Effect of ceramide on intracellular glutathione determines apoptotic or necrotic cell death of JB6 tumor cells.

Selective induction of cell death is a means to remove unwanted cell populations from a tissue or organ. Understanding the signaling events responsible for mediating cell death by cytokines, such as tumor necrosis factor-alpha (TNFalpha) are key to the development of pharmacologic inducers of this response. Ceramide has been implicated as a secondary messenger for TNFalpha-induced cell death, but many of the intracellular effects of ceramide are not fully understood. Recent reports suggest that ceramide signaling may involve oxidative stress. To explore the relationship between TNF sensitivity and ceramide signaling, two genetic variants of mouse JB6 RT101 epidermal tumor cells, one resistant and one sensitive to TNFalpha-induced cytotoxicity, were treated with C2-ceramide. Treatment with 20 microM ceramide induced apoptosis and this was quickly followed by oncotic necrosis in the TNFalpha-sensitive JB6 (TNFs) cells. The same concentration of ceramide induced apoptosis, but not oncotic necrosis of the TNFalpha resistant JB6 (TNFr) cells. The basal level of glutathione was significantly higher in TNFr cells than in TNFs cells. Treatment with 20 microM ceramide decreased cellular glutathione in TNFs cells by 50%, in contrast to an insignificant decrease in the TNFr cells. A significant increase in reactive oxygen was noted in TNFs cells treated with 10 or 20 microM ceramide. Furthermore, pretreatment with the antioxidant N-acetylcysteine or with glutathione monoethylester delayed the onset of ceramide-induced oncotic necrosis, but did not inhibit apoptosis. Our results suggest that the severity of the decrease in glutathione appears to determine whether cells undergo just apoptosis or also oncotic necrosis. They also suggest that ceramide-induced oncotic necrosis is modulated by a decline in cellular glutathione and an elevation of reactive oxygen. These results suggest that a decrease in cellular redox potential determines susceptibility to ceramide-dependent killing pathways.     

The natural marine anhydrophytosphingosine, Jaspine B, induces apoptosis in melanoma cells by interfering with ceramide metabolism

Marine environment has frequently afforded a variety of biologically active compounds with strong anticancer and cytotoxic properties. In the present study, the mechanism of action of Jaspine B, an anhydrophytosphingosine derivative isolated from the marine sponge Jaspis sp., was investigated. Jaspine B was able to dose- and time-dependently decrease the viability of murine B16 and human SK-Mel28 melanoma cells. On these cells, Jaspine B treatment triggered cell death by typical apoptosis as illustrated by phosphatidylserine externalization, the release of cytochrome c and caspase processing. These effects were associated with increased intracellular ceramide levels owing to perturbed ceramide metabolism. Indeed, Jaspine B exposure strongly inhibited the activity of sphingomyelin synthase (SMS), an enzyme that converts de novo ceramide into the membrane lipid sphingomyelin. Moreover, whereas Jaspine B-induced cell death was enhanced in SMS1-depleted cells, it was strongly inhibited in cells that stably overexpress human SMS1. Finally, the cytotoxic effects of Jaspine B truncated analogs were also shown to be dependent on SMS activity.Altogether, Jaspine B is able to kill melanoma cells by acting on SMS activity and consequently on ceramide formation, and may represent a new class of cytotoxic compounds with potential applications in anticancer melanoma therapy.     

The cisplatin-irradiation combination suggests that apoptosis is not a major determinant of clonogenic death

It is commonly believed that tumor cells treated with anticancer agents, chemotherapy and/or radiation, die by apoptosis and that tumors which do not undergo apoptosis are resistant to treatment. In this study, we investigated the molecular basis underlying cisplatin cytotoxicity in the murine teratocarcinoma F9 cell line to see whether irradiation enhances cisplatin-induced cytotoxicity. We compared the apoptosis induced by chemo and/or radiotherapy with other cellular effects such as cell survival, clonogenic capability, cell cycle perturbation, expression of p53 and p53-related mRNAs, and necrosis. When combined with radiation, a clear additive cytotoxic effect of cisplatin was demonstrated. We found that both cisplatin and radiation induced cell death, but the level of induced apoptosis was low and there was no correlation with the results of the clonogenic assays: we noted a difference between cytotoxic effects in the clonogenic assay and the extent of apoptosis by fluorescence-activated cell sorter analysis, suggesting that cell killing reflected not only apoptosis but also cell cycle arrest, and that apoptosis, cell kinetics and clonogenicity suppression were independent processes.     

