Changes in elemental concentrations in LNCaP cells are associated with a protective effect of neuropeptides on etoposide-induced apoptosis

One of the mechanisms that has been put forward for the development of the androgen-resistant status is neuroendocrine differentiation. Neuroendocrine cells secrete neuropeptides that may represent one of the possible molecular bases by which hormone-dependent prostate cancer cells could escape treatment. LNCaP prostate cancer cells were treated with either etoposide or neuropeptides. Morphological changes related to apoptosis and cell viability were assessed. Changes in intracellular ion content were quantitatively analyzed by electron probe X-ray microanalysis. Etoposide treatment consistently induces a decrease in K and an increase in Na, which are inhibited by bombesin or calcitonin. The Na/K ratio increased markedly after exposure to etoposide, and both bombesin and calcitonin blocked this increase. Etoposide also caused changes in the intracellular P and S concentrations that to a large extent could be blocked by neuropeptides. These results support the hypothesis that neuropeptides confer anti-apoptotic capabilities onto non-neuroendocrine cells in close proximity to neuroendocrine cells.     

The Na+/H+ exchanger controls deoxycholic acid-induced apoptosis by a H+-activated, Na+-dependent ionic shift in esophageal cells

Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.     

(+)-Medioresinol leads to intracellular ROS accumulation and mitochondria-mediated apoptotic cell death in Candida albicans

The phytochemical (+)-Medioresinol, a furofuran type lignan identification and isolation on the stem bark of Sambucus williamsii, which is a folk medicinal plant used in traditional medicine. (+)-Medioresinol is known to possess a lesishmanicidal activity and cardiovascular disease risk reduction but its antifungal effects have not yet been identified. In this study, to confirm (+)-Medioresinol's antifungal properties and mode of action, we observed morphological and physiological change in Candida albicans. In cells exposed to (+)-Medioresinol, arrested the cell cycle and intracellular reactive oxygen species (ROS) which is a major cause of apoptosis were increased. The increase of ROS induced oxidative stress and the mitochondria dysfunction which causes release of pro-apoptotic factors. We investigated a series of characteristic cellular changes of apoptosis by using various apoptosis detection methods. We report here for the first time that (+)-Medioresinol has effects on mitochondria and induced the accumulation of ROS in C. albicans cells. We demonstrated that one of the important features of apoptosis, mitochondrial membrane depolarization is caused by ROS. Substantially, we investigated the release of cytochrome c, which is one of the factors of metacaspase activity. We also show that the effects of (+)-Medioresinol are mediated at an early stage in apoptosis acting on the plasma membrane phosphatidylserine externalization. In addition, (+)-Medioresinol induced apoptotic morphological changes, showing the reduced cell size (low FSC) and enhanced intracellular density (high SSC). In late stage of confirmation of diagnostic markers in yeast apoptosis include the effects of nucleus morphological change, DNA fragmentation and condensation by influence of oxidative stress. These apoptotic phenomena represent that oxidative stress and mitochondria dysfunctions by inducing the phytochemical (+)-Medioresinol must be an important factors of the apoptotic process in C. albicans. These results support the elucidation of the underlying antifungal mechanisms of (+)-Medioresinol.     

Changes in intracellular sodium, chlorine, and potassium concentrations in staurosporine-induced apoptosis

Ion gradients across the plasma membrane, fundamentally K(+), play a pivotal role in the execution phase of apoptosis. However, little is known about other monovalent anions (Cl(-)) or cations (Na(+)) in apoptosis. In addition, the relationship between changes in total ion composition and morphological and biochemical events are poorly understood. We investigated simultaneous changes in sodium (Na), chlorine (Cl), and potassium (K) concentrations in stauroporine-induced apoptosis by quantitative electron probe X-ray microanalysis (EPXMA) in single cells. Apoptotic cells identified unequivocally from the presence of chromatin condensation in backscattered electron images were characterized by an increase in intracellular Na, a decrease in intracellular Cl and K concentrations, and a decrease in K/Na ratio. The ouabain-sensitive Rb-uptake assay demonstrated a net decrease in Na(+)/K(+)-ATPase activity, suggesting that increases in Na and decreases in K and the K/Na ratio in apoptotic cells were related with inhibition of the Na(+)/K(+)-ATPase pump. These changes in diffusible elements were associated with externalization of phosphatidyl serine and oligonucleosomal fragmentation of DNA. This alteration in ion homeostasis and morphological hallmarks of apoptosis occur in cells that have lost their inner mitochondrial transmembrane potential and before the plasma membrane becomes permeable.     

