Therapeutic potential of AIF-mediated caspase-independent programmed cell death

Resistance to anticancer drugs is often related to deficient cell death execution pathways in cancer cells. Apoptosis, which denotes a form of cell death executed by caspases, was traditionally considered as the only physiological and programmed form of cell death. However, recent evidence indicates that programmed cell death (PCD) can occur in complete absence of caspase activation. Indeed, a large number of caspase-independent models are now defined and a key protein implicated in this type of PCD, apoptosis-inducing factor (AIF), has been identified. AIF is a mitochondrial protein with two faces looking in opposite life/death directions. Recently, the identification of five different isoforms allowed a better characterization of AIFs life/mitochondrial versus death/nuclear functions, as well as definition of its pro-apoptotic region and some of its nuclear partners. Importantly, much work on caspase-independent PCD has revealed that AIF participates in more PCD systems than initially thought. A wider molecular knowledge of AIF, and of the caspase-independent PCDs in which it is involved, are key to provide new insights into the role of PCD. There is no doubt that these insights will lead to the development of more selective and efficient drugs against cancer, degenerative diseases, and other pathological disorders implicating AIF.     

Aminopyrimidoisoquinolinequinone (APIQ) redox cycling is potentiated by ascorbate and induces oxidative stress leading to necrotic-like cancer cell death

Several phenylaminopyrimidoisoquinolinequinones (APIQs) were tested for their cytotoxicity against different cancer cell lines (K562, T24, HepG2) in the presence or absence of ascorbate. Ascorbate enhanced the toxic effects of quinones with first half-wave potential E(I) (1/2) values in the range of -480 to -660?mV. Phenylaminoquinones that were unsubstituted at position 6 exhibited greater cytotoxic activity than did their 6-methyl-substituted analogues. Two groups of compounds were further selected, namely 8-10 and 20-22, to study the cellular mechanisms involved in quinone cytotoxicity. Indeed, these compounds have the same range of redox potentials but differed considerably in their capacity to induce cell death. In the presence of ascorbate, the cell demise induced by compounds 8-10 was not caspase-3 dependent, as shown by the lack of activation of caspase-3 and the absence of cleaved PARP fragments. In addition, an index of ER stress (eIF2α phosphorylation) was activated by these compounds. Quinones 8-10 decreased the cellular capacity to reduce MTT dye and caused marked ATP depletion. Taken together, our results show that ascorbate enhances quinone redox-cycling and leads to ROS formation that inhibits cell proliferation and provokes caspase-independent cell death. Interestingly, we also observed that quinone 8 had a rather selective effect given that freshly isolated peripheral blood leukocytes from human healthy donors were more resistant than human leukemia K562 cells.     

Diphenyl diselenide induces apoptotic cell death and modulates ERK1/2 phosphorylation in human neuroblastoma SH-SY5Y cells

Diphenyl diselenide (PhSe)₂ is a synthetic organoselenium compound displaying glutathione peroxidase-like activity. Protective and antioxidant potential of (PhSe)₂ have been extensively investigated in in vivo and in vitro studies. In spite of this, there is a lack of studies addressed to the investigation of potential cytotoxic effect and signaling pathways modulated by this compound. Herein, we aimed to analyze the effects of 24-h treatment with (PhSe)₂ on cell viability and a possible modulation of signaling pathways in human neuroblastoma cell line SH-SY5Y. For this purpose, cells were incubated with (PhSe)₂ (0.3–30 μM) for 24 h and cell viability, apoptotic cell death and modulation of MAPKs (ERK1/2 and p38^MAPK), and PKC substrates phosphorylation was determined. (PhSe)₂ treatment significantly decreased cell viability and increased the number of apoptotic cells with induction of PARP cleavage. An increase in ERK1/2 phosphorylation was observed at (PhSe)₂ 3 μM. In contrast, higher concentrations of the chalcogenide inhibited ERK1/2, p38^MAPK and PKC substrate phosphorylation. Pre-treatment with ERK1/2 inhibitor, U0126, increased cell susceptibility to (PhSe)₂. Together, these data indicate a cytotoxic potential of (PhSe)₂ in a neuronal cell line, which appears to be mediated by the ERK1/2 pathway.     

