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Epithelial ovarian cancer remains the leading cause of mortality among all gynecologic malignancies owing to recurrence and ultimate development of chemotherapy resistance in the majority of patients. In the chemotherapy-resistant ovarian cancer preclinical model, we investigated whether AZD6738 (an ataxia telangiectasia and Rad3-related (ATR) inhibitor) could synergize with belotecan (a camptothecin analog and topoisomerase I inhibitor). In vitro, both chemotherapy-resistant and chemotherapy-sensitive ovarian cancer cell lines showed synergistic anti-proliferative activity with a combination treatment of belotecan and AZD6738. The combination also demonstrated synergistic tumor inhibition in mice with a chemotherapy-resistant cell line xenograft. Mechanistically, belotecan, a DNA-damaging agent, increased phospho-ATR (pATR) and phospho-Chk1 (pChk1) in consecutive order, indicating the activation of the DNA repair system. This consequently induced G2/M arrest in the cell cycle analysis. However, when AZD6738 was added to belotecan, pATR and pChk1 induced by belotecan alone were suppressed again. A cell cycle analysis in betotecan showed a sub-G1 increase as well as a G2/M decrease, representing the release of G2/M arrest and the induction of apoptosis. In ascites-derived primary cancer cells from both chemotherapy-sensitive and -resistant ovarian cancer patients, this combination was also synergistic, providing further support for our hypothesis. The combined administration of ATR inhibitor and belotecan proved to be synergistic in our preclinical model. This combination warrants further investigation in a clinical trial, with a particular aim of overcoming chemotherapy resistance in ovarian cancer.

Background/aim: Oxysterol plays important physiological roles in diverse biological processes including apoptosis. However, the mechanisms underlying oxysterol-induced apoptosis remain unknown. 25-hydroxycholesterol (25-HC) is an oxysterol synthesized by cholesterol 25-hydroxylase from cholesterol during sterol metabolism. The aim of present study was to investigate 25-HC-induced apoptosis and associated signalling pathways in FaDu cells, which is originated form human head and neck squamous cell carcinoma cells., Materials and Methods: 25-HC-induced apoptosis was investigated by cell cytotoxicity assay using MTT, cell viability assay using cell LIVE/DEAD cell viability assay, haematoxylin & eosin staining, nuclear staining, fluorescence-activated cell sorting, western blotting using specific antibodies associated with extrinsic and intrinsic apoptosis pathways, and caspase-3/-7 activity assay in FaDu cells., Results: 25-HC dose-dependently decreased the viability of FaDu cells and up-regulated apoptotic events, such as alteration in morphology, and nuclear condensation. Flow cytometric analysis showed an increase in apoptotic population upon 25-HC treatment, suggesting that 25-HC induces apoptosis in FaDu cells. Moreover, 25-HC-induced apoptosis in FaDu cells was dependent on the activation of caspases by Fas antigen ligand-triggered death receptor-mediated extrinsic pathway and mitochondria-dependent intrinsic pathway via mitogen activated protein kinases., Conclusion: