Triterpenoid CDDO-Im downregulates PML/RARalpha expression in acute promyelocytic leukemia cells

The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation and apoptosis of diverse human tumor cells. In the present study, we examined the effects of the CDDO imidazolide imide (CDDO-Im) on the NB4 acute promyelocytic leukemia (APL) cell line and primary APL cells. The results show that CDDO-Im selectively downregulates expression of the PML/retinoic receptor alpha fusion protein by a caspase-dependent mechanism and sensitizes APL cells to the differentiating effects of all-trans retinoic acid (ATRA). CDDO-Im treatment of APL cells was also associated with disruption of redox balance and activation of the extrinsic apoptotic pathway. In concert with these results, CDDO-Im sensitizes APL cells to arsenic trioxide (ATO)-induced apoptosis. Our findings indicate that CDDO-Im may be effective in the treatment of APL by: (i) downregulation of PML/RARalpha; (ii) enhancement of ATRA-induced differentiation; and (iii) sensitization of ATO-induced APL cell death.     

The novel triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) potently enhances apoptosis induced by tumor necrosis factor in human leukemia cells

Tumor necrosis factor (TNF) is a potent activator of the nuclear factor-kappaB (NF-kappaB) pathway that leads to up-regulation of anti-apoptotic proteins. Hence, TNF induces apoptosis in the presence of inhibitors of protein or RNA synthesis. We report that a novel triterpenoid, 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) inhibits NF-kappaB-mediated gene expression at a step after translocation of activated NF-kappaB to the nucleus. This effect appears specific for the NF-kappaB pathway as CDDO does not inhibit gene expression induced by the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). CDDO in combination with TNF caused a dramatic increase in apoptosis in ML-1 leukemia cells that was associated with activation of caspase-8, cleavage of Bid, translocation of Bax, cytochrome c release, and caspase-3 activation. Experiments with caspase inhibitors demonstrated that caspase-8 was an initiator of this pathway. TNF also induced a transient activation of c-Jun N-terminal kinase (JNK), which upon addition of CDDO was converted to a sustained activation. The activation of JNK was also dependent on caspase-8. Sustained activation of JNK is frequently pro-apoptotic, yet inhibition of JNK did not prevent Bax translocation or cytochrome c release, demonstrating its lack of involvement in CDDO/TNF-induced apoptosis. Apoptosis was acutely induced by CDDO/TNF in every leukemia cell line tested including those that overexpress Bcl-x(L), suggesting that the mitochondrial pathway is not required for apoptosis by this combination. These results suggest that the apoptotic potency of the CDDO/TNF combination occurs through selective inhibition of NF-kappaB-dependent anti-apoptotic proteins, bypassing potential mitochondrial resistance mechanisms, and thus may provide a basis for the development of novel approaches to the treatment of leukemia.     

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-imidazolide alters transforming growth factor beta-dependent signaling and cell migration by affecting the cytoskeleton and the polarity complex

The anti-tumor synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-imidazolide (CDDO-Im) ectopically activates the transforming growth factor beta (TGFbeta)-Smad pathway and extends the duration of signaling by an undefined mechanism. Here we show that CDDO-Imdependent persistence of Smad2 phosphorylation is independent of Smad2 phosphatase activity and correlates with delayed TGFbeta receptor degradation and trafficking. Altered TGFbeta trafficking parallels the dispersal of EEA1-positive endosomes from the perinuclear region of CDDO-Im-treated cells. The effect of CDDO-Im on the EEA1 compartment led to an analysis of the cytoskeleton, and we observed that CDDO-Im alters microtubule dynamics by disrupting the microtubule-capping protein, Clip-170. Interestingly, biotinylated triterpenoid was found to localize to the polarity complex at the leading edge of migrating cells. Furthermore, CDDO-Im disrupted the localization of IQGAP1, PKCzeta, Par6, and TGFbeta receptors from the leading edge of migrating cells and inhibited TGFbeta-dependent cell migration. Thus, the synthetic triterpenoid CDDO-Im interferes with TGFbeta receptor trafficking and turnover and disrupts cell migration by severing the link between members of the polarity complex and the microtubule network.     