beta-Lapachone-induced apoptosis in human prostate cancer cells: involvement of NQO1/xip3.

beta-Lapachone (beta-lap) induces apoptosis in various cancer cells, and its intracellular target has recently been elucidated in breast cancer cells. Here we show that NAD(P)H:quinone oxidoreductase (NQO1/xip3) expression in human prostate cancer cells is a key determinant for apoptosis and lethality after beta-lap exposures. beta-Lap-treated, NQO1-deficient LNCaP cells were significantly more resistant to apoptosis than NQO1-expressing DU-145 or PC-3 cells after drug exposures. Formation of an atypical 60-kDa PARP cleavage fragment in DU-145 or PC-3 cells was observed after 10 microM beta-lap treatment and correlated with apoptosis. In contrast, LNCaP cells required 25 microM beta-lap to induce similar responses. Atypical PARP cleavage in beta-lap-treated cells was not affected by 100 microM zVAD-fmk; however, coadministration of dicoumarol, a specific inhibitor of NQO1, reduced beta-lap-mediated cytotoxicity, apoptosis, and atypical PARP cleavage in NQO1-expressing cells. Dicoumarol did not affect the more beta-lap-resistant LNCaP cells. Stable transfection of LNCaP cells with NQO1 increased their sensitivity to beta-lap, enhancing apoptosis compared to parental LNCaP cells or vector-alone transfectants. Dicoumarol increased survival of beta-lap-treated NQO1-expressing LNCaP transfectants. NQO1 activity, therefore, is a key determinant of beta-lap-mediated apoptosis and cytotoxicity in prostate cancer cells.     

Neuropeptides, apoptosis and ion changes in prostate cancer. Methods of study and recent developments

It has been suggested that neuroendocrine (NE) cells provide paracrine stimuli for the propagation of local carcinoma cells and that NE differentiation is associated with the progression of prostate cancer toward an androgen-independent state. Apoptosis comprises a critical intracellular defense mechanism against tumorigenic growth and is associated with a number of changes in the elemental content of the cell. The neuropeptides bombesin and calcitonin, which inhibit etoposide-induced apoptosis, also inhibit the etoposide-induced elemental changes in prostate carcinoma cells. This important fact strengthens the link between apoptosis and changes in the intracellular elemental content. This protective effect on etoposide-induced apoptosis appears to be quite similar in androgen-dependent and androgen-independent cell lines. This confirms that neuropeptides confer antiapoptotic capabilities on non-neuroendocrine cells in close proximity to neuroendocrine cells. It can therefore be speculated that certain neuroendocrine peptides can increase the survival and further growth of neighboring cells and may thereby contribute to the aggressive clinical course of prostate tumors containing neuroendocrine elements. In addition, this correlation provides an objective basis for the study of neuropeptide target points and may be helpful for alternative therapeutic protocols using neuropeptide inhibitors in the treatment of patients with advanced prostatic carcinoma. The culture techniques described were, thus, designed in order to achieve two important goals. First, the development of an in vitro model that allows an approach to neuroendocrine differentiation in prostate cancer and its role in apoptosis blockage. Second, the method has been designed in order to permit rapid cryofixation of intact cell monolayers for subsequent x-ray microanalysis.     

Qualitative and Quantitative Analysis of ROS-Mediated Oridonin-Induced Oesophageal Cancer KYSE-150 Cell Apoptosis by Atomic Force Microscopy

