H(2)O(2) generation and DNA ladder formation. The levels of NAD(+), a PARP substrate, were significantly decreased in HL-60 cells after a 3-h incubation with TAS-103. The decreases in NAD(+) levels preceded both increases in DeltaPsim and DNA ladder formation. Inhibitors of NAD(P)H oxidase prevented TAS-103-induced apoptosis, suggesting that NAD(P)H oxidase is the primary enzyme mediating H(2)O(2) formation. Expression of the antiapoptotic protein, Bcl-2, in BJAB cells drastically inhibited TAS-103-induced apoptosis, confirming that H(2)O(2) generation occurs upstream of mitochondrial permeability transition. Therefore, these findings indicate that DNA cleavage by TAS-103 induces PARP hyperactivation and subsequent NAD(+) depletion, followed by the activation of NAD(P)H oxidase. This enzyme mediates O(2)(-)-derived H(2)O(2) generation, followed by the increase in DeltaPsim and subsequent caspase-3 activation, leading to apoptosis.

Microcin E492, a channel-forming bacteriocin from Klebsiella pneumoniae, induces apoptosis in some human cell lines.

The cytotoxic effect of microcin E492, a low-molecular-mass channel-forming bacteriocin (7,887 Da) produced by a strain of Klebsiella pneumoniae, was characterized in HeLa cells. At low (5 microg/ml) and intermediate (10 microg/ml) concentrations, microcin E492 induced biochemical and morphological changes typical of apoptosis, such as cell shrinkage, DNA fragmentation, extracellular exposure of phosphatidylserine, caspase activation, and loss of mitochondrial membrane potential. Treatment with zVAD-fmk, a general caspase inhibitor, completely blocked the cytotoxic effect of this bacteriocin. At higher microcin concentrations (>20 microg/ml) a necrotic phenotype was observed. Induction of apoptosis by microcin E492 was associated with the release of calcium from intracellular stores, probably after microcin-triggered ion channel formation. Microcin E492 also presented a cytotoxic effect on Jurkat and RJ2.25 cells, but had no effect on KG-1 cells nor on a primary culture of human tonsil endothelial cells, suggesting that there is a specific interaction of the bacteriocin with components of the target cell surface. This report describes a bacteriocin that has the capacity to induce apoptosis in human cell lines.