Cholesterol-derived oxysterol, 25-HC has potential anti-cancer function in FaDu cells and may have potential properties for the discovery of anti-cancer agents., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The adaptor-related protein complex 5 subunit mu 1 (AP5M1) is an evolutionally conserved protein with ubiquitous expression in human tissues. However, the major function of AP5M1 in living organisms is unclear owing to few published studies. Here, we demonstrate that AP5M1 is a potent apoptosis-inducing molecule in cervical cancer cells. We also found that AP5M1 upregulated the level of BAX protein, a key pro-apoptotic B cell lymphoma (BCL)-2 family member regulating mitochondrial apoptotic cell death pathway. Moreover, AP5M1 completely lost its apoptotic activity in BAX-knockout or -knockdown cells, indicative of its functional dependence on BAX. Comparative analysis of cervical tissues from patients with cervical carcinoma and non-cancer control revealed a prominent downregulation in AP5M1 expression with a concomitant downregulation in BAX expression; AP5M1 and BAX mRNA expression levels in cervical tissues exhibited a strong positive correlation (r = 0.97). Thus, we identified AP5M1 as a previously unrecognized apoptotic protein that governs BAX expression and revealed the association between AP5M1 and malignancy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The 2-(4-morpholinoanilino)-6-cyclohexylaminopurine (reversine) acts as a chemopreventive agent and induces apoptotic cell death in various cancer cells. However, the anticancer effects of reversine on osteosarcoma cells are not clearly established., Objective: The purpose of this study was to investigate the effect of reversine on cell proliferation and induction of apoptosis in human osteosarcoma cells., Methods: Cell viability assay, histological analysis, DAPI staining, caspase activation analysis, flow cytometric analysis and immunoblotting were carried out in MG-63 osteosarcoma cells., Results: Reversine inhibited the growth of cells in a dose-dependent manner and induced nuclear condensation and fragmentation. Reversine-treated cells showed caspase-3/7 activation and increased apoptosis versus control cells. FasL, a death ligand associated with extrinsic apoptotic signaling pathways, was significantly up-regulated by reversine treatment. Moreover, the caspase-8, a part of the extrinsic apoptotic pathway, was activated by reversine treatments. Expressions of anti-apoptotic factors such as Bcl-2 and Bcl-xL, components of the mitochondria dependent intrinsic apoptosis pathway, significantly decreased following reversine treatment. The expressions of pro-apoptotic factors such as BAX, BAD and caspase-9 increased by reversine treatments. In addition, reversine activated caspase-3 and Poly (ADP-ribose) polymerase (PARP) to induce cell death. The Z-VAD-fmk significantly inhibited cell death through the suppression of caspase-3 expression in MG-63 cells treated with reversine., Conclusion: These results suggest that the reversine may inhibit cell proliferation and induce apoptotic cell death in MG-63 osteosarcoma cells through both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway, and may have potential properties for the discovery of anti-cancer agents.