Urothelial cells malignantly transformed by exposure to cadmium (Cd(+2)) and arsenite (As(+3)) have increased resistance to Cd(+2) and As(+3)-induced cell death.

This laboratory has shown that both Cd(+2) and As(+3) can malignantly transform human urothelial cells. The present study examined metal resistance and the mechanism of cell death when the parental and malignantly transformed UROtsa cells were exposed to Cd(+2) and As(+3). It was shown that the malignantly transformed UROtsa cells were more resistant to the toxic effects of both metals. The assessment of the mode of cell death demonstrated that the parental UROtsa cells died by both apoptosis and necrosis when exposed to either metal. It was shown that apoptosis was the more prominent mechanism of cell death, accounting for over 50% of cell death. Apoptotic cell death was determined by the observation of fragmented nuclei using 4',6-diamidino-2-phenylindole staining, the formation of a DNA ladder, and the detection of cleaved caspase-3 and caspase-9 products in the cell lysates. Necrotic cell death was determined by measuring the release of lactate dehydrogenase into the growth medium. It was determined that the extent of apoptosis of the malignantly transformed UROtsa cells was decreased and that the extent of necrosis was increased compared to the parental UROtsa cells. These observations are consistent with in vivo studies which suggest that As(+3) can act as a tumor promoter during the regeneration of the bladder urothelium. The present in vitro studies suggest that As(+3)-induced cytotoxicity could set the stage for tissue repair due to its own inherent toxicity to normal urothelium, and then subsequently act as a tumor promoter during the regeneration process through the stimulation of the regrowth of cells that have gained increased resistance to As(+3).     

The Anti-Cancer Potency and Mechanism of a Novel Tumor-Activated Fused Toxin,DLM

Melittin, which acts as a membrane-disrupting lytic peptide, is not only cytotoxic to tumors, but also vital to normal cells. Melittin had low toxicity when coupled with target peptides. Despite significant research development with the fused toxin, a new fused toxin is needed which has a cleavable linker such that the fused toxin can release melittin after protease cleavage on the tumor cell surface. We describe a novel fused toxin, composed of disintegrin, uPA (urokinase-type plasminogen activator)-cleavable linker, and melittin. Disintegrin is a single strand peptide (73 aa) isolated from Gloydius Ussuriensis venom. The RGD (Arg-Gly-Asp) site of disintegrin dominates its interaction with integrins on the surface of the tumor cells. uPA is over-expressed and plays an important role in tumor cell invasiveness and metastatic progression. The DLM (disintegrin-linker-melittin) linker is uPA-cleavable, enabling DLM to release melittin. We compared binding activity of our synthesized disintegrin with native disintegrin and report that DLM had less binding activity than the native form. uPA-cleavage was evaluated in vitro and the uPA-cleavable linker released melittin. Treating tumors expressing uPA with DLM enhanced tumor cell killing as well as reduced toxicity to erythrocytes and other non-cancerous normal cells. The mechanism behind DLM tumor cell killing was tested using a DNA ladder assay, fluorescent microscopy, flow cytometry, and transmission electron microscopy. Data revealed tumor cell necrosis as the mechanism of cell death, and the fused DLM toxin with an uPA-cleavable linker enhanced tumor selectivity and killing ability.     

3-Nitrofluoranthene (3-NF) but not 3-aminofluoranthene (3-AF) elicits apoptosis as well as programmed necrosis in Hepa1c1c7 cells

In this study, we show that the environmental pollutant, 3-nitrofluoranthene (3-NF) but not its amine form, 3-aminofluoranthene (3-AF), induces apoptosis as well as regulated necrosis with necroptotic features in Hepa1c1c7 cells. Upon exposure to 3-NF, both typical apoptotic and necrotic cells were observed. A large number of the cells exhibited a characteristic partial nuclear chromatin condensation. Cycloheximide completely attenuated 3-NF-induced cell death. Activation of caspase-8, -9, and -3 were observed. Moreover, Z-VAD-FMK decreased the apoptotic cells, whereas the number of propidium iodide (PI)-positive cells with partial chromatin condensation was reduced by Nec-1, an inhibitor of receptor interacting protein (RIP-1). Cyp1a1, but not nitric oxide synthase (NOS), appears to be involved in activation of 3-NF to reactive metabolites. Increase in the number as well as size of lysosomes, myelinosomes, and activation of autophagy were also observed. 3-NF induced phosphorylation of ERK1/2, JNK and p38 MAPKs. Interestingly, while inhibitors of ERK1/2 and JNK reduced apoptotic as well as necrotic cell death, the p38 inhibitor, SB202190 reduced only the necrotic cell death. Taken together, 3-NF elicits both apoptosis and a caspase-independent programmed cell death (PCD) with autophagic characteristics. Conversely, with 3-AF, no apparent cytotoxic effects besides a reduction in cell proliferation was observed.     