High levels of intracellular reactive oxygen species (ROS) in cells is recognized as one of the major causes of cancer cell apoptosis and has been developed into a promising therapeutic strategy for cancer therapy. However, whether apoptosis associated biophysical properties of cancer cells are related to intracellular ROS functions is still unclear. Here, for the first time, we determined the changes of biophysical properties associated with the ROS-mediated oesophageal cancer KYSE-150 cell apoptosis using high resolution atomic force microscopy (AFM). Oridonin was proved to induce ROS-mediated KYSE-150 cell apoptosis in a dose dependent manner, which could be reversed by N-acetylcysteine (NAC) pretreatment. Based on AFM imaging, the morphological damage and ultrastructural changes of KYSE-150 cells were found to be closely associated with ROS-mediated oridonin-induced KYSE-150 cell apoptosis. The changes of cell stiffness determined by AFM force measurement also demonstrated ROS-dependent changes in oridonin induced KYSE-150 cell apoptosis. Our findings not only provided new insights into the anticancer effects of oridonin, but also highlighted the use of AFM as a qualitative and quantitative nanotool to detect ROS-mediated cancer cell apoptosis based on cell biophysical properties, providing novel information of the roles of ROS in cancer cell apoptosis at nanoscale.     

Modulation of intracellular signaling pathways to induce apoptosis in prostate cancer cells

An understanding of the molecular pathways defining the susceptibility of prostate cancer, especially refractory prostate cancer, to apoptosis is the key for developing a cure for this disease. We previously demonstrated that up-regulating Ras signaling, together with suppression of protein kinase C (PKC), induces apoptosis. Dysregulation of various intracellular signaling pathways, including those governed by Ras, is the important element in the development of prostate cancer. In this study, we tested whether it is possible to modulate the activities of these pathways and induce an apoptotic crash among them in prostate cancer cells. Our data showed that DU145 cells express a high amount of JNK1 that is phosphorylated after endogenous PKC is suppressed, which initiates caspase 8 cleavage and cytochrome c release, leading to apoptosis. PC3 and LNCaP cells contain an activated Akt. The inhibition of PKC further augments Akt activity, which in turn induces ROS production and the accumulation of unfolded proteins in the endoplasmic reticulum, resulting in cell death. However, the concurrent activation of JNK1 and Akt, under the condition of PKC abrogation, dramatically augment the magnitude of apoptosis in the cells. Thus, our study suggests that Akt, JNK1, and PKC act in concert to signal the intracellular apoptotic machinery for a full execution of apoptosis in prostate cancer cells.     

Redox modulation of human prostate carcinoma cells by selenite increases radiation-induced cell killing

Although selenium compounds have been extensively studied as chemopreventative agents for prostate cancer, little is known about the potential use of selenium compounds for chemotherapy. We have shown that selenite inhibits cell growth and induces apoptosis in androgen-dependent LAPC-4 prostate cancer cells. LAPC-4 cells were more sensitive to selenite-induced apoptosis than primary cultures of normal prostate cells. Selenite-induced apoptosis in LAPC-4 cells correlated with a decrease in the Bcl-2:Bax expression ratio. Selenite-induced oxidative stress and apoptosis are dependent upon its reaction with reduced GSH. LAPC-4 cells treated with selenite showed decreased levels of total GSH and increased concentrations of GSSG. Thus, selenite altered the intracellular redox status toward an oxidative state by decreasing the ratio of GSH:GSSG. Because increased levels of Bcl-2 and GSH are associated with radioresistance, we examined the ability of selenite to sensitize prostate cancer cells to gamma-irradiation. Both LAPC-4 and androgen-independent DU 145 cells pretreated with selenite showed increased sensitivity to gamma-irradiation as measured by clonogenic survival assays. Importantly, selenite-induced radiosensitization was observed in combination with a clinically relevant dose of 2 Gy. These data suggest that altering the redox environment of prostate cancer cells with selenite increases the apoptotic potential and sensitizes them to radiation-induced cell killing.     

AFM-detected apoptotic changes in morphology and biophysical property caused by paclitaxel in Ishikawa and HeLa cells

