Including Cell Viability Assays Research Articles

Sodium orthovanadate inhibits growth and triggers apoptosis of human anaplastic thyroid carcinoma cells in vitro and in vivo.

Vanadium and its compounds exhibit concentration- and time-dependent anticancer effects on various types of tumor; however, the effects of sodium orthovanadate (SOV) on anaplastic thyroid carcinoma (ATC) have not yet been reported. In the present study, the anticancer effects of SOV on ATC were evaluated. In vitro experiments, including cell viability assays, plate colony formation assays, cell cycle analysis and apoptosis analysis were used to study the role of SOV in ATC. Using in vivo experiments, the effects of SOV on the growth and apoptosis of an ATC-xenograft tumor were studied by comparing the SOV-treatment with the control group. The results revealed that treatment of the human ATC cell line 8505C with SOV inhibited cell viability, induced G2/M phase cell cycle arrest, stimulated apoptosis and reduced mitochondrial membrane potential in a concentration-dependent manner. These findings were confirmed in vivo in a nude mouse ATC xenograft model. In conclusion, the present study demonstrated that SOV inhibited human ATC by regulating proliferation, cell cycle progression and apoptosis, thus suggesting that SOV may be considered a novel option for the treatment of ATC.

Nanomedicine-Assisted Combination Therapy of NSCLC: New Platinum-Based Anticancer Drug Synergizes the Therapeutic Efficacy of Ganetespib.

Purpose: K-RAS is the most common mutated oncogene associated with Non-Small-Cell Lung Cancer (NSCLC). So far, there are no promising chemotherapies for the direct inhibition of K-RAS, and considered to be undruggable. In this work, we have introduced a new platinum-based cyanoximate complex, Pt(MCO)2, as an anti-cancer drug to enhance the therapeutic efficacy of Hsp90 inhibitor drug, ganetespib for the combination therapy of NSCLC.Methods: We have synthesized polyacrylic acid (PAA)-coated magnetic nanoparticles (MNPs) and used as drug delivery system. These MNPs were decorated with folic acid in order to target folate receptor-expressing NSCLC. The individual and combination of drugs as well as an optical dye DiI were co-encapsulated successfully inside the PAA-coating of MNPs to evaluate synergistic treatment option for NSCLC. The magnetic resonance (MR) and optical imaging modalities assisted for the monitoring drug loading and NSCLC treatment.Results: To evaluate the therapeutic efficacy of these customized MNPs, various cell-based assays including cell viability, apoptosis and necrosis, cell migration, comet and ROS experiments were performed. Results showed minimal toxicity for functional MNPs with no therapeutic drug and more than 60% cell death within 48 h of treatment, when single drug was encapsulated. Importantly, more than 90% cells were dead when both drugs were delivered. Overall, the results indicated that the Pt(MCO)2 drug enhances the therapeutic efficacy of ganetespib by more than 30% toxicity towards the targeted treatment of NSCLC, while showed minimal toxicity to the normal healthy tissues.Conclusion: We successfully developed new dual-modal magnetic nanomedicines for the rapid and controlled release of combination of drugs for the effective treatment of NSCLC. The MR and fluorescence modalities help monitoring the delivery of drugs, where the new platinum-based drug Pt(MCO)2 synergizes the therapeutic efficacy of ganetespib.

Fabrication and cytotoxicity assessment of cellulose nanofibrils using Bassia eriophora biomass

Cellulose nanofibrils (CNs) are eco-friendly, biodegradable, biocompatible, renewable, cost-effective, and possess excellent mechanical properties. We fabricated CNs from Bassia eriophora biomass, and the structure and morphology were investigated by transmission electron microscopy that revealed 2–6 μm long fibrillated structures with diameters of 15–40 nm. CNs biocompatibility was assessed using in vitro based assays, including cell viability assay, AO/EB staining, Hoechst staining, JC-1 staining, and gene expression analysis. The assessment of cellular and nuclear morphologies of human mesenchymal stem cells (hMSCs) showed that CNs do not affect cell viability and morphology. JC-1 staining results revealed that CNs do not cause mitochondrial membrane potential of hMSCs. Cell-based in vitro assays revealed that CNs are biocompatible even at high concentrations. The CNs effect on cell cycle regulated gene expression was studied that results suggested that CCND1 and CCND3 gene expression levels increased slightly, when compared with control. But CCNG1, CYCS3, and CCNC1 genes has no significant difference was observed. Overall, our results suggested that CNs can be used for tissue engineering and regenerative medicine.

