Mechanism of ricin-induced apoptosis in human cervical cancer cells

The mechanism of ricin-induced apoptosis in human cervical cancer cell line HeLa was studied. The present study demonstrated that ricin induces apoptosis of human cervical cancer cells (HeLa) in a time dependent manner with an IC(50) for cell viability of 1 microg/ml. Ricin treatment resulted in a time dependent increase in LDH leakage, DNA fragmentation, percent apoptotic cells, generation of reactive oxygen species and depletion of intracellular glutathione levels. DNA agarose gel electrophoresis showed typical oligonucleosomal length DNA fragmentation. Additionally, DNA diffusion assay was performed to confirm DNA damage and apoptosis. Ricin activated caspase-3 as evidenced by both proteolytic cleavage of procaspase-3 into 20 and 18 kDa subunits, and increased protease activity. Caspase activity was maximum at 4h and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the 85 kDa cleavage product. Ricin-induced caspase-3 activation also resulted in cleavage of DNA fragmentation factor-45 (DFF45/ICAD) and DFF40 or caspase-activated DNase in HeLa cells. Activation of caspase-3, cleavage of PARP and DNA fragmentation was blocked by pre-treatment with caspase-3 specific inhibitor Ac-DEVD-CHO (100 microM) and broad-spectrum caspase inhibitor Z-VAD-FMK (40 microM). Ricin-induced DNA fragmentation was inhibited by pre-treatment with PARP inhibitors 3-aminobenzamide (100 microM) and DPQ (10 microM). Our results indicate that ricin-induced cell death was mediated by generation of reactive oxygen species and subsequent activation of caspase-3 cascade followed by down stream events leading to apoptotic mode of cell death.     

Increased cytotoxicity of an unusual DNA topoisomerase II inhibitor compound C-1305 toward HeLa cells with downregulated PARP-1 activity results from re-activation of the p53 pathway and modulation of mitotic checkpoints

Our previous studies have shown that murine fibroblast cells, in which PARP-1 gene was inactivated by gene disruption, are extremely sensitive to triazoloacridone compound C-1305, an inhibitor of DNA topoisomerase II with unusual properties. Here, we show that pharmacological inhibition of PARP-1 activity by its inhibitor compound NU1025, sensitizes human cervical carcinoma HeLa cells to compound C-1305 compared to treatment with drug alone. Cytotoxic effect of drug/NU1025 of other topoisomerase II inhibitors varied depending on the dose of PARP-1 inhibitor. Increased cytotoxicity of topoisomerase II inhibitor/NU1025 combinations was attributable to the re-activation of the p53 pathway in drug-treated HeLa cells. This lead to a more stringent cell cycle checkpoint control during G2 and M and enhanced cell death by mitotic catastrophe induced by drug/NU1025 combinations. Interestingly, treatment of HeLa cells with NU1025 alone also increased p53 expression. This effect is, at least in part, related to the inhibition of proteasome activity by drug treatments. Together, our results show that concomitant inhibition of topoisomerase II and PARP-1 leads to the synergistic cytotoxic effect toward tumor cells that may be important for combination therapies with NU1025 and topoisomerase II inhibitors. We also confirmed our earlier work and show the important role of PARP-1 activity in the maintenance of the G2 arrest induced by DNA damaging drugs. Finally, based on our studies we propose that NU1025 and possibly other inhibitors of PARP-1 may be used as non-genotoxic agents to activate p53 in tumor cells with non-functional p53 pathways.     

Induction of apoptosis in human hepatoblastoma cells by tetrandrine via caspase-dependent Bid cleavage and cytochrome c release

