Comparison of the rapid pro-apoptotic effect of trans-ß-nitrostyrenes with delayed apoptosis induced by the standard agent 5-fluorouracil in colon cancer cells

Trans-beta-nitrostyrene (TBNS) has been reported to be a potent inhibitor of protein phosphatases PTB1 and PP2A and to display a pro-apoptotic effect even in multidrug resistant tumour cells. Here we compared the anti-tumour potential of TBNS with 5-fluorouracil (5-FU) as the standard chemotherapeutic agent for colorectal cancer in LoVo cells. Resistance to 5-FU based therapy might be a consequence of 5-FU's delayed effect requiring long-term effective concentrations in the tumour tissue. Thus, alternatives like platin containing drugs with a more rapid effect have been introduced recently. Compared to 5-FU TBNS displayed a faster cytotoxic and pro-apoptotic effect. A 50% decrease in viability was observed already after 8 h with TBNS while 5-FU displayed no significant effect before 48 h. DNA fragmentation and caspase-3 assays confirmed the more rapid apoptotic effect of TBNS. Since apoptosis affects individual cells these results about a rapidly induced apoptosis were further studied on a single cell level in microscopic assays of caspase-3 and caspase-8 activation. Adducts of trans-beta-nitrostyrene displayed an anti-tumour effect comparable to TBNS which suggests the possibility of creating adducts with optimised tissue targeting. Finally, the calculation of a drug combination index displayed a synergistic effect for the combination of TBNS and 5-FU in Lovo as well as in HT-29 and HCT116 colon cancer cells.     

Drug resistance to 5-FU linked to reactive oxygen species modulator 1

While acute oxidative stress triggers cell apoptosis or necrosis, persistent oxidative stress induces genomic instability and has been implicated in tumor progression and drug resistance. In a previous report, we demonstrated that reactive oxygen species modulator 1 (Romo1) expression was up-regulated in most cancer cell lines and suggested that increased Romo1 expression might confer chronic oxidative stress to tumor cells. In this study, we show that enforced Romo1 expression induces reactive oxygen species (ROS) production in the mitochondria leading to massive cell death. However, tumor cells that adapt to oxidative stress by increasing manganese superoxide dismutase (MnSOD), Prx I, and Bcl-2 showed drug resistance to 5-FU. To elucidate the relationship between 5-FU-induced ROS production and Romo1 expression, Romo1 siRNA was used to inhibit 5-FU-triggered Romo1 induction. Romo1 siRNA treatment efficiently blocked 5-FU-induced ROS generation, demonstrating that 5-FU treatment stimulated ROS production through Romo1 induction. Based on these results we suggest that cellular adaptive response to Romo1-induced ROS is another mechanism of drug resistance to 5-FU and Romo1 expression may provide a new clinical implication in drug resistance of cancer chemotherapy.     

Effects of 5-Fluorouracil on Morphology,Cell Cycle,Proliferation, Apoptosis,Autophagy and ROS Production in Endothelial Cells and Cardiomyocytes

Antimetabolites are a class of effective anticancer drugs interfering in essential biochemical processes. 5-Fluorouracil (5-FU) and its prodrug Capecitabine are widely used in the treatment of several solid tumors (gastro-intestinal, gynecological, head and neck, breast carcinomas). Therapy with fluoropyrimidines is associated with a wide range of adverse effects, including diarrhea, dehydration, abdominal pain, nausea, stomatitis, and hand-foot syndrome. Among the 5-FU side effects, increasing attention is given to cardiovascular toxicities induced at different levels and intensities. Since the mechanisms related to 5-FU-induced cardiotoxicity are still unclear, we examined the effects of 5-FU on primary cell cultures of human cardiomyocytes and endothelial cells, which represent two key components of the cardiovascular system. We analyzed at the cellular and molecular level 5-FU effects on cell proliferation, cell cycle, survival and induction of apoptosis, in an experimental cardioncology approach. We observed autophagic features at the ultrastructural and molecular levels, in particular in 5-FU exposed cardiomyocytes. Reactive oxygen species (ROS) elevation characterized the endothelial response. These responses were prevented by a ROS scavenger. We found induction of a senescent phenotype on both cell types treated with 5-FU. In vivo, in a xenograft model of colon cancer, we showed that 5-FU treatment induced ultrastructural changes in the endothelium of various organs. Taken together, our data suggest that 5-FU can affect, both at the cellular and molecular levels, two key cell types of the cardiovascular system, potentially explaining some manifestations of 5-FU-induced cardiovascular toxicity.     