Targeting TRAIL death receptors

The natural occurring tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induces apoptosis following binding to the two TRAIL death receptors (DRs). Its recombinant form and monoclonal antibodies against the TRAIL DRs induce cell death in a wide variety of tumor cell lines and xenografts without causing toxicity to normal cells and are therefore potential attractive anticancer agents. These agents are currently in early clinical development. The phase 1 and 2 studies showed until now limited toxicity and tumor responses have been observed. Ongoing studies focus especially on combination of these agents with other targeted therapies or cytotoxic therapies. In this review, we summarize current knowledge on these agents and highlight their potential role in the intrinsically chemotherapy-resistant glioblastomas. In addition, we discuss the mechanisms to sensitize tumors cells to rhTRAIL by combination with the proteasome inhibitor bortezomib.     

Cell cycle arrest,extracellular matrix changes and intrinsic apoptosis in human melanoma cells are induced by Boron Neutron Capture Therapy

Boron Neutron Capture Therapy (BNCT) involves the selective accumulation of boron carriers in tumor tissue followed by irradiation with a thermal or epithermal neutron beam. This therapy is therefore a cellular irradiation suited to treat tumors that have infiltrated into healthy tissues. BNCT has been used clinically to treat patients with cutaneous melanomas which have a high mortality. Human normal melanocytes and melanoma cells were treated with BNCT at different boronophenylalanine concentrations for signaling pathways analysis. BNCT induced few morphological alterations in normal melanocytes, with a negligible increase in free radical production. Melanoma cells treated with BNCT showed significant extracellular matrix (ECM) changes and a significant cyclin D1 decrease, suggesting cell death by necrosis and apoptosis and cell cycle arrest, respectively. BNCT also induced a significant increase in cleaved caspase-3 and a decrease in the mitochondrial electrical potential with selectivity for melanoma cells. Normal melanocytes had no significant differences due to BNCT treatment, confirming the data from the literature regarding the selectivity of BNCT. The results from this study suggest that some signaling pathways are involved in human melanoma treatment by BNCT, such as cell cycle arrest, ECM changes and intrinsic apoptosis.     

Troglitazone does not protect rat pancreatic beta cells against free fatty acid-induced cytotoxicity

Thiazolidinediones are a novel class of antidiabetic drugs that reduce insulin resistance through interaction with nuclear peroxisome proliferator-activated receptor (PPAR)gamma. One of these agents, troglitazone, was also proposed to protect beta cells against FFA-induced toxicity, but this effect has not yet been directly demonstrated. We recently reported in vitro conditions under which free fatty acids (FFA) cause beta cell death by necrosis or apoptosis. The present study investigates whether troglitazone (10 microM) interferes with this FFA-induced toxicity. Addition of this compound did not protect against oleate- or palmitate-induced toxicity. On the contrary, it increased palmitate-induced necrosis during the first two days of culture, and elevated (increase by 10-20%, P<0.05) both oleate- and palmitate-induced apoptosis after 8 days. These results do not support the view that troglitazone exerts a direct protective effect on beta cells that are exposed to cytotoxic FFA concentrations. They instead indicate that the agent may sensitize pancreatic beta cells to FFA-induced damage, raising the possibility that its use facilitates the deleterious effect of increased FFA levels on the pancreatic beta cell mass.     