Intracellular thiol depletion causes mitochondrial permeability transition in ebselen-induced apoptosis

Ebselen, a selenoorganic compound, has recently been shown to display a novel property of inducing apoptosis through rapid depletion of intracellular thiols in human hepatoma cells, HepG(2). The present study was thus designed to explore the mechanism of how ebselen triggers apoptosis upon depletion of intracellular thiols. The results demonstrated that ebselen treatment triggered mitochondrial permeability transition rather rapidly as revealed by redistribution of calcein green fluorescence from cytosol into mitochondria. Ebselen treatment also caused a dose- and time-dependent loss of mitochondrial membrane potential (MMP) and release of cytochrome c. Pretreatment with N-acetylcysteine, a precursor of intracellular reduced glutathione (GSH) synthesis, significantly attenuated the ebselen-induced MMP disruption and subsequently inhibited the apoptosis. In contrast, pretreatment with buthionine sulfoximine, a specific inhibitor of intracellular GSH synthesis, significantly augmented the ebselen-induced MMP alteration, and enhanced the apoptosis. Although ebselen treatment significantly increased the intracellular superoxide radical and calcium concentrations, superoxide dismutase, and BAPTA (a calcium chelator), however, failed to prevent ebselen-induced MMP loss and apoptosis. Neither caspase-9 nor caspase-3 activation was detected in ebselen-treated cells. Z-VAD-FMK, a general caspase inhibitor, also had no effect on ebselen-induced MMP decrease and apoptosis. The overall findings thus suggest that mitochondrial permeability transition resulted from intracellular thiol depletion is a critical event in ebselen-induced apoptosis.     

Evidence supporting a role for calcium in apoptosis induction by the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a novel anticancer agent that induces apoptosis in tumor cells. The cytotoxic stress underpinning CDDO-induced apoptosis has not been established. This study compared and contrasted the effects of CDDO on COLO 16 human skin cancer cells and their respiration-deficient (rho(0)) clones to elucidate the stress signal responsible for initiating apoptosis. CDDO promoted apoptosis in COLO 16 cells in a dose- and time-dependent manner. The rho(0) clones appeared to be more sensitive to CDDO-induced apoptosis implying that the disruption of mitochondrial respiration was not directly associated with triggering cell death. After a 4-h exposure to CDDO, mitochondrial inner transmembrane potential-sensitive dyes revealed mitochondrial hyperpolarization in the COLO 16 cells and mitochondrial depolarization in the rho(0) clones. Electron microscopy illustrated that this exposure also promoted mitochondrial condensation, endoplasmic reticulum dilation, and chromatin condensation in the COLO 16 cells. Endoplasmic reticulum dilation and chromatin condensation were also observed in the rho(0) clones, but the mitochondria in these cells were markedly swollen implying that the disruption of intracellular Ca(2+) homeostasis was associated with cell death. A Ca(2+)-sensitive dye confirmed that CDDO increased cytoplasmic free Ca(2+) in the COLO 16 cells, their rho(0) clones, as well as in malignant breast and lung epithelial cells. A cell-permeant Ca(2+) chelator reduced the CDDO-induced increase in cytoplasmic free Ca(2+), and inhibited caspase activation, the development of apoptotic morphology, and DNA fragmentation in the COLO 16 cells, implying that Ca(2+) played a pivotal role in signaling the initiation of apoptosis.     

The novel triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid induces apoptosis of human myeloid leukemia cells by a caspase-8-dependent mechanism.

The oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a multifunctional molecule that induces growth inhibition and differentiation of human myeloid leukemia cells. The present studies demonstrate that CDDO treatment results in apoptosis of U-937 and HL-60 myeloid leukemia cells. Similar to 1-beta-D-arabinofuranosylcytosine (ara-C), another agent that inhibits growth and induces apoptosis of these cells, CDDO induced the release of mitochondrial cytochrome c and activation of caspase-3. Overexpression of Bcl-X(L) blocked cytochrome c release, caspase-3 activation, and apoptosis in ara-C-treated cells. By contrast, CDDO-induced release of cytochrome c, and activation of caspase-3 were diminished only in part by Bcl-X(L). In concert with these findings, we demonstrate that CDDO, but not ara-C, activates caspase-8 and thereby caspase-3 by a cytochrome c-independent mechanism. The results also show that CDDO-induced cytochrome c release is mediated by caspase-8-dependent cleavage of Bid. These findings demonstrate that CDDO induces apoptosis of myeloid leukemia cells and that this novel agent activates an apoptotic signaling cascade distinct from that induced by the cytotoxic agent ara-C.     