Flavone protects HBE cells from DNA double-strand breaks caused by PM2.5

Ambient air particulate matter 2.5 (PM2.5) contains many harmful components that can enter the circulatory system and produce reactive oxygen species (ROS) in body. Oxidative stress and DNA damage induced by ROS may affect any cellular macromolecule and lead to DNA double-strand breaks (DSBs). Flavonoids, widely distributed in some herbs and berries, have been proved having anti-oxidative or anti-cancer efficacy. In this study, we investigated whether Flavone, a kind of flavonoids, can protect human bronchial epithelial cells (HBE) from DSBs caused by PM2.5 and how this function is probably implemented. We found that cells exposed to PM2.5 obviously induced viability inhibition, DNA damage and part of apoptosis. However, Flavone treatment prior to PM2.5 apparently improved cell viability, and mitigated the formation of 8-hydroxy-2-deoxyguanosine, the expression of DNA damage-relative protein and cell apoptosis. Our studies demonstrated that PM2.5 induced oxidative DSBs while Flavone ameliorated the DNA damage and increased cell viability probably through influencing DNA repair mechanism of cells.     

Photodynamic therapy regulates fate of cancer stem cells through reactive oxygen species

Photodynamic therapy (PDT) is an effective and promising cancer treatment. PDT directly generates reactive oxygen species (ROS) through photochemical reactions. This oxygen-dependent exogenous ROS has anti-cancer stem cell (CSC) effect. In addition, PDT may also increase ROS production by altering metabolism, endoplasmic reticulum stress, or potential of mitochondrial membrane. It is known that the half-life of ROS in PDT is short, with high reactivity and limited diffusion distance. Therefore, the main targeting position of PDT is often the subcellular localization of photosensitizers, which is helpful for us to explain how PDT affects CSC characteristics, including differentiation, self-renewal, apoptosis, autophagy, and immunogenicity. Broadly speaking, excess ROS will damage the redox system and cause oxidative damage to molecules such as DNA, change mitochondrial permeability, activate unfolded protein response, autophagy, and CSC resting state. Therefore, understanding the molecular mechanism by which ROS affect CSCs is beneficial to improve the efficiency of PDT and prevent tumor recurrence and metastasis. In this article, we review the effects of two types of photochemical reactions on PDT, the metabolic processes, and the biological effects of ROS in different subcellular locations on CSCs.     

Combinations of 5-fluorouracil with UCN-01 or staurosporine

The action of 5-Fluorouracil (5-FU) is mediated by inhibition of thymidylate synthase (TS), which is regulated by cell cycle proteins controlled by protein phosphorylation. We studied the effects of staurosporine and its analogue UCN-01, inhibitors of protein kinase C (PKC) on 5-FU cytotoxicity in Lovo colon cancer cells. Each drug contributes equally to the cell cycle effects of the 5-FU combinations. In sequential drug administration, the cell cycle distribution was determined by the first drug. Simultaneous 5-FU combinations induced additive effects in induction of apoptosis. When staurosporine was used as the second drug, induction of apoptosis was 2-fold higher than the sum of both drugs alone. Based on induction of apoptosis 5-FU addition prior to the PKC inhibitors seemed preferable.     