Chrysophanol-induced necrotic-like cell death through an impaired mitochondrial ATP synthesis in Hep3B human liver cancer cells

Liver cancer is the most common form of cancer in Taiwan and it usually responds to chemotherapy. However, patients often have side effects to the chemotherapeutic drugs. Thus new agents are urgently required to treat liver cancer. Chrysophanol, one of the anthraquinone derivatives, was reported to inhibit some human cancer cell growth which may be due to the induction of apoptosis similar to other anthraquinone derivatives though such actions have not been reported. In the present study, we reported that chrysophanol inhibits cell growth in Hep3B liver cancer cells based on the following observations: 1) induc cell morphological changes; 2) decreased percentage of viable cells; 3) induced S phase arrest of cell cycle progression; 4) induced DNA damage as measured by comet assay and DAPI staining. Chrysophanol-induced cell death however, seems to be related to necrotic processes rather than typical apoptosis. Chrysophanol induced reactive oxygen species and Ca(2+) production and decreased mitochondrial membrane potential (ΔΨm) and ATP levels in Hep3B cells. No effects were observed on known protein regulators of apoptosis such as Bax and Bcl-2. Chrysophanol-induced cell death took place independently of caspase-8 and -9. Based on our findings, we propose that chrysophanol reduces cellular ATP levels causing a drop in energy resulting in necrotic-like cell death.     

Exposure to mixtures of endosulfan and zineb induces apoptotic and necrotic cell death in SH-SY5Y neuroblastoma cells, in vitro

A number of epidemiological studies have demonstrated a strong association between the incidence of Parkinson's disease and pesticide exposure. Earlier it was demonstrated that exposure to the pesticides endosulfan and zineb, alone and in combination, caused neurodegeneration in vivo. It was hypothesized that these pesticides cause neurotoxicity, in part, by enhancing apoptotic cell death. SH-SY5Y human neuroblastoma cells, which retain a catecholaminergic phenotype, were exposed to endosulfan, zineb or a combination of these chemicals, in vitro. For mixture studies, concentrations of pesticides (100 microM each) were chosen based on LC(25) (lethal concentration) that would result in minimum cell death. Exposure to a mixture of pesticides exhibited significantly (P < or = 0.05) higher toxicity than each one alone. Both pesticides were found to cause apoptotic cell death that was concentration (50-400 microM) dependent. A flow cytometric (7-aminoactinomycin D) assay was used to distinguish live, early apoptotic and late apoptotic/necrotic populations. Exposure to mixtures of the pesticides enhanced both early apoptosis and late apoptosis/necrosis compared with either chemical alone. Visual evaluation using a DNA ladder assay and a fluorescence Annexin V/PI assay confirmed the contribution of both apoptotic and necrotic processes. These findings suggest that the cytotoxicity of endosulfan and zineb, both individually and in mixtures, is associated with the occurrence of early and late apoptotic/necrotic processes in SH-SY5Y human neuroblastoma cells and support the contention that pesticide-induced neuronal cell death leading to neurodegenerative disease may, at least in part, be associated with early and late apoptosis of dopaminergic neurons.     

Enkephalin degradation by enkephalinergic neuroblastoma cells

Summary Degradation of tritiated [Leu⁵]enkephalin was studied in cultures of neuroblastoma cells (clone N1E-115). Incubation of cells in suspension revealed Tyr as the main tritiated metabolite; however, Tyr-Gly-Gly and Tyr-Gly were detectable as well. In a crude membrane preparation of the neuroblastoma cells the level of Tyr is reduced to 13% and that of Tyr-Gly to 10% of the initial value, whereas Tyr-Gly-Gly is increased to about 5 times the initial value. Of the degraded enkephalin, 66% was accounted for by the formation of Tyr, 30% by the formation of Tyr-Gly-Gly and 4% by the formation of Tyr-Gly. The production of Tyr was inhibited by bestatin, an inhibitor of aminopeptidases, and that of Tyr-Gly-Gly by captopril, an inhibitor of angiotensin-converting-enzyme. The results prove the ability of neuroblastoma cells (N1E-115) to degrade enkephalin by amino-peptidase and the membrane-bound angiotensin-converting-enzyme.     