The apoptosis of cancer cells is associated with changes in the important cell properties including morphology, surface roughness and stiffness. Therefore, the changes in morphology and biophysical properties can be a good way of evaluating the anticancer activity of a drug. This study examined the effect of paclitaxel on the properties of Ishikawa and HeLa cells using atomic force microscopy (AFM), and the relationship between the changes in morphology and the biophysical properties and apoptosis was discussed. The viability and proliferation of the cells were analyzed using the methylthiazol tetrazolium (MTT) method and a TUNEL assay to confirm cellular apoptosis due to a paclitaxel treatment. AFM observations clearly showed the apoptotic morphological and biophysical changes in Ishikawa and HeLa cells. After the paclitaxel treatment, the cell membrane was torn and holed, the surface roughness was increased, and the stiffness was decreased. These changes were observed more apparently after a 24 h treatment and in Ishikawa cells compared to HeLa cells. The MTT and TUNEL assays results revealed the Ishikawa cells to be more sensitive to paclitaxel than HeLa cells and definite apoptosis occurred after a 24 h treatment. These results showed good agreement with the AFM results. Therefore, research on the morphological and biophysical changes by AFM in cancer cells will help to evaluate the anticancer activities of the drugs.     

Parasporin-2 from a New Bacillus thuringiensis 4R2 Strain Induces Caspases Activation and Apoptosis in Human Cancer Cells

In previous studies, parasporin-2Aa1, originally isolated from Bacillus thuringiensis strain A1547, was shown to be cytotoxic against specific human cancer cells but the mechanisms of action were not studied. In the present study, we found that proteinase K activated parasporin-2Aa1 protein isolated from a novel B. thuringiensis strain, 4R2, was specifically cytotoxic to endometrial, colon, liver, cervix, breast and prostate cancer. It showed no toxicity against normal cells. Upon treatment with proteinase K-activated parasporin-2Aa1, morphological changes were observed and western blot analysis revealed the cleavage of poly (ADP-Ribose) polymerase, caspase-3 and caspase-9 in cancer cell lines exclusively, indicative of programmed cell death, apoptosis. Flow cytometry analyses,using propidium iodide and annexin V, as well as a caspases 3/7 assay confirmed apoptosis induction. Further analyses were performed to study survival pathways, including AKT, XIAP, ERK1/2 and PAR-4, a known inducer of apoptosis. These results indicate that parasporin-2Aa1 is a selective cytotoxic protein that induces apoptosis in various human cancer cell lines from diverse tissues.     

Sesquiterpenoids from the Resina Commiphora Promoting the Apoptotic Activity of PC-3 Prostate Cancer Cells

Four new germacrane-type sesquiterpenes commiphoranes M1-M4 ( 1 - 4 ) together with eighteen sesquiterpenes were isolated from the Resina Commiphora. The structures and relative configurations of new substances were determined by using spectroscopic methods. Biological activity investigation revealed that nine compounds including 7 , 9 , 14 , 16 , (+)- 17 , (−)- 17 , 18 , 19 , and 20 could induce the apoptosis of prostate cancer originated PC-3 cells, through classic apoptosis signaling pathway, even using flow cytometry showed that the compound (+)- 17 caused apoptosis of PC-3 cells more than 40 %, suggesting their potential therapeutic application in the development of novel drugs against prostate cancer.     

The rhizome of Trillium tschonoskii Maxim. extract induces apoptosis in human lung cancer cells

Trillium tschonoskii Maxim. has been used to treat several diseases including cancers in folk medicine. However, the mechanisms responsible for T. tschonoskii extract-induced apoptosis are not clear. This study was mainly undertaken to identify the major biochemical changes in a lung cancer cell line upon treatment with an T. tschonoskii extract (TTME), and to investigate the functional relationship between these changes. The n-butanol extract was used to evaluate the mechanism of induction of apoptosis in A549 human lung cancer cells and its effects on mitochondrial function and production of reactive oxygen species (ROS). The n-butanol extract of T. tschonoskii has cytotoxic, antiproliferative, and morphological effects on the lung cancer cell line. T. tschonoskii mainly leads to apoptosis of cancer cells with a concomitant increase in the release of cytochrome c and a loss of mitochondrial membrane potential in a dose-dependent manner. A rapid increase in the level of intracellular ROS and an accumulation of cells in the G2/M and S phase of the cell cycle were also observed in treated cells. These observations suggest that the n-butanol extract of T. tschonoskii has promising anticancer activities, which could be useful in cancer treatment.     