Selenocystine potentiates cancer cell apoptosis induced by 5-fluorouracil by triggering reactive oxygen species-mediated DNA damage and inactivation of the ERK pathway

5-Fluorouracil (5-FU)-based chemotherapy as a first-line treatment is quite limited, because of its inefficiency and clinical resistance to it. The search for chemosensitizers that could augment its efficiency and overcome the drug resistance to 5-FU has kindled great interest among scientists. Selenocystine (SeC), a naturally occurring selenoamino acid, displayed broad-spectrum anticancer activity in our previous studies. This study demonstrates that SeC acts as an effective enhancer of 5-FU-induced apoptosis in A375 human melanoma cells through induction of mitochondria-mediated apoptosis with the involvement of DNA damage-mediated p53 phosphorylation and ERK inactivation. Pretreatment of the cells with SeC significantly enhanced 5-FU-induced loss of mitochondrial membrane potential (∆ψ_m) by regulating the expression levels of Bcl-2 family proteins. SeC and 5-FU in combination also triggered cell oxidative stress through regulation of the intracellular redox system and led to DNA damage and inactivation of ERK and AKT. Moreover, inhibitors of ERK and AKT effectively enhanced the apoptotic cell death induced by the combined treatment. However, pretreatment of the cells with glutathione reversed the apoptosis induced by SeC and 5-FU and recovered the expression of ERK and AKT inactivation, which revealed the important role of reactive oxygen species in cell apoptosis and regulation of ERK and AKT pathways. Taken together, our results suggest that a strategy of using SeC and 5-FU in combination could be a highly efficient way to achieve anticancer synergism.     

Enhancement of radiation-induced cell killing and DNA double-strand breaks in a human tumor cell line using nanomolar concentrations of aclacinomycin A.

Several agents that induce differentiation have previously been shown to induce the terminal differentiation of leukemic cells and enhance the radiosensitivity of certain solid tumor cell lines in vitro using millimolar concentrations. We now report that aclacinomycin A (ACM), a potent inducer of leukemic cell differentiation in vitro, can significantly enhance the radiosensitivity of a human colon tumor cell line (Clone A) at a concentration of 10 nM. Based on colony-forming efficiency, the maximum increase in radiosensitivity was found using 15 nM ACM for 3 days with a dose enhancement factor of 1.4 at a surviving fraction of 0.10. This treatment increased cell doubling time, but had no effect on cell-cycle phase distribution. To gain insight into the mechanisms responsible for this radiosensitization, gamma-ray-induced DNA single- and double-strand breaks were examined. Aclacinomycin A had no effect on the induction of DNA single-strand breaks but significantly enhanced the formation of gamma-ray-induced DNA double-strand breaks. The rate or extent of repair of the induced double-strand breaks was not influenced by ACM treatment. These data suggest that ACM, at achievable plasma concentrations, can enhance the radiosensitivity of a human tumor cell line by increasing the initial level of radiation-induced DNA double-strand breaks.     

Reactive oxygen species mediate doxorubicin induced p53-independent apoptosis

Doxorubicin (DOX) is a common anticancer drug. The mechanisms of DOX induced apoptosis and the involvement of reactive oxygen species (ROS) in apoptotic signaling were investigated in p53-null human osteosarcoma Saos-2 cells. Accumulation of pre-G1 phase cells and induction of DNA laddering, which are the hallmarks of apoptosis, were detected in cells at 48 h upon DOX treatment. Furthermore, DOX increased the intracellular hydrogen peroxide and superoxide levels, followed by mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, prior to DNA laddering in Saos-2 cells. In addition, DOX treatment also upregulated Bax and downregulated Bcl-2 levels in the cells. The role of ROS in DOX induced cell death was confirmed by the suppression effect of catalase on DOX induced ROS formation, mitochondrial cytochrome c release, procaspase-3 cleavage, and apoptosis in Saos-2 cells. The catalase treatment however only suppressed DOX induced Bax upregulation but had no effect on Bcl-2 downregulation. Results from the present study suggested that ROS might act as the signal molecules for DOX induced cell death and the process is still functional even in the absence of p53.     