The resistance of tumor cells to apoptosis-inducing anticancer agents is regarded as a major impediment for the treatment of cancer patients. This study aimed to examine the possibility whether a necrosis-inducing peptide containing the mitochondria-targeting domain (MTD) of NOXA kills tumor cells that are resistant to apoptosis-inducing anticancer agents. To examine this possibility, we established doxorubicin-resistant (Dox-Res) cells by treating CT26 cells with increasing amounts of doxorubicin. The apoptosis resistance of the Dox-Res CT26 cells was confirmed by measuring the cell viability and activation of caspases. We showed that the MTD-containing peptide fused to eight arginine residues (R8:MTD), a necrosis-inducing peptide, induced necrosis in the Dox-Res CT26 cells, together with a cytosolic calcium spike, reactive oxygen species production, and the release of high mobility group box 1 into the media. Moreover, we demonstrated the killing effect of R8:MTD in tumor tissues generated using the Dox-Res CT26 cells in a mouse model. Therefore, our results suggest that MTD-containing peptides may provide an alternative tool for the elimination of relapsed tumor cells that are not responsive to apoptosis-inducing anticancer agents.

Apoptosis and oxidative stress are essential for the pathogenesis of acute liver failure and fulminant hepatic failure. Human placental hydrolysate (hPH) has been reported to possess antioxidant and anti‑inflammatory properties. In the present study, the protective effects of hPH against D‑galactosamine (D‑GalN)‑ and lipopolysaccharide (LPS)‑induced hepatocyte apoptosis were investigated in vivo. In addition, the molecular mechanisms underlying the anti‑apoptotic activities of hPH against D‑GalN‑induced cell death in vitro were examined. Male Sprague‑Dawley rats were injected with D‑GaIN/LPS with or without the administration of hPH. Rats were sacrificed 24 h after D‑GaIN/LPS intraperitoneal injection, and the blood and liver samples were collected for future inflammation and hepatotoxicity analyses. Changes in cell viability, apoptosis protein expression, mitochondrial mass, mitochondrial membrane potential, reactive oxygen species generation, and the levels of proteins and mRNA associated with a protective mechanism were determined in HepG2 cells pretreated with hPH for 2 h prior to D‑GalN exposure. The findings suggested that hPH treatment effectively protected against D‑GalN/LPS‑induced hepatocyte apoptosis by reducing the levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, interleukin‑6, and tumor necrosis factor‑α, and increasing the level of proliferating cell nuclear antigen. It was also found that hPH inhibited the apoptotic cell death induced by D‑GalN. hPH activated the expression of antioxidant enzymes, including superoxide dismutase, glutathione peroxidase, and catalase, which were further upregulated by the Kelch‑like ECH2‑associated protein 1‑p62‑nuclear factor‑erythroid 2‑related factor 2 pathway, a component of oxidative stress defense mechanisms. Furthermore, hPH markedly reduced cytosolic and mitochondrial reactive oxygen species and rescued mitochondrial loss and dysfunction through the reduction of damage‑regulated autophagy modulator, p53, and C/EBP homologous protein. Collectively, hPH exhibited a protective role in hepatocyte apoptosis by inhibiting oxidative stress and maintaining cell homeostasis. The underlying mechanisms may be associated with the inhibition of endoplasmic reticulum stress and minimization of the autophagy progress.

Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4), human breast carcinoma (MDA-MB231), and human glioma (U87MG) cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926). We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5) expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Serum deprivation or withdrawal induces apoptosis in endothelial cells, resulting in endothelial cell dysfunction that is associated with cardiovascular disease. However, there is still limited information on the role of miRNA in serum deprivation-induced apoptosis. Here we found that serum deprivation increased caspase-dependent apoptosis through miRNA-101-3p downregulation, without altering expression of its host gene RNA 3'-terminal phosphate cyclase-like 1, which was highly correlated with suppressed expression levels of Dicer and Argonaute 2 (Ago2), indicating that miR-101-3p is post-transcriptionally elevated in serum-deprived conditions. The decreased miR-101-3p caused elevated Bim expression by targeting its 3'-untranslated region (3'-UTR). This resulted in activation of the intrinsic pathway of apoptosis via interaction with Bcl-2, decreased mitochondrial membrane potential, cytochrome c release, mitochondrial reactive oxygen species (ROS) production, and caspase activation. These events were abrogated by miR-101-3p mimic and the proapoptotic Bim siRNA, which suggest a determinant role of the miR-101-3p/Bim axis in serum deprivation-induced apoptosis. The apoptosis induced by miR-101-3p-mediated Bim expression is mediated by both caspase-3 and -1, which are activated by two distinct intrinsic mechanisms, cytochrome c release and ROS-induced inflammasome activation, respectively. In other words, the antioxidant inhibited endothelial cell death mediated by caspase-1 that activated caspase-7, but not caspase-3. These findings provide mechanistic insight into a novel function of miR-101-3p in serum withdrawal-induced apoptosis triggered by activating two different intrinsic or mitochondrial apoptosis pathways, implicating miR-101-3p as a therapeutic target that limits endothelial cell death associated with vascular disorders.