Intracellular glutathione depletion and reactive oxygen species generation are important in α-hederin-induced apoptosis of P388 cells

alpha-Hederin, a pentacyclic triterpene saponin isolated from the seeds of Nigella sativa, was recently reported to have potent in vivo antitumor activity against LL/2 (Lewis Lung carcinoma) in BDF1 mice. In this study we observed that alpha-hederin caused a dose- and time-dependent increase in apoptosis of murine leukemia P388 cells. In order to evaluate the possible mechanisms for apoptosis, the effects of alpha-hederin on intracellular thiol concentration, including reduced glutathione (GSH), and protein thiols, and the effects of pretreatment with N-acetlycysteine (NAC), a precursor of intracellular GSH synthesis, or buthionine sulfoxime (BSO), a specific inhibitor of intracellular GSH synthesis, on alpha-hederin-induced apoptosis were investigated. It was found that alpha-hederin rapidly depleted intracellular GSH and protein thiols prior to the occurrence of apoptosis. NAC significantly alleviated alpha-hederin-induced apoptosis, while BSO augmented alpha-hederin-induced apoptosis significantly. The depletion of cellular thiols observed after alpha-hederin treatment caused disruption of mitochondrial membrane potential (deltapsi(m)) and subsequently increased the production of reactive oxygen species (ROS) in P388 cells at an early time point. Bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, and cyclosporin (CsA) attenuated the alpha-hederin-induced loss of deltapsi(m), and ROS production. Thus, oxidative stress after alpha-hederin treatment is an important event in alpha-hederin-induced apoptosis. As observed in this study, permeability transition of mitochondrial membrane occurs after depletion of GSH and precedes a state of reactive oxygen species (ROS) generation. Further, we observed that alpha-hederin caused the release of cytochrome c from the mitochondria to cytosol, leading to caspase-3 activation. Our findings thus demonstrate that changes in intracellular thiols and redox status leading to perturbance of mitochondrial functions are important components in the mechanism of alpha-hederin-induced cell death.     

Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism

Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity.     

Neuroprotection against neuroblastoma cell death induced by depletion of mitochondrial glutathione

Mitochondrial glutathione pool is vital in protecting cells against oxidative stress as the majority of the cellular reactive oxygen species are generated in mitochondria. Oxidative stress is implicated as a causative factor in neuronal death in neurodegenerative disorders. We hypothesized that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptotic death of SK-N-SH (human neuroblastoma) cells and investigated the neuroprotective strategies against GSH depletion. SK-N-SH cells were treated with two distinct inhibitors of glutathione metabolism: L-buthionine-(S, R)-sulfoximine (BSO) and ethacrynic acid (EA). EA treatment caused depletion of both the total and mitochondrial glutathione (while BSO had no effect on mitochondrial glutathione), enhanced rotenone-induced ROS production, and reduced the viability of SK-N-SH cells. Glutathione depletion by BSO or EA demonstrated positive features of mitochondria-mediated apoptosis in neuroblastoma cell death. Prevention of apoptosis by Bcl2 overexpression or use of antioxidant ebselen did not confer neuroprotection. Co-culture with U-87 (human glioblastoma) cells protected SK-N-SH cells from the cell death. Our data suggest that depletion of mitochondrial glutathione leads to mitochondrial dysfunction and apoptosis. The study indicates that preventing mitochondrial glutathione depletion could become a novel strategy for the development of neuroprotective therapeutics in neurodegenerative disorders.     

Triterpenoid CDDO-Me blocks the NF-kappaB pathway by direct inhibition of IKKbeta on Cys-179

The novel oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) and the C-28 methyl ester (CDDO-Me) induce apoptosis of human tumor cells by disruption of redox balance and are currently in clinical trials. The present studies show that CDDO and CDDO-Me block tumor necrosis factoralpha-induced targeting of NF-kappaB p65 to the nucleus. CDDO-Me also blocked tumor necrosis factor alpha-induced phosphorylation of IkappaBalpha. In concert with these results, we found that CDDO-Me inhibits IkappaBalpha kinasebeta (IKKbeta) activity in cells. In support of a direct mechanism, CDDO-Me inhibited recombinant IKKbeta activity in vitro. The results also demonstrate that (i) CDDO and CDDO-Me form adducts with IKKbeta, but not IKKbeta with mutation of Cys-179 to Ala, and (ii) CDDO-Me inhibits IKKbeta by a mechanism dependent on oxidation of Cys-179. These findings indicate that CDDO and CDDO-Me directly block IKKbeta activity and thereby the NF-kappaB pathway by interacting with Cys-179 in the IKKbeta activation loop.     