Tetrandrine, a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, induces apoptosis in human T-cell lines, lung carcinoma and hepatoblastoma cells. However, the mechanisms by which tetrandrine inhibits tumor cell growth are poorly understood. The purpose of the present study was to investigate the intracellular signaling mechanism of tetrandrine-induced apoptosis in HepG2 cells. The induction of apoptosis was determined by morphological analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Treatment of cells with tetrandrine caused the upregulation of p53, downregulation of Bcl-X(L), cleavage of Bid and Bax, and release of cytochrome c, which were accompanied by activation of caspases 9, 3 and 8. The activation of caspases 9 and 3 preceded that of caspase 8. A broad-spectrum caspase inhibitor and a caspase 8-specific inhibitor completely blocked tetrandrine-induced Bid processing, cytochrome c release, activation of caspase 3, and cell death. These findings and data showing the early release of cytochrome c, cleavage of Bid and downregulation of Bcl-X(L) suggest that the mitochondrial pathway is primarily involved in tetrandrine-induced apoptosis. The activation of caspase 8 after early caspases 9 and 3 activation might act as an amplification loop for activation of upstream signals such as Bid cleavage or cytochrome c release. These data suggest that tetrandrine may constitute a plausible therapeutic for hepatocellular carcinoma.     

A novel adenosine analog, thio-Cl-IB-MECA, induces G0/G1 cell cycle arrest and apoptosis in human promyelocytic leukemia HL-60 cells

Human A3 adenosine receptor (A3AR) agonists have been shown to play important roles in several physiological and pathological processes, including growth inhibition of human cancer cells. On this line, we recently found that a novel adenosine analog, 2-chloro-N6-(3-iodobenzyl)-4'-thioadenosine-5'-N-methyluronamide (thio-Cl-IB-MECA) was a potent human A3AR agonist, and is superior to a known agonist Cl-IB-MECA [Jeong LS, Jin DZ, Kim HO, Shin DH, Moon HR, Gunaga P, et al. J Med Chem 2003;46:3775]. Here, we report that a novel A3AR agonist, thio-Cl-IB-MECA inhibited the growth of human promyelocytic leukemia HL-60 cells by arresting cell cycle and induction of apoptosis. Thio-Cl-IB-MECA induced the cell cycle arrest of G0/G1 in the early time and at lower concentration (up to 25 microM). At higher concentration (50 microM), the apoptotic cell deaths were manifested by observation of the increase of sub-G0 phase of cell cycle distribution, DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. In addition, the down-regulation of checkpoint protein cyclin D1 and c-myc by thio-Cl-IB-MECA was well correlated with the arrest of cell cycle transition of G1 to S phase. Further study revealed that the growth inhibitory activity of thio-Cl-IB-MECA is also related with the modulation of Wnt signaling pathway. The levels of beta-catenin, phosphorylated forms of GSK-beta and Akt were down-regulated by the treatment of thio-Cl-IB-MECA (10 nM) in a time-dependent manner, providing one of plausible mechanistic evidence for the involvement of the Wnt signaling pathway in the HL-60 cell growth inhibitory effects by thio-Cl-IB-MECA. These results suggest that a novel A3AR agonist, thio-Cl-IB-MECA can down-regulate Wnt signaling, inhibit proliferation and induce apoptosis in HL-60 leukemia cells, and thus provide the possibility of this compound in the potential therapeutic value of the treatment of leukemia.     

Role of tissue transglutaminase in GTP depletion-induced apoptosis of insulin-secreting (HIT-T15) cells

The role of tissue transglutaminase (tTG), a calcium-dependent and GTP-modulated enzyme, in apoptotic death induced by GTP depletion in islet beta-cells was investigated. GTP depletion and apoptosis were induced by mycophenolic acid (MPA) in insulin-secreting HIT-T15 cells. MPA treatment increased in situ tTG activity (but not protein levels) in a dose- and time-dependent manner in parallel with the induction of apoptosis. MPA-induced increases of both tTG activity and apoptosis were entirely blocked by co-provision of guanosine but not adenosine. MPA-enhanced tTG activity could be substantially reduced by co-exposure to monodansylcadaverine or putrescine (tTG inhibitors), and largely blocked by lowering free Ca(2+) concentrations in the culture medium. However, MPA-induced cell death was either not changed or was only slightly reduced under these conditions. By contrast, a pan-caspase inhibitor (Z-VAD-FMK) entirely prevented apoptosis induced by MPA, but did not block the enhanced tTG activity, indicating that GTP depletion can induce apoptosis and activate tTG either independently or as part of a cascade of events involving caspases. Importantly, the morphological changes accompanying apoptosis could be markedly prevented by tTG inhibitors. These findings suggest that the effect of the marked increase in tTG activity in GTP depletion-induced apoptosis of insulin-secreting cells may be restricted to some terminal morphological changes.     