Tauroursodeoxycholic acid attenuates gentamicin-induced cochlear hair cell death in vitro

Gentamycin is one of the most clinically used aminoglycoside antibiotics which induce intrinsic apoptosis of hair cells. Tauroursodeoxycholic acid (TUDCA) is known as safe cell-protective agent in disorders associated with apoptosis. We aimed to investigate the protective effects of TUDCA against gentamicin-induced ototoxicity. House Ear Institute-Organ of Corti 1(HEI-OC1) cells and explanted cochlear tissue were treated with gentamicin and TUDCA, followed by serial analyses including cell viability assay, hair cell staining, qPCR, ELISA and western blotting to determine the cell damage by the parameters relevant to cell apoptosis and endoplasmic reticulum stress. TUDCA significantly attenuated gentamicin-induced cell damage in cultured HEI-OC1 cells and explanted cochlear hair cells. TUDCA alleviated gentamicin-induced cell apoptosis, supported by the decreased Bax/Bcl2 ratio compared with that of gentamicin treated alone. TUDCA decreased gentamicin-induced nitric oxide production and protein nitration in both models. In addition, TUDCA suppressed gentamicin-induced endoplasmic reticulum stress as reflected by inversing the expression levels of Binding immunoglobulin protein (Bip), CCAAT/-enhancer-binding protein homologous protein (CHOP) and Caspase 3. TUDCA attenuated gentamicin-induced hair cell death by inhibiting protein nitration activation and ER stress, providing new insights into the new potential therapies for sensorineural deafness.

In situ preparation of water-soluble ginsenoside Rh2-entrapped bovine serum albumin nanoparticles: in vitro cytocompatibility studies.

The present study investigates a simple and convenient one-step procedure for the preparation of bovine serum albumin (BSA)-Rh2 nanoparticles (NPs) at room temperature. In this work, ginsenoside Rh2 was entrapped within the BSA protein to form BSA-Rh2 NPs to enhance the aqueous solubility, stability, and therapeutic efficacy of Rh2. The physiochemical characterization by high-performance liquid chromatography, nuclear magnetic resonance, Fourier transform infrared spectroscopy, field emission transmission electron microscopy, dynamic light scattering, and thermogravimetric analysis confirmed that the prepared BSA-Rh2 NPs were spherical, highly monodispersed, and stable in aqueous systems. In addition, the stability of NPs in terms of different time intervals, pHs, and temperatures (20°C–700°C) was analyzed. The results obtained with different pHs showed that the synthesized BSA-Rh2 NPs were stable in the physiological buffer (pH 7.4) for up to 8 days, but degraded under acidic conditions (pH 5.0) representing the pH inside tumor cells. Furthermore, comparative analysis of the water solubility of BSA-Rh2 NPs and standard Rh2 showed that the BSA nanocarrier enhanced the water solubility of Rh2. Moreover, in vitro cytotoxicity assays including cell viability assays and morphological analyses revealed that Rh2-entrapped BSA NPs, unlike the free Rh2, demonstrated better in vitro cell viability in HaCaT skin cell lines and that BSA enhanced the anticancer effect of Rh2 in A549 lung cell and HT29 colon cancer cell lines. Additionally, anti-inflammatory assay of BSA-Rh2 NPs and standard Rh2 performed using RAW264.7 cells revealed decreased lipopolysaccharide-induced nitric oxide production by BSA-Rh2 NPs. Collectively, the present study suggests that BSA can significantly enhance the therapeutic behavior of Rh2 by improving its solubility and stability in aqueous systems, and hence, BSA-Rh2 NPs may potentially be used as a ginsenoside delivery vehicle in cancer and inflammatory cell lines.

Biphasic Polyurethane/Polylactide Sponges Doped with Nano-Hydroxyapatite (nHAp) Combined with Human Adipose-Derived Mesenchymal Stromal Stem Cells for Regenerative Medicine Applications.