Ebselen induces apoptosis in HepG(2) cells through rapid depletion of intracellular thiols

Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one, is a synthetic seleno-organic compound with antioxidant capability. In the present study, we systematically examined the ability of ebselen to induce apoptosis in a human hepatoma cell line, HepG(2). Ebselen-induced apoptosis was evaluated by (i) TdT-mediated dUTP nick end labeling assay; (ii) analysis of sub-G1 cells; (iii) cell morphology, including cell size and granularity examination; and (iv) DNA gel electrophoresis. The results showed that ebselen was able to induce typical apoptosis in HepG(2) cells in a dose- and time-dependent manner. In order to explore the possible mechanisms involved in ebselen-induced apoptosis, the effect of ebselen on intracellular thiol concentrations including reduced glutathione (GSH) and protein thiols and the effect of N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) pretreatment on ebselen-induced apoptosis were investigated. It was found that (i) ebselen rapidly depleted intracellular GSH and protein thiols, moreover, the depletion preceded the occurrence of apoptosis; (ii) NAC, a precursor of intracellular GSH synthesis, significantly alleviated ebselen-induced apoptosis; and (iii) BSO, a specific inhibitor of intracellular GSH synthesis, augmented ebselen-induced apoptosis significantly. Taken together, the present study demonstrates that ebselen is able to induce apoptosis in HepG(2) cells, most probably through rapid depletion of intracellular thiols.     

Involvement of glutathione in the cytotoxicity of 9-isothiocyanatoacridine

Isothiocyanates (ITCs) are phytochemicals with promising cancer-preventive potential. To elucidate the mechanism of cytotoxicity of ITCs, their accumulation by cells and the role of intracellular glutathione, fluorescent 9-isothiocyanatoacridine (AcITC) was synthesized. The kinetic parameters for the reactions of AcITC with thiols were estimated and the influence of AcITC on human chronic myeloid leukemia cell line (K562) in regard to intracellular glutathione was studied. Cytotoxicity was evaluated by MTT assay, IC(50)=29.2 +/- 2.5 microM (48 h incubation). This acridine derivative was able to induce apoptosis of cells (morphological changes of cells and DNA fragmentation were observed) at least within certain dose that only decreased the level of intracellular glutathione, excessive doses (completely depleted intracellular pool of glutathione) induced necrosis rather than apoptosis. Our results indicated that apoptosis of leukemia cells induced by ITC is possible only if intracellular glutathione is not entirely depleted.     

tert-Butylhydroperoxide-induced toxicity in isolated hepatocytes: contribution of thiol oxidation and lipid peroxidation

Incubation of isolated rat hepatocytes with tert-butylhydroperoxide resulted in marked cytotoxicity preceded by intracellular glutathione depletion and extensive lipid peroxidation. Addition of antioxidants delayed, but did not prevent, this toxicity. A significant decrease in protein-free sulfhydryl groups also occurred in the presence of tert-butylhydroperoxide; direct oxidation of protein thiols and mixed disulfide formation with glutathione were responsible for this decrease. The involvement of protein thiol depletion in tert-butylhydroperoxide-induced cytotoxicity is suggested by our observation that administration of dithiothreitol, which caused re-reduction of the oxidized sulfhydryl groups and mixed disulfides, efficiently protected the cells from toxicity. Moreover, depletion of intracellular glutathione by pretreatment of the hepatocytes with diethyl maleate accelerated and enhanced the depletion of protein thiols induced by tert-butylhydroperoxide and potentiated cell toxicity even in the absence of lipid peroxidation.     

Involvement of mitochondrial dysfunction in human islet amyloid polypeptide-induced apoptosis in INS-1E pancreatic beta cells: An effect attenuated by phycocyanin

Misfolded human islet amyloid polypeptide (hIAPP) in pancreatic islets is associated with the loss of insulin-secreting beta cells in type 2 diabetes. Insulin secretion impairment and cell apoptosis can be due to mitochondrial dysfunction in pancreatic beta cells. Currently, there is little information about the effect of hIAPP on mitochondrial function. In this study, we used INS-1E rat insulinoma beta cells as a model to investigate the role of mitochondria in hIAPP-induced apoptosis and the protective effects of phycocyanin (PC). We demonstrated that hIAPP induced apoptosis in INS-1E cells was associated with the disruption of mitochondrial function, as evidenced by ATP depletion, mitochondrial mass reduction, mitochondrial fragmentation and loss of mitochondrial membrane potential (ΔΨ(m)). Further molecular analysis showed that hIAPP induced changes in the expression of Bcl-2 family members, release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into cytosol, activation of caspases and cleavage of poly (ADP-ribose) polymerase. Interestingly, the hIAPP-induced mitochondrial dysfunction in INS-1E cells was effectively restored by co-treatment of PC. Moreover, there was crosstalk between the extrinsic and intrinsic apoptotic pathways as demonstrated by cleavage of Bid by caspase-8 in the apoptotic process triggered by hIAPP. Taken together, we demonstrated for the first time the involvement of mitochondrial dysfunction in hIAPP-induced INS-1E cell apoptosis. Attenuation of mitochondrial dysfunction provides a mechanism for the protective effects of PC.     