The mechanisms of oxidative DNA damage and apoptosis induced by norsalsolinol, an endogenous tetrahydroisoquinoline derivative associated with Parkinson's disease

Tetrahydroisoquinoline (TIQ) derivatives are putative neurotoxins that may contribute to the degeneration of dopaminergic neurons in Parkinson's disease. One TIQ, norsalsolinol (NorSAL), is present in dopamine-rich areas of human brain, including the substantia nigra. Here, we demonstrate that NorSAL reduces cell viability and induces apoptosis via cytochrome c release and caspase 3 activation in SH-SY5Y human neuroblastoma cells. Cytochrome c release, caspase 3 activation, and apoptosis induction were all inhibited by the antioxidant N -acetylcysteine. Thus, reactive oxygen species (ROS) contribute to apoptosis induced by NorSAL. Treatment with NorSAL also increased levels of oxidative damage to DNA, a stimulus for apoptosis, in SH-SY5Y. To clarify the mechanism of intracellular DNA damage, we examined the DNA damage caused by NorSAL using ³²P-5'-end-labeled isolated DNA fragments. NorSAL induced DNA damage in the presence of Cu(II). Catalase and bathocuproine, a Cu(I) chelator, inhibited this DNA damage, suggesting that ROS such as the Cu(I)-hydroperoxo complex derived from the reaction of H₂O₂ with Cu(I), promote DNA damage by NorSAL. In summary, NorSAL-generated ROS induced oxidative DNA damage, which led to caspase-dependent apoptosis in neuronal cells.     

Involvement of both caspase-dependent and -independent pathways in apoptotic induction by hexaminolevulinate-mediated photodynamic therapy in human lymphoma cells

Photodynamic therapy (PDT) is a cancer treatment based on the interaction of a photosensitizer, light and oxygen. PDT with the endogenous photosensitizer, protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) or its derivatives is a modification of this treatment modality with successful application in dermatology. However, the mechanism of cell destruction by ALA-PDT has not been elucidated. In this study a human T-cell lymphoma Jurkat cell line was treated with PDT using hexaminolevulinate (HAL, hexylester of ALA). Four hours following treatment nearly 80% of the cells exhibited typical apoptotic features. Mitochondrial pro-apoptotic proteins were evaluated by Western blots in subcellular fractionated samples. PDT caused cytosolic translocation of cytochrome c and nuclear redistribution of apoptosis-inducing factor (AIF), but the release of mitochondrial Smac/DIABLO, Omi/HtrA2 and EndoG was not observed. The release of cytochrome c was followed by the cleavage of caspase-9 and caspase-3 as well as its downstream substrates, together with oligonucleosomal DNA fragmentation. The pan-caspases inhibitor, z-VAD.fmk, prevented oligonucleosomal DNA fragmentation, but failed to inhibit PDT-mediated apoptosis. The apoptotic induction by AIF-mediated caspase-independent pathway was also found after HAL-PDT with large-scale DNA fragmentation in the presence of z-VAD.fmk. These results demonstrate that cytochrome c-mediated caspase-dependent pathway and AIF-induced caspase-independent pathway are simultaneously involved in the apoptotic induction by PDT. When the cytochrome c-induced caspase-dependent pathway is blocked, the cells go into apoptosis via AIF-mediated pathway, clearly demonstrating that the cytochrome c-mediated caspase-dependent pathway is not required for such apoptotic induction. This finding may have an impact on improved PDT effectiveness.     