Acrylamide induces cell death in neural progenitor cells and impairs hippocampal neurogenesis

Acrylamide (ACR) is a well-known neurotoxin in mammalian species that causes neuropathy characterized by ataxia and skeletal muscle weakness. Therefore, ACR-mediated axon damage in the central and peripheral nervous systems is considered to be central-peripheral axonopathy. However, the molecular mechanisms underlying ACR's toxicity to neural progenitor cells are unknown. This study investigated the adverse effects of ACR on mouse multipotent neural progenitor cells and adult hippocampal neurogenesis. ACR significantly reduced the proliferation of neural progenitor cells, and high ACR concentrations induced apoptotic and necrotic cell death. We found that elevated intracellular levels of reactive oxygen species were involved in ACR-mediated cytotoxicity. Interestingly, the administration of ACR to young mice resulted in a significant decrease in the number of newly generated cells in the dentate gyrus of the hippocampus, suggesting an impairment of adult neurogenesis. These results suggest that ACR's deleterious effects on the central nervous system are due to the death of neural progenitor cells and impaired adult neurogenesis.     

Effects of thimerosal on NGF signal transduction and cell death in neuroblastoma cells.

Signaling through neurotrophic receptors is necessary for differentiation and survival of the developing nervous system. The present study examined the effects of the organic mercury compound thimerosal on nerve growth factor signal transduction and cell death in a human neuroblastoma cell line (SH-SY5Y cells). Following exposure to 100 ng/ml NGF and increasing concentrations of thimerosal (1 nM-10 microM), we measured the activation of TrkA, MAPK, and PKC-delta. In controls, the activation of TrkA MAPK and PKC-delta peaked after 5 min of exposure to NGF and then decreased but was still detectable at 60 min. Concurrent exposure to increasing concentrations of thimerosal and NGF for 5 min resulted in a concentration-dependent decrease in TrkA and MAPK phosphorylation, which was evident at 50 nM for TrkA and 100 nM for MAPK. Cell viability was assessed by the LDH assay. Following 24-h exposure to increasing concentrations of thimerosal, the EC50 for cell death in the presence or absence of NGF was 596 nM and 38.7 nM, respectively. Following 48-h exposure to increasing concentrations of thimerosal, the EC50 for cell death in the presence and absence of NGF was 105 nM and 4.35 nM, respectively. This suggests that NGF provides protection against thimerosal cytotoxicity. To determine if apoptotic versus necrotic cell death was occurring, oligonucleosomal fragmented DNA was quantified by ELISA. Control levels of fragmented DNA were similar in both the presence and absence of NGF. With and without NGF, thimerosal caused elevated levels of fragmented DNA appearing at 0.01 microM (apoptosis) to decrease at concentrations >1 microM (necrosis). These data demonstrate that thimerosal could alter NGF-induced signaling in neurotrophin-treated cells at concentrations lower than those responsible for cell death.     

Pannarin inhibits cell growth and induces cell death in human prostate carcinoma DU-145 cells

In the course of our continuing search for new natural anticancer compounds for treatment and/or prevention of prostate cancer, our laboratory has focused its search on poorly investigated lichen metabolites, sphaerophorin, pannarin and epiphorellic acid-1. To this end, we treated DU-145, a cell line resembling the last stage of prostate carcinoma, with different concentrations (6-50 micromol/l) of these compounds for 72 h. Our data clearly evidenced that these lichen metabolites inhibit the growth of human prostate carcinoma DU-145 cells, but pannarin exhibits a higher effect. Our data show an induction of apoptotic death of advanced prostate cancer cells by sphaerophorin, pannarin and epiphorellic acid-1. In fact, a significant (P<0.001) increase in caspase-3 enzyme activity occurred in DU-145 cells treated with all lichen compounds at 12 and 25 micromol/l concentrations, correlated to a high DNA fragmentation, but without the disruption of the plasma membrane, as evaluated by the percentage of lactic dehydrogenase release. Alternatively, we found a low, but significant (P<0.01) lactic dehydrogenase release at higher concentrations (50 micromol/l), suggesting that in these experimental conditions sphaerophorin, pannarin and epiphorellic acid-1 induce necrosis in DU-145 cells, through the increase in reactive oxygen species generation. The experimental evidence is further confirmed by caspase-3 activity results, evidencing a reduction in the activity of this protease at a higher concentration, 50 micromol/l.     