Role of intracellular thiol depletion, mitochondrial dysfunction and reactive oxygen species in Salvia miltiorrhiza-induced apoptosis in human hepatoma HepG2 cells.

Recent studies have demonstrated that induction of apoptosis is related to the cell growth inhibition potential of Salvia Miltiorrhiza (SM), a traditional herbal medicine. In the present study, we further explore the mechanistic pathway involved in SM-induced apoptosis in human hepatoma HepG2 cells. A rapid decline of intracellular glutathione (GSH) and protein thiol content was found in SM-treated cells. Moreover. SM exposure resulted in mitochondrial dysfunction as demonstrated by: (i) the onset of mitochondrial permeability transition (MPT); (ii) the disruption of mitochondrial membrane potential (MMP); and (iii) the release of cytochrome c from mitochondria into the cytosol. Subsequently, elevated level of intracellular reactive oxygen species (ROS) was observed prior to the onset of DNA fragmentation. However, no caspase-3 cleavage was observed throughout the whole period of SM treatment, while a caspase-3-independent poly(ADP-ribose) polymerase (PARP) cleavage was noted at the late stage in SM-induced apoptosis. Pretreatment of cells with N-acetylcysteine (NAC), the GSH synthesis precursor, conferred complete protection against MMP loss, ROS generation and apoptosis induced by SM. MPT inhibitors, cyclosporin A plus trifluoperazine, partially restored intracellular GSH content, and reduced SM-induced ROS formation and subsequently inhibited cell death. Moreover, antioxidants NAC, deferoxamine and catalase had little effect on GSH depletion and mitochondrial dysfunction, yet still were able to completely protect cells from SM-induced apoptosis. Taken together, our results suggest that SM deplete intracellular thiols, which, in turn, causes MPT and subsequent increase in ROS generation, and eventually apoptotic cell death.     

Rapid and transient intracellular oxidative stress due to novel macrosphelides trigger apoptosis via Fas/caspase-8-dependent pathway in human lymphoma U937 cells

The ability of the derivatives of macrosphelides (MS) core (simplified 16-membered core structure of natural MS) to induce apoptosis in human lymphoma U937 cells was investigated. Of the five compounds examined, MS core with ketones at 8 and 14 positions (MS5) showed the highest potency to induce apoptosis, while another, MS3 with one ketone, was minimal potent. MS5 was found to induce apoptosis in the U937 cells in a time- and dose-dependent fashion, as confirmed by DNA fragmentation analysis. MS5 treated cells showed increase in intracellular reactive oxygen species (ROS), glutathione depletion, Bid activation and lipid peroxidation. Pretreatment of cells with pancaspase inhibitor resulted in the complete inhibition of MS5-induced apoptosis. N-Acetyl-l-cysteine (NAC) pretreatment resulted in the increase in glutathione concentration, reduction of intracellular ROS, complete inhibition of DNA fragmentation, mitochondrial membrane potential (MMP) collapse, Fas externalization and caspase-8 activation. Furthermore, MS5-induced oxidative stress also triggered transient increase in intracellular calcium ion ([Ca2+]i) concentration which was completely inhibited by NAC. Pretreatment with an intracellular Ca2+ chelator, BAPTA-AM reduced MS5-induced DNA fragmentation and caspase-8 activation while it has marginal effects on MMP collapse. Taken together our present data showed that a rapid increase in intracellular ROS by MS5 triggers apoptosis via the Fas/caspase-8-mediated mitochondrial pathway suggesting that the presence of diketone makes the compound more potent to induce apoptosis. These characteristics of MS5 will make it useful for therapeutic applications of targeted apoptosis.     