Constituents of Corydalis heterocarpa and their anti-proliferative effects on human cancer cells

Two new coumarins, 1 and 2, along with four known coumarins (3–6) have been isolated from Corydalis heterocarpa. On the basis of spectroscopic and chemical methods, compounds 1 and 2 were elucidated as (2′S,7′S)-O-2-methylbutanoyl-columbianetin and (2′S)-columbianetin-3′-sulfate, respectively. The anti-proliferative activity against human cancer cells of compounds 1–6 isolated from C. heterocarpa was evaluated using a MTT assay and by mRNA expression of several factors related to apoptosis. Among them, compound 2 exerted the more potent anti-proliferative activity compared with the other compounds treated. The potent inhibitory effect of compound 2 was produced by induction of apoptosis through activating Bax, p53 and p21 expressions.     

Leptomycin B-induced apoptosis is mediated through caspase activation and down-regulation of Mcl-1 and XIAP expression, but not through the generation of ROS in U937 leukemia cells

Leptomycin B (LMB), which is originally isolated from Streptomyces, possesses anti-tumor properties in vivo and in vitro. Though it was previously reported that LMB induces cell cycle arrest and p53-mediated apoptosis in certain cancer cells, however, the mechanism by which LMB induces apoptosis remains poorly understood. Here, we investigated the mechanisms of apoptosis induced by LMB in U937 cells. Treatment with LMB concentration-dependently induced cytotoxicity and apoptosis in U937 cells that correlated temporally with activation of caspases and down-regulation of Mcl-1 and XIAP. LMB did not change the expressions of Bcl-2 or Bax. A broad spectrum caspase inhibitor, z-VAD-fmk, blocked caspase-3 activation and elevated the survival in LMB-treated U937 cells, suggesting that caspase-3 activation is critical for LMB-induced apoptosis. Interestingly, Bcl-2 overexpression that blocked cytochrome c release by LMB effectively attenuated the apoptotic response to LMB, suggesting that LMB-induced apoptosis is mediated through the mitochondrial pathway. Antioxidants or antioxidant enzymes had no effects on LMB-induced apoptosis. Data of flow cytometry analysis using 2',7'-dichlorofluorescein-diacetate further revealed no reactive oxygen species (ROS) generation by LMB, indicating that apoptosis induced by LMB is ROS-independent. However, the apoptotic response to LMB was not shown in U937 cells pretreated with the sulfhydryl group-containing antioxidant N-acetylcysteine (NAC). Further analysis suggested that NAC directly binds LMB and abolishes the apoptotic effects of LMB. Collectively, these findings suggest that LMB potently induces apoptosis in U937 cells, and LMB-induced apoptosis in U937 cells is related with cytochrome c release, activation of caspases, and selective down-regulation of Mcl-1 and XIAP.     

Effects of ruthenium red on accumulation and cytotoxicity of m-AMSA, vinblastine and daunorubicin in leukemia cells

Effects of ruthenium red on accumulation and cytotoxicity of m-AMSA, vinblastine and daunorubicin were examined in the P388 murine leukemia cell line and in an adriamycin-resistant subline, P388/ADR, which is cross-resistant to all three agents. Ruthenium red increased m-AMSA accumulation by both P388 and P388/ADR cells; the extent of this effect was a function of the concentration of both agents. Uptake enhancement occurred within 5 min of exposure of cells to ruthenium red and was readily reversed when cells were suspended in fresh medium. A 24-hr exposure to ruthenium red was needed to affect vinblastine or daunorubicin accumulation, and the effect was substantially less than that observed with m-AMSA. Ruthenium red protected P388 cells from m-AMSA toxicity. These data, together with reports indicating a protective effect of ruthenium red against vinblastine and anthracycline toxicity, suggest that the dye promotes tight binding of m-AMSA and other agents to cellular loci on which toxic effects are not exerted.     