Cartilage and bone tissue injuries are common targets in regenerative medicine. The degeneration of cartilage tissue results in tissue loss with a limited ability to regenerate. However, the application of mesenchymal stem cells in the course of such condition makes it possible to manage this disorder by improving the structure of the remaining tissue and even stimulating its regeneration. Nevertheless, in the case of significant tissue loss, standard local injection of cell suspensions is insufficient, due to the low engraftment of transplanted cells. Introduction of mesenchymal stem cells on the surface of a compatible biomaterial can be a promising tool for inducing the regeneration by both retaining the cells at the desired site and filling the tissue gap. In order to obtain such a cell-biomaterial hybrid, we developed complex, biphasic polymer blend biomaterials composed of various polyurethane (PU)-to-polylactide (PLA) ratios, and doped with different concentrations of nano-hydroxyapatite (nHAp). We have determined the optimal blend composition and nano-hydroxyapatite concentration for adipose mesenchymal stem cells cultured on the biomaterial. We applied biological in vitro techniques, including cell viability assay, determination of oxidative stress factors level, osteogenic and chondrogenic differentiation potentials as well as cell proteomic analysis. We have shown that the optimal composition of biphasic scaffold was 20:80 of PU:PLA with 20% of nHAp for osteogenic differentiation, and 80:20 of PU:PLA with 10% of nHAp for chondrogenic differentiation, which suggest the optimal composition of final biphasic implant for regenerative medicine applications.

Repositioning of anti-viral drugs as therapy for cervical cancer.

Increase in expression of eIF4E (Eukaryotic translation initiation factor 4E) protein is mediated by oncogenic proteins of Human Papilloma Virus (HPV). Increased expression of eIF4E plays an important role in HPV induced carcinogenesis. Ribavirin and Indinavir are known inhibitors of eIF4E activity. The effect of the drugs on HeLa cells was assessed by in vitro assays including cell viability using MTT and Neutral red assay, apoptotic potential using Caspase-3, Caspase-8 and Caspase-9 activity assays and MMP-2 and MMP-9 secretion by determination of Gelatinase activity. The in vivo effect of Ribavirin treatment on tumor volume was assessed in human xenograft in immunocompromised C57BL/6 mice. In vitro analyses indicate that Ribavirin and Indinavir reduce viability of HeLa cells, induce apoptosis and decrease secretion of MMPs. Treatment with Ribavirin at a dose of 50mg/kg and 100mg/kg daily led to significant decrease in tumor volume in vivo. The study thus provides evidence that Ribavirin and Indinavir can be explored as therapy against HPV-18 induced cervical cancer.

Colchicine induces apoptosis in HT‑29 human colon cancer cells via the AKT and c-Jun N-terminal kinase signaling pathways.

Colchicine is a natural compound, which belongs to the botanical family Colchicaceae and prevents growth of cancer cells via antimitotic activity by interacting with microtubules. Although numerous studies have demonstrated that the effect of colchicine on cell apoptosis is mediated by the activation of caspase‑3, the signaling pathways involved in the process remain unknown. In the current study, evidence is presented regarding the missing information using HT‑29 human colon cancer cells. The effect of colchicine on apoptosis in HT‑29 cells and the apoptosis‑associated signaling pathways were determined using various methods, including cell viability assay, Annexin V/propidium idodide (PI) binding, PI staining, Hoechst 33342 staining, mitochondrial membrane potential (Δψm) assay, reactive oxygen species (ROS) assay and western blot analysis. Colchicine was observed to induce a dose‑dependent reduction in cell viability in HT‑29 cells and early apoptosis occurred when the cells were treated with 1 µg/ml colchicine. Furthermore, colchicine treatment induced a loss of Δψm, increased ROS production, activated caspase‑3, upregulated BAX expression and downregulated Bcl‑2 expression, which evidenced the colchicine activity on apoptosis, potentially by acting via the intrinsic apoptotic signaling pathway. Colchicine increased phosphorylation of p38, although not phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase, which indicates that colchicine activates the p38 signaling pathway in order to induce cell apoptosis. Therefore, colchicine exhibited significant growth inhibition of the HT‑29 colon cancer cell line and induced apoptosis in the cells via the mitochondrial pathway, which is regulated by p38 signaling pathways.

Protective Effects of Berry Extracts on Hydrogen Peroxide-induced Rat Brain Neuronal Cell Damage in vitro

The purpose of this study was to evaluate the antioxidative activities and protective effects of boysenberry, black currant, and blueberry extracts on neuronal cells. We found that berry extracts had different levels of antioxidant efficacy in various assays, including cell viability assays using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT). In addition, reactive oxygen species (ROS) were measured using 2',7'–dichlorofluorescein diacetate (DCF-DA) and flow cytometry analysis. Fruit extracts possessed strong antioxidant activities as measured using the malondialdehyde (MDA) assays. These extracts effectively inhibited lipid peroxidation. Black currant fruit extract significantly reduced intracellular ROS accumulation resulting from hydrogen peroxide treatment in PC12 cells. Black currant fruit more effectively inhibited lactate dehydrogenase (LDH) release into the medium than other tested fruit extracts did. The present results indicate that fruit extracts have the potential to protect neurons from hydrogen peroxide-induced cell damage and should be considered as a prospective functional food and therapeutic product.