A Synthetic Triterpenoid CDDO-Im Inhibits Tumorsphere Formation by Regulating Stem Cell Signaling Pathways in Triple-Negative Breast Cancer

Triple-negative breast cancer is associated with poor prognosis because of a high rate of tumor recurrence and metastasis. Previous studies demonstrated that the synthetic triterpenoid, CDDO-Imidazolide (CDDO-Im) induced cell cycle arrest and apoptosis in triple-negative breast cancer. Since a small subpopulation of cancer stem cells has been suggested to be responsible for drug resistance and metastasis of tumors, our present study determined whether the effects of CDDO-Im in triple-negative breast cancer are due to the inhibition of a cancer stem cell subpopulation. CDDO-Im treatment markedly induced cell cycle arrest at G2/M-phase and apoptosis in the triple-negative breast cancer cell lines, SUM159 and MDA-MB-231. Because SUM159 cells were more sensitive to CDDO-Im than MDA-MB-231 cells, the effects of CDDO-Im on the cancer stem cell subpopulation were further investigated in SUM159 cells. SUM159 cells formed tumorspheres in culture, and the cancer stem cell subpopulation, CD24−/EpCAM+ cells, was markedly enriched in SUM159 tumorspheres. The CD24−/EpCAM+ cells in SUM159 tumorspheres were significantly inhibited by CDDO-Im treatment. CDDO-Im also significantly decreased sphere forming efficiency and tumorsphere size in both primary and secondary sphere cultures. PCR array of stem cell signaling genes showed that expression levels of many key molecules in the stem cell signaling pathways, such as Notch, TGF-β/Smad, Hedgehog and Wnt, were significantly down-regulated by CDDO-Im in SUM159 tumorspheres. Protein levels of Notch receptors (c-Notch1, Notch1 and Notch3), TGF-β/Smad (pSmad2/3) and Hedgehog downstream effectors (GLI1) also were markedly reduced by CDDO-Im. In conclusion, the present study demonstrates that the synthetic triterpenoid, CDDO-Im, is a potent anti-cancer agent against triple-negative breast cancer cells by targeting the cancer stem cell subpopulation.     

Role of glutathione in augmenting the anticancer activity of pyrroloquinoline quinone (PQQ)

Abstract

Pyrroloquinoline quinone (PQQ), a bacterial redox co-factor and antioxidant, is highly reactive with nucleophilic compounds present in biological fluids. PQQ induced apoptosis in human promonocytic leukemia U937 cells and this was accompanied by depletion of the major cellular antioxidant glutathione and increase in intracellular reactive oxygen species (ROS). Treatment with glutathione (GSH) or N-acetyl-L-cysteine (NAC) did not spare PQQ toxicity but resulted in a 2–5-fold increase in PQQ-induced apoptosis in U937 cells. Cellular GSH levels increased following treatment by NAC alone but were severely depleted by co-treatment with NAC and PQQ. This was accompanied by an increase in intracellular ROS. Alternatively, depletion of glutathione also resulted in increased PQQ cytotoxicity. However, the cells underwent necrosis as evidenced by dual labeling with annexin V and propidium iodide. PQQ-induced cytotoxicity is thus critically regulated by the cellular redox status. An increase in GSH can augment apoptosis and its depletion can switch the mode of cell death to necrosis in the presence of PQQ. Our data suggest that modulation of intracellular GSH can be used as an effective strategy to potentiate cytotoxicity of quinones like PQQ.     

Destabilization and denaturation of cellular protein by glutathione depletion

This investigation tested the hypothesis that depletion of intracellular glutathione, in contrast to its oxidation, could lead to non-native oxidation of protein thiols, thereby trapping proteins in an unstable conformation. Chinese hamster cells were exposed to the α,β-unsaturated dicarboxylic acid diethylmaleate in order to produce rapid gluthathione (GSH) depletion without oxidation. Measurement of the fluorescence of oxidized 2′,7′-dichlorofluorescein diacetate indicated that reactive oxygen species accumulated in GSH depleted cells. Glutathione depletion was found to alter protein thiol/disulfide exchange ratios such that 17 to 23 nmol of protein SH/mg protein underwent oxidation. Differential scanning calorimetry (DSC) of glutathione depleted cells yielded a profile of specific heat capacity versus temperature that was characteristic of cells containing destabilized and denatured protein. In addition, cells depleted of glutathione exhibited a two-fold increase in NP-40 insoluble protein. These results indicate that depletion of intracellular glutathione caused oxidation of protein thiols, protein denaturation and aggregation and provide a mechanism to explain how GSH depletion can initiate stress responses.