XIAP inhibitor embelin induces autophagic and apoptotic cell death in human oral squamous cell carcinoma cells

Embelin is an active ingredient of traditional herbal remedies for cancer and other diseases. Recently, it has been suggested that autophagy may play an important role in cancer therapy. However, little data are available regarding the role of autophagy in oral cancers. Therefore, we conducted this study to examine whether Embelin modulates autophagy in Ca9‐22. Our results showed that Embelin had anticancer activity against the Ca9‐22 human tongue squamous cell, and we observed that autophagic vacuoles were formed by MDC and AO. We also analyzed Embelin‐treated Ca9‐22 cells for the presence of biochemical markers and found that it directly affected the conversion of LC3‐II, the degradation of p62/SQSTM1, full‐length cleavage formation of ATG5‐ATG12 complex and Beline‐1, and caspase activation. Rescue experiments using an autophagy inhibitor showed Embelin‐induced cell death in Ca9‐22, confirming that autophagy acts as a pro‐death signal. Furthermore, Embelin exhibited anticancer activity against Ca9‐22 via both autophagy and apoptosis. These findings suggest that Embelin may potentially contribute to oral cancer treatment and provide useful information for the development of a new therapeutic agent.     

Overexpression of NQO1 protects human SK-N-MC neuroblastoma cells against dopamine-induced cell death

NAD(P)H quinone oxidoreductase 1 (NQO1) can metabolize dopamine-derived quinones (DAQ) and absence of NQO1 due to the NQO1*2 polymorphism has been suggested to be a risk factor for Parkinson's disease. In order to define whether NQO1 plays a protective role in dopamine toxicity, we have examined the potential role of NQO1 in the SK-N-MC human neuroblastoma cell line. SK-N-MC cells were stably transfected with NQO1 to generate stable clones with NQO1 enzymatic activity of 245 nmol/mgmin while vector control and parental cells had NQO1 activities of less than 12 nmol/mgmin. Incubation of dopamine for 24 h in both parental and vector control SK-N-MC cells resulted in 85% and 72% cell death as assessed by annexin-V/propidium iodide analysis. In agreement, 88% and 84% of parental and vector control cells, respectively underwent loss of mitochondrial membrane potential (MMP) assessed by tetramethylrhodamine ethyl ester. In contrast, NQO1-transfected cells were resistant to dopamine toxicity and both cell death and loss of MMP were markedly abrogated in NQO1-transfected SK-N-MC cells. When dopamine was added to medium, oxygen uptake could be detected indicating autoxidation with concomitant formation of oxygen radicals and quinones. However, dopamine-induced cell death was not affected by the inclusion of either superoxide dismutase or catalase suggesting that superoxide and hydrogen peroxide were not involved in toxicity. Quinones formed in medium may exert toxicity extracellularly or intracellularly but the protective role of NQO1 argues for an intracellular mechanism. In summary, transfection of SK-N-MC cells with NQO1 protects against dopamine-induced toxicity.     

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

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

Malondialdehyde-acetaldehyde-haptenated protein induces cell death by induction of necrosis and apoptosis in immune cells

Recent studies have demonstrated that circulating antibodies against malondialdehyde-acetaldehyde (MAA)-haptenated proteins are significantly increased in patients with alcohol-induced cirrhosis and hepatitis and correlate with the severity of liver damage. Additionally, when proteins are haptenated with MAA, they become highly immunogenic in vivo in the absence of adjuvants. However, the mechanism(s) of this immunogenicity are currently unknown. Initial in vitro studies on the effects of MAA-modified proteins on cells demonstrated an increase in cell death at concentrations that were cell type specific and time-dependent. Since immunogenicity due to cell death has been described, we investigated the mechanism(s) by which cell death was occurring. Assessment of cell death in splenocytes after 1 h found significant levels of apoptosis as compared to controls. After 5 h, a significant and dose-dependent necrosis occurred in which cells were exposed to >62.5 microg/ml (43.1 mM) MAA-haptenated protein. After 24 h, exposure to >31.3 microg/ml (21.6 mM) MAA-haptenated protein resulted in significant levels of necrosis, although DNA laddering studies found apoptosis was occurring as well. Morphological changes in the cells were observed by light microscopy that correlated with a "low" forward scatter population by flow cytometry. Since necrosis has been implicated in enhancing both primary and secondary immune responses, and necrosis was predominantly occurring in response to MAA-haptenated proteins, a possible mechanism by which the immunogenicity of MAA modification of proteins in vivo may occur is suggested. Specifically, MAA modification of self proteins may result in the death of various cell types, most likely those in the liver. These necrotic materials may induce anti-MAA antibodies and other auto antibodies, whose levels may then correlate with the severity of ALD.     