R115777 (Zarnestra)/Zoledronic acid (Zometa) cooperation on inhibition of prostate cancer proliferation is paralleled by Erk/Akt inactivation and reduced Bcl-2 and bad phosphorylation

Zoledronic acid (ZOL) has proved activity in bone metastases from prostate cancer through inhibition of mevalonate pathway and of prenylation of intracellular proteins. We have reported that ZOL synergizes with R115777 farnesyltransferase inhibitor (FTI, Zarnestra) in inducing apoptosis and growth inhibition on epidermoid cancer cells. Here, we have studied the effects of the combination of these agents in prostate adenocarcinoma models and, specifically, on androgen-independent (PC3 and DU145) and -dependent (LNCaP) prostate cancer cell lines. We have found that ZOL and R115777 were synergistic in inducing both growth inhibition and apoptosis in prostate adenocarcinoma cells. These effects were paralleled by disruption of Ras-->Erk and Akt survival pathways, consequent decreased phosphorylation of both mitochondrial bcl-2 and bad proteins, and caspase activation. Finally, ZOL/R115777 combination induced cooperative effects also in vivo on tumor growth inhibition of prostate cancer xenografts in nude mice with a significant survival increase. These effects were paralleled by enhanced apoptosis and inactivation of both Erk and Akt. In conclusions, the combination between ZOL and FTI leads to enhanced anti-tumor activity in human prostate adenocarcinoma cells likely through a more efficacious inhibition of ras-dependent survival pathways and consequent bcl-related proteins-dependent apoptosis.     

Juglone,isolated from Juglans mandshurica Maxim,induces apoptosis via down-regulation of AR expression in human prostate cancer LNCaP cells

Juglone is a natural compound which has been isolated from Juglans mandshurica Maxim. Recent studies have shown that juglone had various pharmacological effects such as anti-viral, anti-bacterial and anti-cancer. However, its anti-cancer activity on human prostate cancer LNCaP cell has not been examined. Thus, the current study was designed to elucidate the molecular mechanism of apoptosis induced by juglone in androgen-sensitive prostate cancer LNCaP cells. MTT assay was performed to examine the anti-proliferative effect of juglone. Occurrence of apoptosis was detected by Hoechst 33342 staining and flow cytometry in LNCaP cells treated with juglone for 24 h. The result shown that juglone inhibited the growth of LNCaP cells in a dose-dependent manner. Morphological changes of apoptotic body formation after juglone treatment were observed by Hoechst 33342 staining. This apoptotic induction was associated with loss of mitochondrial membrane potential, and caspase-3, -9 activation. Moreover, we found that juglone significantly inhibited the expression levels of androgen receptor (AR) and prostate-specific antigen (PSA) in a dose-dependent manner, as well as abrogated up-regulation of AR and PSA genes with and/or without dihydrotestosterone (DHT). Take together, our results demonstrated that juglone might induce the apoptosis in LNCaP cell via down-regulation of AR expression. Therefore, our results indicated that juglone may be a potential candidate of drug for androgen-sensitive prostate cancer.     

Regulation of circGOLPH3 and its binding protein CBX7 on the proliferation and apoptosis of prostate cancer cells

To clarify the mechanism of circGOLPH3 regulation on prostate cancer cells, we performed an overexpression and interference circGOLPH3 assay in prostate cancer cells PC-3 and then evaluated cellular viability, proliferation, cell cycle, and apoptosis of prostate cancer cells by MTT, CCK8, Edu stain, TUNEL stain, and flow cytometry. Binding proteins of CircGOLPH3 were identified by RNA pull-down, mass spectrometry, and RNA-binding protein immunoprecipitation (RIP) assays. The expressions of CircGOLPH3 and CBX7 were measured by qRT-PCR. The results showed that after overexpression of circGOLPH3, the proliferative capacity and the viability of PC-3cells were significantly improved, whereas apoptosis was inhibited. CircGOLPH3 could bind to the CBX7 protein that was highly expressed in the PC-3 cell. Additionally, a functional test on CBX7 showed that the CBX7 overexpression notably improved the proliferative capacity and the viability of PC-3 cells and decreased cellular apoptosis, which was consistent with the effects of circGOLPH3. The validated the present study that circGOLPH3 and its binding protein CBX7 can promote prostate cancer cell proliferation and inhibit apoptosis.     