Cellular nucleic acid binding protein suppresses tumor cell metastasis and induces tumor cell death by downregulating heterogeneous ribonucleoprotein K in fibrosarcoma cells

BackgroundCellular nucleic acid binding protein (CNBP) has been implicated in vertebrate craniofacial development and in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human diseases by controlling cell proliferation and survival to mediate neural crest expansion. CNBP has been found to bind single-stranded nucleic acid and promote rearrangements of nucleic acid secondary structure in an ATP-independent manner, acting as a nucleic acid chaperone. MethodsA variety of methods were used, including cell viability assays, wound-scratch assays, chemotaxis assays, invasion assays, circular dichroic (CD) spectroscopy, NMR spectroscopy, chromatin immunoprecipitation, expression and purification of recombinant human CNBP, electrophoretic mobility shift assay (EMSA), surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET) analyses, luciferase reporter assay, Western blotting, and isothermal titration calorimetry (ITC). ResultsUp-regulation of CNBP induced human fibrosarcoma cell death and suppressed fibrosarcoma cell motility and invasiveness. It was found that CNBP transcriptionally down-regulated the expression of heterogeneous ribonucleoprotein K (hnRNP K) through its conversion of a G-rich sequence into G-quadruplex in the promoter of hnRNP K. G-quadruplex stabilizing ligand tetra-(N-methyl-4-pyridyl) porphyrin (TMPyP4) could interact with and stabilize the G-quadruplex, resulting in downregulation of hnRNP K transcription. ConclusionsCNBP overexpression caused increase of cell death and suppression of cell metastasis through its induction of G-quadruplex formation in the promoter of hnRNP K resulting in hnRNP K down-regulation. General significanceThe present result provided a new solution for controlling hnRNP K expression, which should shed light on new anticancer drug design and development.

Luminol As In Situ Light Source in Meso-Tetraphenylporphyrin-Mediated Photodynamic Therapy

The light sources used in current photodynamic therapy are mainly lasers or light emitting diodes, which are not suitable to treat large-volume tumors and those located in the inner body. To overcome the limitation, we propose an in situ light source to activate the photosensitizer and kill the cancer cells directly. In the present work, we use luminol as light source and meso-tetraphenylporphyrin as the photosensitizer. According to the results, cells incubated with meso-tetraphenylporphyrin, subsequently triggered by luminol, decreased significantly in assays including cell viability and cytotoxicity, while the other groups showed only minor differences. The flow cytometric and fluorescent microscopy analysis showed similar results as well. In the analysis of cell death pathway, cell shrinkage was noticed after photodynamic therapy treatment, which might refer to apoptosis. Briefly, we suggest that luminol is a promising light source in meso-tetraphenylporphyrin-mediated photodynamic therapy for its greater penetration depth and well matched emission wavelength.

In vitro biocompatibility of solid lipid nanoparticles

This study was undertaken to address the current deficient knowledge of cellular response to solid lipid nanoparticles (SLNs) exposure. We investigated the cytotoxicity of several SLNs formulations in two fibroblast cell lineages, Vero and MDCK. Several methods were used to explore the mechanisms involved in this cytotoxic process, including cell viability assays, flow cytometry and ROS generation assessment. Among nanoparticles tested, two of them (F4 and F5) demonstrated more cytotoxic effects in both cell lineages. The cell viability assays suggested that F4 and F5 interfere in cell mitochondrial metabolism and in lysosomal activity. In addition, F5 decreased the percentage of MDCK cells in G0/G1 and G2/M phases, with a marked increase in the Sub/G1 population, suggesting DNA fragmentation. Regarding F4, although IC50 was higher (~700μg/mL), this formulation affected mitochondrial membrane potential for Vero cells. However, the IC50 of F5 was around 250μg/mL, suggesting the effect of SDS (sodium dodecyl sulfate) present in the formulation. In summary, the nanoparticles tested here appears to be biocompatible, with the exception of F5. Further studies are required to elucidate the in vivo effects of these nanoscale structures, in order to evaluate or predict the connotation of their increased and widespread use.