Methamphetamine induces striatal cell death followed by the generation of new cells and a second round of cell death in mice

Our laboratory has been investigating the impact of a neurotoxic exposure to methamphetamine (METH) on cellular components of the striatum post-synaptic to the dopaminergic terminals. A systemic bolus injection of METH (30 mg/kg, ip) induces the production of new cells in the striatum during a period lasting from 24-48 hours after METH. The newly generated cells arise from dormant striatal progenitors and not from the subventricular zone. The newly generated cells display glial phenotypes and begin to die 24 hours after birth, or 2.5 days post-METH. The protracted phase of cell death lasts for at least three months post-METH at which time the bulk of the newly generated cells have disappeared. The METH-induced production of new cells is associated with enlarged striatal volume (up to 50% larger than controls in some animals). As the newly generated cells die over a period of three months, the enlarged striatal volume normalizes. In conclusion, a neurotoxic dose of METH induces the generation of new cells in the striatum associated with enlarged striatal volume. The new cells die over three months post-METH and the enlarged striatal volume returns to control levels. This observation is significant because studies involving METH users show striatal enlargement and the normalization of striatal volume in METH users who have been abstinent for up to 20 months.     

Diazene JK-279 induces apoptosis-like cell death in human cervical carcinoma cells.

Diazene N-phenyl-2-(2-pyridinyl)diazenecarboxamide (JK-279) is a newly synthesized compound, cytotoxic for several tumor cell lines and their drug-resistant sublines. In human cervical carcinoma cells (HeLa), this compound reduced intracellular glutathione content and increased sensitivity to cisplatin. The aim of the present study was to elucidate the molecular mechanisms involved in the cytotoxic effect of diazene JK-279 on HeLa cells. Cytotoxicity was determined by the MTT method. Flow cytometry analysis showed that diazene JK-279 induces G(2)/M phase arrest, mediated by the increase in p21 expression, and accompanied by an alteration in the expression of survivin. The highest concentration of JK-279 altered nuclear morphology in intact cells, showing "apoptosis-like" features. No cleavage of procaspase-3, procaspase-9 and PARP, or altered expression of apoptotic proteins Bcl-2 and Bax were detected. At the same time, PS externalization and internucleosomal DNA cleavage were observed. Partial necrosis was detected as well. Our results demonstrate that cytotoxicity of diazene JK-279 is mostly the consequence of caspase-independent cell death, which is in some aspects "apoptosis-like". Taking into account the multiplicity of mechanisms used by cancer cells to prevent apoptosis, the drugs (like diazene JK-279) that would activate alternative cell death pathways could provide a useful tool for new types of cancer therapy.     

The broad-spectrum caspase inhibitor Boc-Asp-CMK induces cell death in human leukaemia cells.

Synthetic caspase inhibitors and particularly broad-spectrum caspase inhibitors can prevent cells from death or at least slow down cell death process and abrogate some apoptotic hallmarks [Kitanaka, C., Kuchino, Y., 1999. Caspase-independent programmed cell death with necrotic morphology. Cell Death and Differentiation 6, 508-515]. However, not all synthetic caspase inhibitors diminish cell death. We have found that the broad-spectrum caspase inhibitor Boc-Asp-CMK induced cell death at micromolar concentrations in human leukaemia cells. Interestingly, low concentrations of Boc-Asp-CMK induced cell death with apoptotic hallmarks. Increasing concentrations of Boc-Asp-CMK led to necrotic cell death. The switch between apoptosis and necrosis seemed to depend upon the degree of inhibition of executioner caspases, including caspase-3/7 with Boc-Asp-CMK. Interestingly, caspase-3 processing was not inhibited even for the highest concentration of Boc-Asp-CMK used. We assume, that toxic properties of Boc-Asp-CMK can be attributed to the chloromethylketone residuum in its molecule, as its analogue Boc-Asp-FMK with fluoromethylketone residuum was more than 13 times less toxic. Our results further indicated that toxicity of Boc-Asp-CMK might arise from its interference with mitochondrial metabolism.     

Stapled peptide induces cancer cell death

Hydrocarbon stapling could enable peptides from the key domains of natural proteins to be used therapeutically. Using the technique on a peptide involved in apoptosis, researchers have succeeded in destroying cancer cells in a mouse model of leukaemia.