This study investigated the effects of 1α, 25-dihydroxyvitamin D 3 -induced cell death and its underlying molecular mechanisms in Ishikawa endometrial carcinoma cells. The effects of 1α, 25-dihydroxyvitamin D 3 on Ishikawa cells were examined by 3-[4,5-dimethylthiazol-2-yl]-2.5-diphenyl-tetrazolium bromide, thiazolyl blue (MTT) assay. 1α, 25-dihydroxyvitamin D 3 was shown to induce programmed cell death in Ishikawa endometrial carcinoma cells by activation of caspase-3 and caspase-9, along with elevation of Bcl-2 and Bcl-xL. Cell viability was reduced by 1α, 25-dihydroxyvitamin D 3 in a concentration-dependent manner up to 2.5 μM. In addition, ezrin phosphorylation increased with the 1α, 25-dihydroxyvitamin D 3 concentration (0-0.5 μM). The protein level of caspase-9 was increased by 1α, 25-dihydroxyvitamin D 3 up to 0.5 μM. This is the first report regarding the efficacy and molecular mechanisms underlying the effects of 1α, 25-dihydroxyvitamin D 3 in endometrial cancer cells. Our findings indicate that 1α, 25-dihydroxyvitamin D 3 induces endometrial cancer cell death in a concentration-dependent manner. Impact statement Up to date, there is no report about the efficacy and molecular underlying mechanisms on the effect of vitamin D 3 in endometrial cancer cells. Our findings indicate that 1α, 25-dihydroxyvitamin D 3. which is an active metabolite of vitamin D 3 , induces Ishikawa endometrial cancer cell death in a concentration-dependent manner by activation of caspase-3 and -9, along with elevation of Bcl-2 and Bcl-xL. In addition, the same concentration of 1α, 25-dihydroxyvitamin D 3 that provoked apoptotic signals caused phosphorylation of ezrin at threonine 567 in a VDR-dependent manner. This study suggests that 1α, 25-dihydroxyvitamin D 3 within the optimal range (0.5 uM) would induce apoptosis through Fas-ezrin-caspase-3, -8, -9 signalling axis which may be a critical cell death regulator in Ishikawa endometrial cancer cell. Further study will be more interesting to address molecular connections or prove this critical optimal concentration range of vitamin D.

Khz (fusion of Ganoderma lucidum and Polyporus umbellatus), isolated from the mycelia of G. lucidum and P. umbellatus, exerts anti‑proliferative effects against malignant cells; however, its activity against human breast cancer cells remains to be elucidated. In the present study, cell proliferation was assessed using a 3-(4,5‑dimethylthiazol‑2‑yl)-2,5‑diphenyltetrazolium bromide assay, and poptosis was examined using annexin V‑propidium iodide staining and flow cytometry. The activation of caspases 7, 8 and 9 were detected in the Khz‑treated cells using western blotting. The results demonstrated that Khz increased the intracellular calcium concentration and induced the production of reactive oxygen species in MCF‑7 breast cancer cells, as determined using flow cytometry. The results also demonstrated that Khz inhibited cell proliferation and induced apoptosis in the MCF‑7 cells. In addition, the mechanism by which Khz induces apoptosis in cancer cells was investigated. Khz induced apoptosis preferentially in transformed cells, with a minimal effect on non‑transformed cells, suggesting its potential as an anticancer therapeutic agent. Oxidative stress is associated with apoptotic and non‑apoptotic cell death, although pro‑oxidative conditions are not a pre‑requisite for apoptosis. Assessment of the activation status of caspases 7, 8 and 9 revealed that the levels of cleaved caspases were significantly increased in the cells treated with Khz. It is widely accepted that calcium signaling is important in apoptosis, and the present study observed an increase in [Ca2+]i in response to Khz treatment. The anti‑proliferative and pro‑apoptotic effects of Khz suggest that this extract may be developed as a potential anticancer agent.

Khz (a fusion mycelium of Ganoderma lucidum and Polyporus umbellatus mycelia) is isolated from ganoderic acid and P. umbellatus and it exerts antiproliferative effects against malignant cells. However, no previous study has reported the inhibitory effects of Khz on the growth of human colon cancer cells. In the present study, we found that Khz suppressed cell division and induced apoptosis in HCT116 cells. Khz cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Khz reduced cell viability and mitochondrial membrane potential levels and it also induced disruption of the mitochondrial membrane potential and increased calcium concentration and reactive oxygen species generation. Khz increased caspase 3, PARP, caspase 7, and caspase 9 levels, but reduced Bcl-2 protein levels. Flow cytometry showed that the percentage of HCT116 cells in the sub-G1 phase of the cell cycle increased in response to Khz treatment.