Oxidative stress induction by (+)-cordiaquinone J triggers both mitochondria-dependent apoptosis and necrosis in leukemia cells

(+)-Cordiaquinone J is a 1,4-naphthoquinone isolated from the roots of Cordia leucocephala that has antifungal and larvicidal effects. However, the cytotoxic effects of (+)-cordiaquinone J have never being explored. In the present study, the effect of (+)-cordiaquinone J on tumor cells viability was investigated, showing IC₅₀ values in the range of 2.7–6.6 μM in HL-60 and SF-295 cells, respectively. Studies performed in HL-60 leukemia cells indicated that (+)-cordiaquinone J (1.5 and 3.0 μM) reduces cell viability and 5-bromo-2-deoxyuridine incorporation after 24 h of incubation. (+)-Cordiaquinone J showed rapid induction of apoptosis, as indicated by phosphatidylserine externalization, caspase activation, DNA fragmentation, morphologic changes, and rapid induction of necrosis, as indicated by the loss of membrane integrity and morphologic changes. (+)-Cordiaquinone J altered the redox potential of cells by inducing the depletion of reduced GSH intracellular content, the generation of reactive oxygen species and the loss of mitochondrial membrane potential. However, pre-treatment of cells with N-acetyl-l-cysteine abolished most of the observed effects related to (+)-cordiaquinone J treatment, including those involving apoptosis and necrosis induction.     

Targeting apoptosis by hydroxymethylacylfulvene in combination with gamma radiation in prostate tumor cells

Hydroxymethylacylfulvene (HMAF) is a novel agent with alkylating activity and is a potent inducer of apoptosis that is currently undergoing Phase II clinical trials for prostate cancer. This study explored the pro-apoptosis and anti-proliferative potential of HMAF in combination with gamma radiation in human prostate tumor cell lines. Apoptosis was assessed based on the generation of fragmented DNA, a terminal transferase flow cytometry assay, and cell morphology. In each of the tumor cell lines examined, radiation alone induced a marginal level of apoptosis, even after a prolonged 48-h incubation after exposure. In contrast, HMAF alone was a potent inducer of apoptosis in prostate tumor cells but not in normal cells. Marked levels of apoptosis in tumor cells were also observed for the combination of HMAF with gamma radiation. When drug treatment preceded irradiation, at least additive levels of apoptosis were observed in both androgen-responsive and androgen-independent cells. The combined treatment with ionizing radiation and HMAF reduced the radiation dose needed for the same level of clonogenic survival up to 2.5-fold. The potentiation of apoptosis and reduction in the clonogenic survival of tumor cells occurred at HMAF concentrations lower than that which reduced survival to 10% and at doses up to 6 Gy. No potentiation of apoptosis or clonogenic inhibition was noted in normal cells. These results suggest that the combination of HMAF with gamma radiation may have clinical utility for treatments of prostate cancer.     

Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen species and mitochondrial membrane depolarization

The anticancer activity of salinomycin has evoked excitement due to its recent identification as a selective inhibitor of breast cancer stem cells (CSCs) and its ability to reduce tumor growth and metastasis in vivo. In prostate cancer, similar to other cancer types, CSCs and/or progenitor cancer cells are believed to drive tumor recurrence and tumor growth. Thus salinomycin can potentially interfere with the end-stage progression of hormone-indifferent and chemotherapy-resistant prostate cancer. Androgen-responsive (LNCaP) and androgen-refractive (PC-3, DU-145) human prostate cancer cells showed dose- and time-dependent reduced viability upon salinomycin treatment; non-malignant RWPE-1 prostate cells were relatively less sensitive to drug-induced lethality. Salinomycin triggered apoptosis of PC-3 cells by elevating the intracellular ROS level, which was accompanied by decreased mitochondrial membrane potential, translocation of Bax protein to mitochondria, cytochrome c release to the cytoplasm, activation of the caspase-3 and cleavage of PARP-1, a caspase-3 substrate. Expression of the survival protein Bcl-2 declined. Pretreatment of PC-3 cells with the antioxidant N-acetylcysteine prevented escalation of oxidative stress, dissipation of the membrane polarity of mitochondria and changes in downstream molecular events. These results are the first to link elevated oxidative stress and mitochondrial membrane depolarization to salinomycin-mediated apoptosis of prostate cancer cells.