Cytotoxic,genotoxic and oxidative stress induced by 1,4-naphthoquinone in B16F1 melanoma tumor cells

Quinones have diverse pharmacological properties including antibacterial, antifungal, antiviral, anti-inflammatory, antipyretic and anticancer activity. The cytotoxic potential of 1,4-naphthoquinone (NQ14) was studied against B16F1 melanoma cells grown in vitro. NQ14 treatment resulted in a concentration-dependent cytotoxicity as indicated by MTT assay and lactate dehydrogenase leakage assay. Depletion in cellular glutathione levels after 1 h incubation with NQ14 correlated with the corresponding increase in reactive oxygen species generation as determined by 2′,7′-dicholorofluorescein diacetate assay suggests the role of oxidative stress in cell death. The frequency of micronucleated binucleate cells increased with increasing doses of NQ14 with a corresponding decrease in the cytokinesis block proliferation index indicating the drug induced genotoxicity and cell division delay. Further, a dose-dependent decrease in the clonogenic cell survival indicated the potential of NQ14 to inhibit cell proliferation contributing to cell death. The cell death after NQ14 treatment may be attributed to apoptosis as seen in DNA ladder pattern along with necrosis as indicated in flow cytometric analysis of Annexin V/PI stained cells. Results of the present study demonstrate the cytotoxic and genotoxic potential of NQ14 by the induction of oxidative stress mediated mechanisms leading to tumor cell kill.     

Inhibitory effects of fusarochromanone on melanoma growth

Fusarochromanone is a toxic metabolite produced by Fusarium equiseti, a fungus present in decaying cereal plants in northern latitudes; it has been detected in various food grains. Fusarochromanone has been shown to have both stimulatory and inhibitory effects on various mammalian cells, depending on the concentration used. Whether these cytotoxic effects can be used in the clinical treatment of tumors remains to be established. Here, we evaluated the effects of fusarochromanone on the growth of human melanoma cells both in vitro and in vivo. In vitro, low concentrations (0.1-1 nmol/l) of fusarochromanone were found to be cytotoxic to many melanoma cell lines. In contrast, growth of normal melanocytes was inhibited only at much higher fusarochromanone concentrations (100-200 nmol/l). In vivo, the growth of melanoma cells implanted subcutaneously in immuno-compromised mice was significantly (P<0.05) reduced by daily administration of fusarochromanone. Immunohistological analyses indicated a significant (P<0.05) increase in the expression of active caspase-3 in tumor masses of mice treated with fusarochromanone, compared with controls. Together, these observations show that fusarochromanone increased apoptosis of tumor cells and reduced tumor growth in vivo. Therefore, the effects of fusarochromanone warrant further investigation as an adjuvant molecule to prevent growth and recurrence of melanomas.     

Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress

Chromosomal DNA dysfunction plays a role in mammalian cell death. Oxidative stress producing reactive oxygen species (ROS) induces chromatin dysfunction such as single- and double-strand DNA fragmentation leading to cell death through apoptosis or necrosis. More than 1 Mbp giant DNA, 200-800 or 50-300 kbp high molecular weight (HMW) DNA and internucleosomal DNA fragments are produced by oxidative stress and by some agents producing ROS during apoptosis or necrosis in several types of mammalian cells. Some nucleases involved in the chromosomal DNA fragmentation in apoptosis or necrosis are classified. ROS-mediated DNA fragmentation is caused and enhanced by polyunsaturated fatty acids (PUFAs) or their hydroperoxides through lipid peroxidation. A reduction of intracellular GSH levels induced by the inhibition of cystein transport or GSH biosynthesis leads to cell death through over production and accumulation of ROS in some types of mammalian cells. The ROS accumulation system has been used as a model of oxidative stress to discuss whether ROS-mediated DNA fragmentation associated with cell death is based on apoptosis or necrosis.     

Down-Regulation of Survivin by Nemadipine-A Sensitizes Cancer Cells to TRAIL-Induced Apoptosis

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family of cytokines. TRAIL selectively induces apoptotic cell death in various tumors and cancer cells, but it has little or no toxicity in normal cells. Agonism of TRAIL receptors has been considered to be a valuable cancer-therapeutic strategy. However, more than 85% of primary tumors are resistant to TRAIL, emphasizing the importance of investigating how to overcome TRAIL resistance. In this report, we have found that nemadipine-A, a cell-permeable L-type calcium channel inhibitor, sensitizes TRAIL-resistant cancer cells to this ligand. Combination treatments using TRAIL with nemadipine-A synergistically induced both the caspase cascade and apoptotic cell death, which were blocked by a pan caspase inhibitor (zVAD) but not by autophagy or a necrosis inhibitor. We further found that nemadipine-A, either alone or in combination with TRAIL, notably reduced the expression of survivin, an inhibitor of the apoptosis protein (IAP) family of proteins. Depletion of survivin by small RNA interference (siRNA) resulted in increased cell death and caspase activation by TRAIL treatment. These results suggest that nemadipine-A potentiates TRAIL-induced apoptosis by down-regulation of survivin expression in TRAIL resistant cells. Thus, combination of TRAIL with nemadipine-A may serve a new therapeutic scheme for the treatment of TRAIL resistant cancer cells, suggesting that a detailed study of this combination would be useful.     