Luminol as the light source for in situ photodynamic therapy

Presently, the light sources used in photodynamic therapy are high intensity lasers or light emitting diodes, making it unsuitable for large-volume tumors and those located deep inside the body. To overcome this limitation, we propose an in situ light source to excite the photosensitizer to generate toxic singlet oxygen and kill tumor cells directly. In this research, luminol served as the in situ light source in 5-aminolevulinic acid-mediated photodynamic treatment of Caco-2 cell cultures. 72h after luminol excitation the viability of the treated cells significantly decreased compared to the control cells in assays including cell viability, cytotoxicity, flow cytometry and fluorescence confocal microscopy. According to the results, we suggested luminol could be used as an in situ light source for 5-aminolevulinic acid-mediated photodynamic therapy. This method would have great potential to extend the application of photodynamic therapy to tumors located deep inside the body.

Colchicine-induced apoptosis in human normal liver L-02 cells by mitochondrial mediated pathways

Colchicine is an alkaloid that has been widely used to treat gout. It also has a curative effect on cancer. Although many studies have shown that its effect on cell apoptosis was mediated by the activation of caspase-3, the pathways involved in the process remained obscure. Here we show some evidence regarding the missing information using human normal liver cells L-02 in our study. The effect of colchicine on apoptosis in L-02 cells and the apoptosis-associated signaling pathways were determined using different tests including cell viability assay, Annexin V and propidium idodide binding, PI staining, Hoechst 33342 staining, mitochondrial membrane potential assay, caspase activity assay and Western blot analysis. We found that colchicine-induced a dose-dependent drop of cell viability in L-02 cells; early apoptosis happened when cells were treated with 0.1μM of colchicine. The colchicine-induced loss of mitochondrial membrane potential, activation of caspase-3 and 9, up-regulation of Bax and down-regulation of Bcl-2 showed an evidence for the colchicine activity on apoptosis, at least, by acting via the intrinsic apoptotic pathway.

Neuroprotective Effect of Rhizoma Atractylodis Macrocephalae Against Excitotoxicity‐induced Apoptosis in Cultured Cerebral Cortical Neurons

Excitotoxicity has been implicated in neurological disorders. This study investigated the neuroprotective effect of the extract from Rhizoma Atractylodis macrocephalae on excitotoxicity-induced neuronal apoptosis in primary cultured cerebral cortical neurons. Excitotoxicity was induced by exposure of cortical neurons to glutamate. Neuronal apoptosis and the protective effect of Rhizoma Atractylodis macrocephalae extract were examined by multi-indices including cell viability assay, morphological features, DNA fragmentation and flow cytometric analysis. After exposure of cultured neurons to glutamate for 24 h, the neurons exhibited marked apoptotic-like death. Co-treatment of the neurons with glutamate and Rhizoma Atractylodis macrocephalae extract significantly elevated the cell viability, and reduced the number of apoptotic cells. These results demonstrate that Rhizoma Atractylodis macrocephalae is an effective neuroprotective agent against glutamate-induced excitotoxicity and may have therapeutic potential in excitotoxicity-mediated diseases.

Lignans and flavonoids as apoptosis inducers

Apoptosis is an important mode of „programmed“ cell death, which involves the genetically determined elimination of cells. As a vital component of normal cell turnover, inappropriate apoptosis is a factor in many human diseases including cancer, where unwanted cells are unable to be eliminated by the body. Many anticancer drugs today act by way of apoptosis induction [1]. In our ongoing search for bioactive molecules from medicinal plants [2], fifteen phenolic compounds, including lignans and flavonoids were assayed for their apoptosis induction activity on (HuH-7) human liver cancer cells. The lignans, including six dibenzylbutanes and one butirolactone were isolated previously from the dichloromethane extract of Pycnanthus angolensis or obtained by derivatization [3,4]. Further studies on the ethyl acetate extract of this species have led to the isolation of seven isoflavones and one flavanone. Their structures were identified based on spectroscopic methods including 2D NMR experiments. Methodology for apoptosis detection included cell viability assays, nuclear morphology evaluation, and general caspase-3-like activity assessments. The compounds tested at a concentration of 20µM showed varying degrees of apoptosis induction activity after 24h exposure. Caspase activity assays confirmed these results.