Induction of apoptosis by phloroglucinol derivative from Ecklonia Cava in MCF-7 human breast cancer cells

Phloroglucinol derivatives, dioxinodehydroeckol (1) and 1-(3′,5′-dihydroxyphenoxy)-7-(2′′,4′′,6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo-1,4-dioxin (2), were isolated from Ecklonia Cava. Their ability to inhibit the proliferation of human breast cancer cells were evaluated by measuring cell death via induction of apoptosis. Compound 1 exerted a higher anti-proliferative activity in human breast cancer cells compared with compound 2. Furthermore, compound 1 induced a significant proliferative inhibition and apoptosis in a dose-dependent manner on MCF-7 human cancer cells. Treatment with compound 1 also induced the increase in caspase (-3 and -9) activity, DNA repair enzyme poly-(ADP-ribose) polymerase (PARP) cleavage, and pro-apoptotic gene and the decrease in anti-apoptotic gene. In addition, NF-κB family and -dependent activated genes were down-regulated by compound 1. These results indicated that the potential inhibitory effect of compound 1 against growth of MCF-7 human breast cancer cells might be associated with induction of apoptosis through NF-κB family and NF-κB dependent pathway. The present results suggest that compound 1 has a promising potential to be used as a valuable chemopreventive agent.     

Reactive oxygen species-mediated induction of apoptosis by a plant alkaloid 6-methoxydihydrosanguinarine in HepG2 cells

We have found in the previous study that 6-methoxydihydrosanguinarine (6ME), a benzophenanthridine alkaloid isolated from Hylomecon species, may have potential as a chemotherapeutic agent. However, the mechanisms of 6ME-induced cell death have not been investigated. The purpose of the present study was to determine the apoptosis-inducing potential of 6ME in human hepatocarcinoma HepG2 cells and the role of reactive oxygen species in 6ME-induced apoptosis. It can be concluded from the results that 6ME inhibits the growth of HepG2 cells in a concentration- and time-dependent manner (IC50=3.8+/-0.2 microM following 6 h incubation). Treatment of HepG2 cells with 6ME resulted in the release of mitochondrial cytochrome c followed by the activation of caspase proteases, and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. 6ME increased the expression of p53 and bax and decreased the expression of bcl-2. The cytotoxic effect of 6ME is mediated by the time-dependent generation of reactive oxygen species. Our results also show that preincubation of HepG2 cells with vitamin C decreased the expression of p53 and bax and inhibited the release of cytochrome c, activation of downstream caspase and the cleavage of poly(ADP-ribose) polymerase, thus inhibiting the apoptosis inducing effect of 6ME.     

SL-01, an oral gemcitabine derivative, inhibited human cancer growth more potently than gemcitabine

SL-01, an oral gemcitabine derivative, was synthesized by introducing the moiety of 3-(dodecyloxycarbonyl)pyrazine-2-carbonyl at the N4-position on the cytidine ring of gemcitabine. Our goal in this study was to evaluate the efficacy of SL-01 on the growth of human cancers with gemcitabine as control. Experiments were performed on human non-small cell lung cancer NCI-H460 and colon cancer HCT-116 both in vitro and in vivo. In vitro assays, SL-01 significantly inhibited the growth of cancer cells as determined by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Further studies indicated that SL-01 induced the cancer cells to apoptosis showing chromatin condensation and externalization of phosphatidylserine. In in vivo studies, we evaluated the efficacy of SL-01 in nude mice bearing human cancer xenografts. SL-01 effectively delayed the growth of NCI-H460 and HCT-116 without significant loss of body weight. Molecular analysis indicated that the high efficacy of SL-01 was associated with its ability to induce apoptosis as evidenced by increase of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining cells, activation of caspase-9, caspase-3 and cleaved poly ADP-ribose polymerase (PARP) in tumor tissues. SL-01 also increased Bax/Bcl-2 ratio in cancer cells. These biological activities of SL-01 were more potential than that of gemcitabine. Based on these in vitro and in vivo results, SL-01 is proposed as a potent oral anticancer agent that may supplant the use of gemcitabine in the clinic.     