NF-kappaB inhibition improves the sensitivity of human glioblastoma cells to 5-aminolevulinic acid-based photodynamic therapy

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are very resistant to all current therapies and are associated with a huge rate of recurrence. In most cases, this type of tumor is characterized by a constitutive activation of the nuclear factor-kappaB (NF-κB). This factor is known to be a key regulator of various physiological processes such as inflammation, immune response, cell growth or apoptosis. In the present study, we explored the role of NF-κB activation in the sensitivity of human glioblastoma cells to a treatment by 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT). 5-ALA is a physiological compound widely used in PDT as well as in tumor photodetection (PDD). Our results show that inhibition of NF-κB improves glioblastoma cell death in response to 5-ALA-PDT. We then studied the molecular mechanisms underlying the cell death induced by PDT combined or not with NF-κB inhibition. We found that apoptosis was induced by PDT but in an incomplete manner and that, unexpectedly, NF-κB inhibition reduced its level. Oppositely PDT mainly induces necrosis in glioblastoma cells and NF-κB is found to have anti-necrotic functions in this context. The autophagic flux was also enhanced as a result of 5-ALA-PDT and we demonstrate that stimulation of autophagy acts as a pro-survival mechanism confering protection against PDT-mediated necrosis. These data point out that 5-ALA-PDT has an interesting potential as a mean to treat glioblastoma and that inhibition of NF-κB renders glioblastoma cells more sensitive to the treatment.     

Crotonaldehyde induces oxidative stress and caspase-dependent apoptosis in human bronchial epithelial cells

Crotonaldehyde is a widespread environmental pollutant and lipid peroxidation product. Crotonaldehyde is a risk factor for many diseases (e.g., chronic pulmonary inflammation). However, its toxicity and its mechanism of action have not been thoroughly investigated. The purpose of this study is to investigate crotonaldehyde-induced oxidative stress and mechanism of cell death in BEAS-2B cells. Crotonaldehyde caused decreases of intracellular reduced glutathione levels and increases of reactive oxygen species in a dose-dependent manner. Crotonaldehyde induced cell death by apoptosis, and gradually transitioned to necrosis at high dose of crotonaldehyde, as demonstrated by Annexin V-FITC/PI staining and cell morphology analysis. Crotonaldehyde-induced ATP decline observed in the study might partially account for the switch from apoptosis to necrosis. Mitochondria membrane potential, cytochrome c release, caspase-9, and caspase-3/7 activity were investigated, and the results suggest that crotonaldehyde-induced apoptosis was activated in a caspase-dependent way. Collectively, these results demonstrate crotonaldehyde induces cell oxidative stress and caspase-dependent apoptosis.     

B16 and cloudman S91 mouse melanoma cells susceptibility to apoptosis after dacarbazine treatment

Considering the necessity of an individual choice of cytostatic drugs for patients with cancer disease and tumor cells' resistance to these compounds, their ability to induction of apoptosis should be investigated. The aim of this study was to determine the influence of dacarbazine (DTIC) on morphology and kinetics of proliferation of B16 and Cloudman S91 cells. It is important to determine the kind of death induced by the DTIC and the effect of a specific concentration. The evaluation of apoptosis and necrosis in these two mouse melanoma cell lines in vitro was performed. Induction of apoptosis was estimated in annexin V binding assay by flow cytometry. DNA content and cell cycle phases were determined by propidium iodide staining. DTIC induced morphological changes typical for apoptosis and necrosis in both cell lines. DTIC caused cell cycle arrest in S and G2/M phase of both cell lines which showed hypertetraploidy. The highest induction of apoptosis was observed in DTIC concentration of 200 microg/mL for B16 cells (11%) and 100 microg/mL for apoptosis Cloudman S91 cells (22.2%). Higher doses of DTIC caused intensification of necrotic process. The B16 melanoma cells are more sensitive to DTIC than the Cloudman S91 cells, however more intensive apoptotic process was detected in Cloudman S91 cells already at lower concentration of DTIC.     