Apoptosis and cell cycle arrest of human colorectal cancer cell line HT-29 induced by vanillin

Background: Vanillin is responsible for the flavor and smell of vanilla, a widely used flavoring agent. Previous studies showed that vanillin could enhance the repair of mutations and thus function as an anti-mutagen. However, its role in cancer, a disease that is closely related to mutation has not yet been fully elucidated. Methods: Hence, this study investigated the cytolytic and cytostatic properties of vanillin against HT-29, a human colorectal cancer cell line. Methods used including cell viability assay, acridine orange (AO)–ethidium bromide (EB) double staining cell morphological analysis, Cell cycle analysis, annexin V–propidium iodide apoptosis test and 5-bromo-2-deoxyuridine (BrdU)-labeling cell proliferation assay. Results: Results showed that apoptosis was induced by vanillin and the IC 50 for HT-29 and NIH/3T3 normal cell lines were 400 μg/ml and 1000 μg/ml, respectively. Different concentrations of vanillin arrest cell cycle at different checkpoints. 5-Bromo-2-deoxyuridine-labeling cell proliferation assay showed that G0/G1 arrest was achieved at lower concentration of vanillin (200 μg/ml) while cell cycle analysis by flow cytometer showed that G2/M arrest occurs at higher concentration of vanillin (1000 μg/ml). Conclusion: Cytolytic and cytostatic effects shown by vanillin showed that it could be a useful colorectal cancer preventive agent. Further in vivo study should be carried out to confirm that similar effects could happen in animals.

Induction of apoptosis in HuH-7 cancer cells by monoterpene and β-carboline indole alkaloids isolated from the leaves of Tabernaemontana elegans

Three known (1–3) and a novel (4) monoterpene indole alkaloids have been isolated from the methanol extract of leaves of Tabernaemontana elegans and their structures were elucidated by a series of spectroscopic experiments, involving NMR, MS, UV, and IR techniques. The isolated monoterpene indole alkaloids along with previously described β-carbolines (5–7) from the same specimen were studied for their apoptosis induction activity in human hepatoma HuH-7 cells. Methodology for apoptosis induction studies included cell viability assays, nuclear morphology assessments, and general caspase-3-like activity assays. The monoterpene indole alkaloids, tabernaemontanine (1) and vobasine (3) showed the most promising apoptosis induction profile in HuH-7 cells.

Microfluidic device for single-cell analysis.

We have developed a novel microfluidic device constructed from poly(dimethylsiloxane) using multilayer soft lithography technology for the analysis of single cells. The microfluidic network enables the passive and gentle separation of a single cell from the bulk cell suspension, and integrated valves and pumps enable the precise delivery of nanoliter volumes of reagents to that cell. Various applications are demonstrated, including cell viability assays, ionophore-mediated intracellular Ca2+ flux measurements, and multistep receptor-mediated Ca2+ measurements. These assays, and others, are achieved with significant improvements in reagent consumption, analysis time, and temporal resolution over macroscale alternatives.

Ceramide-induced cell death in the prostate cancer cell line LNCaP has both necrotic and apoptotic features.

Prostate cancer is the second leading cause of cancer death in men. The most common treatment of prostate cancer is androgen ablation therapy which leads to regression of the tumor due to increased cell death. However, at later stages, the tumor becomes resistant to androgen ablation. Ceramide is a lipid second messenger that mediates cell death in prostate cancer cells. Previous studies suggested that ceramide may cause either apoptosis or growth arrest in the androgen-responsive prostate cancer cell line LNCaP. However, the molecular details of ceramide-induced cell death in LNCaP cells remain to be elucidated. To investigate the mechanisms of cell death in LNCaP cells, we used various methods, including cell viability assays, fluorescence image analysis, internucleosomal DNA fragmentation analysis, Western blotting, and protein kinase assays. Ceramide caused LNCaP cell death without exhibiting typical signs of apoptosis, such as internucleosomal DNA fragmentation and poly(ADP)-ribose-polymerase (PARP) proteolysis. In addition, the general caspase inhibitor z-VAD-fmk did not alter ceramide-induced cell death in LNCaP cells, whereas it efficiently inhibited thapsigargin-induced apoptosis under similar conditions. However, ceramide treatment of LNCaP cells resulted in nuclear fragmentation, which is characteristic of apoptosis. Ceramide induced a strong and prolonged activation of c-Jun N-terminal Kinase (JNK) that correlated very well with the time course of cell death. Whereas the PKC inhibitor bisindolylmaleimide prevented phorbol ester-induced apoptosis in LNCaP cells, it did not affect ceramide-induced cell death. These results suggest that LNCaP cell death induced by ceramide progresses through a novel pathway that is more necrotic than apoptotic.