Molecular mechanism of gossypol-induced cell growth inhibition and cell death of HT-29 human colon carcinoma cells

Gossypol, a male contraceptive drug, has been demonstrated to have antiproliferative and antimetastatic effects on many kinds of cancer cells in vitro. HT-29 human carcinoma cell line is one of the most susceptible cell lines to gossypol-induced cell death. Here, it is shown that treatment of HT-29 cells with gossypol not only induces cell cycle arrest on the G0/G1 phase, but also induces apoptosis. With a serial of Western blot analysis, it is revealed that gossypol-induced cell cycle arrest is involved in P21 up-regulation and cyclin D1 down-regulation; gossypol-induced apoptosis triggers down-regulation of anti-apoptosis Bcl-2 members: Bcl-X(L), Bag-1 and Mcl-1, up-regulation of pro-apoptosis Bcl-2 member Bak, activation of caspase-3, -6, -7, -8, and -9, up-regulation of Apaf-1, release of cytochrome c (cyto-c) from mitochondria, and activation of both DFF45 and PARP. Taken together, gossypol-induced cell death initiates extensive alterations of cell cycle and apoptosis proteins. Gossypol-induced apoptosis of HT-29 cells is through first the mitochondrial pathway, then the death receptor pathway, and the mitochondria pathway is, at least in part, involved in cyto-c release.     

Phototoxicity and cytotoxicity of fullerol in human lens epithelial cells

The water-soluble, hydroxylated fullerene [fullerol, nano-C60(OH)22-26] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have assessed fullerol's potential ocular toxicity by measuring its cytotoxicity and phototoxicity induced by UVA and visible light in vitro with human lens epithelial cells (HLE B-3). Accumulation of nano-C60(OH)22-26 in the cells was confirmed spectrophotometrically at 405 nm and cell viability estimated using MTS and LDH assays. Fullerol was cytotoxic to HLE B-3 cells maintained in the dark at concentrations higher than 20 microM. Exposure to either UVA or visible light in the presence of >5 microM fullerol-induced phototoxic damage. When cells were pretreated with non-toxic antioxidants: 20 microM lutein, 1 mM N-acetyl cysteine, or 1 mM l-ascorbic acid prior to irradiation, only the singlet oxygen quencher-lutein significantly protected against fullerol photodamage. Apoptosis was observed in lens cells treated with fullerol whether or not the cells were irradiated, in the order UVA>visible light>dark. Dynamic light scattering (DLS) showed that in the presence of the endogenous lens protein alpha-crystallin, large aggregates of fullerol were reduced. In conclusion, fullerol is both cytotoxic and phototoxic to human lens epithelial cells. Although the acute toxicity of water-soluble nano-C60(OH)22-26 is low, these compounds are retained in the body for long periods, raising concern for their chronic toxic effect. Before fullerols are used to deliver drugs to the eye, they should be tested for photo- and cytotoxicity in vivo.     

Toxic effects of copper-based antineoplastic drugs (Casiopeinas) on mitochondrial functions

To elucidate some of the subcellular and biochemical mechanisms of toxicity of metal-based antineoplastic drugs, mitochondria and cells were exposed to Casiopeinas), a new class of copper-based compounds with high antineoplastic activity. The rates of respiration and swelling, the H(+) gradient, and the activities of succinate (SDH) and 2-oxoglutarate dehydrogenases (2-OGDH) and ATPase were measured in mitochondria isolated from rat liver, kidney, heart, and hepatoma AS-30D. Also, oligomycin-sensitive respiration and ATP content in hepatoma AS-30D cells were determined. Casiopeinas) (CS) II-gly and III-i inhibited the rates of state 3 and uncoupled respiration in mitochondria. CS II was 10 times more potent than CS III. The sensitivity to CS II was 4-5-fold higher in mitochondria incubated with 2-OG than with succinate. Thus, at low concentrations (< or =10 nmol (mg protein)(-1); 10 microM), CS II disturbed mitochondrial functions only when 2-OG was present, due to a specific inhibition of 2-OGDH. At high concentrations (> or =15nmol (mg protein)(-1)), CS II-induced stimulation of basal respiration, followed by a strong inhibition, which correlated with K(+)-dependent swelling and cytochrome c release, respectively; K(+)-channel openers induce a similar mitochondrial response. Mitochondria from liver, kidney and hepatoma showed a similar sensitivity towards CS II, whereas heart mitochondria were more resistant. Oxidative phosphorylation and ATP content were also decreased in tumor cells by CS II. The data suggested that CS affected several different mitochondrial sites, bringing about inhibition of respiration and ATP synthesis, which could compromise energy-dependent processes such as cellular duplication.     