Targeting the protein ubiquitination machinery in melanoma by the NEDD8-activating enzyme inhibitor pevonedistat (MLN4924)

Background The neddylation pathway conjugates NEDD8 to cullin-RING ligases and controls the proteasomal degradation of specific proteins involved in essential cell processes. Pevonedistat (MLN4924) is a selective small molecule targeting the NEDD8-activating enzyme (NAE) and inhibits an early step in neddylation, resulting in DNA re-replication, cell cycle arrest and death. We investigated the anti-tumor potential of pevonedistat in preclinical models of melanoma. Methods Melanoma cell lines and patient-derived tumor xenografts (PDTX) treated with pevonedistat were assessed for viability/apoptosis and tumor growth, respectively, to identify sensitive/resistant models. Gene expression microarray and gene set enrichment analyses were performed in cell lines to determine the expression profiles and pathways of sensitivity/resistance. Pharmacodynamic changes in treated-PDTX were also characterized. Results Pevonedistat effectively inhibited cell viability (IC₅₀ < 0.3 μM) and induced apoptosis in a subset of melanoma cell lines. Sensitive and resistant cell lines exhibited distinct gene expression profiles; sensitive models were enriched for genes involved in DNA repair, replication and cell cycle regulation, while immune response and cell adhesion pathways were upregulated in resistant models. Pevonedistat also reduced tumor growth in melanoma cell line xenografts and PDTX with variable responses. An accumulation of pevonedistat-NEDD8 adduct and CDT1 was observed in sensitive tumors consistent with its mechanism of action. Conclusions This study provided preclinical evidence that NAE inhibition by pevonedistat has anti-tumor activity in melanoma and supports the clinical benefits observed in recent Phase 1 trials of this drug in melanoma patients. Further investigations are warranted to develop rational combinations and determine predictive biomarkers of pevonedistat.     

Apoptosis of Hela cells induced by extract from Cremanthodium humile.

Cremanthodium humile (C. humile) is a traditional herbal medicine for treatment of inflammation. Based on initial screening results, the purpose of this study was to evaluate the cytotoxic effect on four human cancer cell lines and one non-cancer cell line (293), then to determine the possible mechanisms of cell death elicited by the extract of C. humile on Hela cells. We have found the ether extract of C. humile (CH-EE) strongly decreased the survival rate of the four human tumor cell lines: Hela, A549, HepG2 and SW480. The cytotoxic effect of CH-EE on 293 was smaller than on tumor cell lines. Flow cytometry assays and nuclear staining showed that CH-EE induced apoptosis in Hela cells. This process was accompanied by the collapse of mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3/7 and -9. Furthermore, CH-EE generated reactive oxygen species (ROS) in Hela cells. These results indicate that CH-EE induces apoptosis in Hela cells through a ROS-mediated mitochondrial dysfunction pathway.     

Tumor cell death induced by the inhibition of mitochondrial electron transport: The effect of 3-hydroxybakuchiol

Changes in mitochondrial ATP synthesis can affect the function of tumor cells due to the dependence of the first step of glycolysis on mitochondrial ATP. The oxidative phosphorylation (OXPHOS) system is responsible for the synthesis of approximately 90% of the ATP in normal cells and up to 50% in most glycolytic cancers; therefore, inhibition of the electron transport chain (ETC) emerges as an attractive therapeutic target. We studied the effect of a lipophilic isoprenylated catechol, 3-hydroxybakuchiol (3-OHbk), a putative ETC inhibitor isolated from Psoralea glandulosa. 3-OHbk exerted cytotoxic and anti-proliferative effects on the TA3/Ha mouse mammary adenocarcinoma cell line and induced a decrease in the mitochondrial transmembrane potential, the activation of caspase-3, the opening of the mitochondrial permeability transport pore (MPTP) and nuclear DNA fragmentation. Additionally, 3-OHbk inhibited oxygen consumption, an effect that was completely reversed by succinate (an electron donor for Complex II) and duroquinol (electron donor for Complex III), suggesting that 3-OHbk disrupted the electron flow at the level of Complex I. The inhibition of OXPHOS did not increase the level of reactive oxygen species (ROS) but caused a large decrease in the intracellular ATP level. ETC inhibitors have been shown to induce cell death through necrosis and apoptosis by increasing ROS generation. Nevertheless, we demonstrated that 3-OHbk inhibited the ETC and induced apoptosis through an interaction with Complex I. By delivering electrons directly to Complex III with duroquinol, cell death was almost completely abrogated. These results suggest that 3-OHbk has antitumor activity resulting from interactions with the ETC, a system that is already deficient in cancer cells.