Role of apoptosis in photodynamic sensitivity of human tumour cell lines

Photodynamic therapy (PDT) using a photosensitizer, such as haematoporphyrin derivative (HpD), in conjunction with visible light is a promising new modality to treat localized cancer. Cell death caused by PDT (through the generation of reactive oxygen species) can occur either by apoptosis (interphase death or as a secondary event following mitosis) and/or necrosis depending on the cell type, concentration and intracellular localization of the sensitizer, and the light dose. Since, apoptosis induced by PDT treatment plays an important role in determining the photodynamic efficacy, in the present work we have investigated the role of apoptotic cell death in relation to the observed differences in sensitivity to HpD-PDT between a human glioma cell line (BMG-1) carrying wild-type tumour suppressor gene p53 and a human squamous carcinoma cell line (4451) with mutated p53. HpD (photosan-3; PS-3) -PDT induced apoptosis was studied by: [A] flow-cytometric analysis of DNA content (sub G0/G1 population); [B] phosphatidylserine externalization (Annexin-V +ve cells); [C] cell size and cytoskeleton reorganization (light-scatter analysis); and [D] fluorescence microscopy (morphological features). PS-3-PDT induced a significantly higher level of apoptosis in BMG-1 cells as compared to 4451 cells. This was dependent on the concentration of PS-3 as well as post-irradiation time in both the cell lines. At 2.5 microg/ml of PS-3 the fraction of BMG-1 cells undergoing apoptosis (60%) was nearly 6 folds higher than 4451 cells (10%). In BMG-1 cells the induction of apoptosis increased with PS-3 concentration up to 5 microg/ml (>80%). However, a decrease was observed at a concentration of 10 microg/ml, possibly due to a shift in the mode of cell death from apoptosis to necrosis. In 4451 cells, on the other hand, the increase in apoptosis could be observed even up to 10 microg/ml of PS-3 (60%). Present results show that the higher sensitivity to PS-3-PDT in glioma cells arise on account of a higher level of apoptosis and suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in certain cell types.     

Subcellular localization of Photofrin determines the death phenotype of human epidermoid carcinoma A431 cells triggered by photodynamic therapy: when plasma membranes are the main targets

Photodynamic therapy (PDT) is a kind of photochemo-therapeutic treatment that exerts its effect mainly through the induction of cell death. Distinct types of cell death may be elicited by different PDT regimes. In this study, the mechanisms involved in the death of human epidermoid carcinoma A431 cells triggered by PDT with Photofrin (a clinically approved photosensitizer) were characterized. Photofrin distributes dynamically in A431 cells; the plasma membranes and Golgi complex are the main target sites of Photofrin after a brief (3 h) and prolonged (24 h) incubation, respectively. Cells with differentially localized Photofrin displayed distinct death phenotypes in response to PDT. The effects of PDT on cells with plasma membrane-localized Photofrin were further studied in details. Cells stopped proliferating post PDT at Photofrin dose >7 micro g/ml, and at higher dose (28 micro g/ml) plasma membrane disruption and cell swelling were observed immediately after PDT. Dramatic alterations of several important signaling events were detected in A431 cells post Photofrin-PDT, including (i) immediate formation of reactive oxygen species (ROS), (ii) rapid activation of c-Jun N-terminal kinase, (iii) delayed activation of caspase-3 and cleavage of polyADP-ribose polymerase and p21-activated kinase 2, and (iv) loss of mitochondrial membrane potential. Intriguingly, the characteristics of typical apoptosis such as phosphatidylserine externalization and DNA fragmentation were not detected in the cell death process caused by this PDT regime. In conclusion, our results show that when plasma membranes are the main targets, Photofrin-PDT can lead to instant ROS formation and subsequent activation of downstream signaling events similar to those elicited by many apoptotic stimuli, but the damage of plasma membranes renders the death phenotype more necrosis like.     

ATR-Chk1 signaling pathway and homologous recombinational repair protect cells from 5-fluorouracil cytotoxicity

5-Fluorouracil (5-FU) has long been a mainstay antimetabolite chemotherapeutic drug for the treatment of major solid tumors, particularly colorectal cancer. 5-FU is processed intracellularly to yield active metabolites that compromise RNA and DNA metabolism. However, the mechanisms responsible for its cytotoxicity are not fully understood. From the phenotypic analysis of mutant chicken B lymphoma DT40 cells, we found that homologous recombinational repair (HRR), involving Rad54 and BRCA2, and the ATR-Chk1 signaling pathway, involving Rad9 and Rad17, significantly contribute to 5-FU tolerance. 5-FU induced γH2AX nuclear foci, which were colocalized with the key HRR factor Rad51, but not with DNA double-strand breaks (DSBs), in a dose-dependent manner as cells accumulated in the S phase. Inhibition of Chk1 kinase by UCN-01 increased 5-FU-induced γH2AX and enhanced 5-FU cytotoxicity not only in wild-type cells but also in Rad54- or BRCA2-deficient cells, suggesting that HRR and Chk1 kinase have non-overlapping roles in 5-FU tolerance. 5-FU-induced Chk1 phosphorylation was significantly impaired in Rad9- or Rad17-deficient cells, and severe γH2AX nuclear foci and DSBs were formed, which was followed by apoptosis. Finally, inhibition of Chk1 kinase by UCN-01 increased 5-FU-induced γH2AX nuclear foci and enhanced 5-FU cytotoxicity in Rad9- or Rad17-deficient cells. These results suggest that Rad9- and Rad17-independent activation of the ATR-Chk1 signaling pathway also significantly contributes to 5-FU tolerance.     