Novel N-thiolated beta-lactam antibiotics selectively induce apoptosis in human tumor and transformed, but not normal or nontransformed, cells

Historically, it has been shown that the beta-lactam antibiotics play an essential role in treating bacterial infections while demonstrating selectivity for prokaryotic cells. We recently reported that certain N-methylthio-substituted beta-lactam antibiotics had DNA-damaging and apoptosis-inducing activities in various tumor cells. However, whether these compounds affect human normal or nontransformed cells was unknown. In the current study, we first show that a lead compound (lactam 1) selectively induces apoptosis in human leukemic Jurkat T, but not in the nontransformed, immortalized human natural killer (NK) cells. Additionally, we screened a library of other N-methylthiolated beta-lactams to determine their structure-activity relationships (SARs), and found lactam 12 to have the highest apoptosis-inducing activity against human leukemic Jurkat T cells, associated with increased DNA-damaging potency. Furthermore, we demonstrate that lactam 12, as well as lactam 1, potently inhibits colony formation of human prostate cancer cells. We also show that lactam 12 induces apoptosis in human breast, prostate, and head-and-neck cancer cells. Finally, lactam 12 induces apoptosis selectively in Jurkat T and simian virus 40-transformed, but not in nontransformed NK and parental normal fibroblast, cells. Our results suggest that there is potential for developing this class of beta-lactams into novel anticancer agents.     

Thioredoxin reductase 1 knockdown enhances selenazolidine cytotoxicity in human lung cancer cells via mitochondrial dysfunction

Thioredoxin reductase (TR1) is a selenoprotein that is involved in cellular redox status control and deoxyribonucleotide biosynthesis. Many cancers, including lung, overexpress TR1, making it a potential cancer therapy target. Previous work has shown that TR1 knockdown enhances the sensitivity of cancer cells to anticancer treatments, as well as certain selenocompounds. However, it is unknown if TR1 knockdown produces similar effect on the sensitivity of human lung cancer cells. To further elucidate the role of TR1 in the mechanism of selenocompounds in lung cancer, a lentiviral microRNA delivery system to knockdown TR1 expression in A549 human lung adenocarcinoma cells was utilized. Cell viability was assessed after 48 hr treatment with the selenocysteine prodrug selenazolidines 2-butylselenazolidine-4(R)-carboxylic acid (BSCA) and 2-cyclohexylselenazolidine-4-(R)-carboxylic acid (ChSCA), selenocystine (SECY), methylseleninic acid (MSA), 1,4-phenylenebis(methylene)selenocyanate (p-XSC), and selenomethionine (SEM). TR1 knockdown increased the cytotoxicity of BSCA, ChSCA, and SECY but did not sensitize cells to MSA, SEM, or p-XSC. GSH and TR1 depletion together decreased cell viability, while no change was observed with GSH depletion alone. Reactive oxygen species generation was induced only in TR1 knockdown cells treated with the selenazolidines or SECY. These three compounds also decreased total intracellular glutathione levels and oxidized thioredoxin, but in a TR1 independent manner. TR1 knockdown increased selenazolidine and SECY-induced mitochondrial membrane depolarization, as well as DNA strand breaks and AIF translocation from the mitochondria. These results indicate the ability of TR1 to modulate the cytotoxic effects of BSCA, ChSCA and SECY in human lung cancer cells through mitochondrial dysfunction.