Combination of 5-fluorouracil and genistein induces apoptosis synergistically in chemo-resistant cancer cells through the modulation of AMPK and COX-2 signaling pathways

5-Fluorouracil (5-FU) is one of the widely used chemotherapeutic drugs targeting various cancers, but its chemo-resistance remains as a major obstacle in clinical settings. In the present study, HT-29 colon cancer cells were markedly sensitized to apoptosis by both 5-FU and genistein compared to the 5-FU treatment alone. There is an emerging evidence that genistein, soy-derived phytoestrogen, may have potential as a chemotherapeutic agent capable of inducing apoptosis or suppressing tumor promoting proteins such as cyclooxygenase-2 (COX-2). However, the precise mechanism of cellular cytotoxicity of genistein is not known. The present study focused on the correlation of AMPK and COX-2 in combined cytotoxicity of 5-FU and genistein, since AMPK is known as a primary cellular homeostasis regulator and a possible target molecule of cancer treatment, and COX-2 as cell proliferation and anti-apoptotic molecule. Our results demonstrated that the combination of 5-FU and genistein abolished the up-regulated state of COX-2 and prostaglandin secretion caused by 5-FU treatment in HT-29 colon cancer cells. These appear to be followed by the specific activation of AMPK and the up-regulation of p53, p21, and Bax by genistein. Under same conditions, the induction of Glut-1 by 5-FU was diminished by the combination treatment with 5-FU and genistein. Furthermore, the reactive oxygen species (ROS) was found as an upstream signal for AMPK activation by genistein. These results suggested that the combination of 5-FU and genistein exert a novel chemotherapeutic effect in colon cancers, and AMPK may be a novel regulatory molecule of COX-2 expression, further implying its involvement in cytotoxicity caused by genistein.     

Photofrin--factor of photodynamic therapy induces apoptosis and necrosis

Photodynamic therapy (PDT) causes irreversible photodamage of tumor and other malignant tissues. The effect of reactive oxygen species generation in the presence of photofrin (HpD) was studied. The studies were performed on endothelial cell line from foetal aorta of calves and on normal fibroblasts cell line (3T3 -Balb) and also on malignant line (A431). The cells were grown in presence of photofrin at different time intervals. Time of interaction of photosensitiser with cells was very important. Short time of exposure of the cells to photofrin induced mostly apoptosis in normal cells and apoptotic or necrotic changes in malignant cells. Longer effect of these factors on cells provoked necrosis. The factors of PDT influence dynamic changes of SOD and CT activity. It was dependent on the intensity of factors. These results strongly suggest that HpD has an effect on generation of ROS, which are a signal for development of morphological changes (apoptosis or necrosis) in normal and malignant cells.     

Induction of autophagy enhances apoptotic cell death via epidermal growth factor receptor inhibition by canertinib in cervical cancer cells

BackgroundIt has been known epidermal growth factor receptor (EGFR) frequently overexpressed in cervical cancer. High levels of EGFR expression in their tumors leads to a poor prognosis and inhibition frequently induces autophagy in cancer cells. This study aimed to investigate whether EGFR inhibition by canertinib induces autophagy and this induction influence the effect of Palladium (Pd) (II) complex and 5-fluorouracil (5-FU) especially in nontoxic doses.MethodsCytotoxicity was evaluated by using SRB assay. Apoptosis, autophagy, and EGFR key markers were determined by flow cytometry, fluorescence staining, and immunoblotting. Colony formation, invasion, and wound healing assays were performed to investigate cell proliferation, invasion, and migration, respectively.ResultsBlocking EGFR by the pan-ErbB tyrosine kinase inhibitor canertinib inhibited cell growth of HeLa cervical cancer cells in combination with Pd(II) complex and 5-FU. Combination of canertinib and Pd(II) complex promotes autophagy and apoptosis of HeLa cancer cells via blockade of the PI3K/AKT and MAPK/ERK pathway, which leads to cervical cancer cell death. ROS accumulation and DNA damage were increased after combinatorial treatment which causes depolarization of the mitochondrial inner membrane and leads to apoptotic cell death. Canertinib combined with Pd(II) complex leads to inhibition of migration and invasion.ConclusionInhibition of EGFR signaling by canertinib in combination with Pd(II) complex promotes apoptosis and autophagy via blockade of the PI3K/AKT and MAPK/ERK.General significanceThe cytotoxic activity of Pd(II) complex and 5-FU on HeLa cells is mediated by EGFR inhibition and autophagy induction, leading to activation of mitochondrial apoptotic cell death.     

AIF suppresses chemical stress-induced apoptosis and maintains the transformed state of tumor cells

Apoptosis-inducing factor (AIF) exhibits reactive oxygen species (ROS)-generating NADH oxidase activity of unknown significance, which is dispensable for apoptosis. We knocked out the aif gene in two human colon carcinoma cell lines that displayed lower mitochondrial complex I oxidoreductase activity and produced less ROS, but showed increased sensitivity to peroxide- or drug-induced apoptosis. AIF knockout cells failed to form tumors in athymic mice or grow in soft agar. Only AIF with intact NADH oxidase activity restored complex I activity and anchorage-independent growth of aif knockout cells, and induced aif-transfected mouse NIH3T3 cells to form foci. AIF knockdown in different carcinoma cell types resulted in lower superoxide levels, enhanced apoptosis sensitivity and loss of tumorigenicity. Antioxidants sensitized AIF-expressing cells to apoptosis, but had no effect on tumorigenicity. In summary, AIF-mediated resistance to chemical stress involves ROS and probably also mitochondrial complex I. AIF maintains the transformed state of colon cancer cells through its NADH oxidase activity, by mechanisms that involve complex I function. On both counts, AIF represents a novel type of cancer drug target.     

Dysregulation of apoptosis by benzene metabolites and their relationships with carcinogenesis

Benzene is a widely recognized human carcinogen, the effect of which is attributed to the production of reactive oxygen species (ROS) from its metabolites. Although there have been many reports on the relationship between DNA damage induced by benzene metabolites and carcinogenesis, only a report approached the subject by examining the benzene-induced dysregulation of apoptosis. Inhibition of apoptosis, aberrantly prolonging cell survival, may contribute to cancer by facilitating the insurgence of mutations and by creating a permissive environment for genetic instability. In this study, we examined the mechanism of antiapoptotic effects by benzene metabolites, p-benzoquinone (BQ) and hydroquinone (HQ), and their relationships with carcinogenesis. BQ and HQ inhibited the apoptotic death of NIH3T3 cells induced by both serum starvation and lack of an extracellular matrix (ECM). An antioxidant agent, N-acetylcysteine, significantly inhibited the antiapoptotic effects induced by benzene metabolites, indicating that the effects were mainly due to the production of ROS. Furthermore, BQ and HQ inhibited the in vitro caspase-3 activation, suggesting that the inhibition of caspase-3 activation due to ROS produced by BQ- and HQ-treatment was related to the suppression of apoptosis. The cells that escaped apoptosis could survive with the addition of serum and attachment to the ECM. Levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine were higher in the cells which survived after BQ- and HQ-treatment than in the normal cells. Furthermore, the cells treated with BQ and HQ showed greater proliferation than normal cells under low-serum conditions and anchorage-independent growth in soft agar. These findings suggested that benzene metabolites induced dysregulation of apoptosis due to caspase-3 inhibition, which contributes to carcinogenesis.