Infection with high-risk human papillomaviruses (HPVs) causes cervical cancer. E6 oncoprotein, an HPV gene product, inactivates the major gatekeeper p53. In contrast, its isoform, TAp73β, has become increasingly important, as it is resistant to E6. However, the intracellular signaling mechanisms that account for TAp73β tumor suppressor activity in cervix are poorly understood. Here, we identified that IER3 is a novel target gene of TAp73β. In particular, TAp73β exclusively transactivated IER3 in cervical cancer cells, whereas p53 and TAp63 failed to do. IER3 efficiently induced apoptosis, and its knockdown promoted survival of HeLa cells. In addition, TAp73β-induced cell death, but not p53-induced cell death, was inhibited upon IER3 silencing. Moreover, etoposide, a DNA-damaging chemotherapeutics, upregulated TAp73β and IER3 in a c-Abl tyrosine kinase-dependent manner, and the etoposide chemosensitivity of HeLa cells was largely determined by TAp73β-induced IER3. Of interest, cervical carcinomas from patients express no observable levels of two proteins. Thus, our findings suggest that IER3 is a putative tumor suppressor in the cervix, and the c-Ab1/p73β/IER3 axis is a novel and crucial signaling pathway that confers etoposide chemosensitivity. Therefore, TAp73β and IER3 induction would be a valuable checkpoint for successful therapeutic intervention of cervical carcinoma patients.

MicroRNA-145 (miR-145) expression is downregulated in several human cancers, but its clinical and functional relevance to ovarian carcinoma has not yet been elucidated. This study addressed the hypothesis that miR-145 serves as a prognostic biomarker and a tumor suppressor that regulates the expression of high-mobility group A2 (HMGA2) oncoprotein in ovarian cancer. Here, we found that low miR-145 expression and HMGA2 overexpression determined by qRT-PCR and immunohistochemistry significantly correlated with advanced stage, lymph node involvement, and distant metastasis in 74 ovarian carcinomas. Low miR-145 expression significantly correlated with tumor recurrence and worse overall survival (HR=8.62, P = 0.039). Transfection of pre-miR-145 resulted in reduced cell growth and migration, and increased apoptosis of ovarian cancer cells by TUNEL, colony forming, and cell migration assays. MiR-145 was found to directly target HMGA2 by luciferase assay and Western blotting. Our findings suggest that miR-145 functions as a tumor suppressor in ovarian cancer and directly targets HMGA2 oncoprotein. Low miR-145 and high HMGA2 expressions are potential biomarkers of poor prognosis of ovarian carcinoma and miR-145 is the more powerful predictor of patient outcome., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Microtubule targeting chemicals are considered excellent antitumor drugs through their binding to tubulin, which affects the instability of microtubules resulting in arrest of cancer cells. The present study was designed to investigate the antitumor effects of viriditoxin (VDT) against human prostate cancer cells. VDT, isolated from Paecilomyces variotii fungus, which was derived from the jellyfish Nemopilema nomurai, offers a new approach for controlling resistant bacterial infections by blocking bacterial cell division proteins. VDT produced dose-dependent cytotoxicity against human prostate cancer cells. Treatment with VDT promoted both apoptosis and autophagy in LNCaP cells. Annexin V/FITC staining indicated that apoptosis occurred in VDT-treated LNCaP cells. DAPI staining revealed morphological changes in the cell nuclei indicative of mitotic catastrophe in LNCaP cells. VDT caused cell growth inhibition via G2/M phase arrest. Moreover, VDT also increased autophagic cell death in LNCaP cells by induction of several autophagy-related proteins such as LC3 II, Atg5, Atg7 and beclin-1 protein, which are essential for autophagy induction. These results were also confirmed by acridine orange staining. This study indicates that VDT could potentially be effective against prostate cancer by promoting multiple modes of growth arrest and cell death coupled with apoptosis and autophagy.

Background: Khz-cp is a crude polysaccharide extract that is obtained after nuclear fusion in Ganoderma lucidum and Polyporus umbellatus mycelia (Khz). It inhibits the growth of cancer cells., Methods: Khz-cp was extracted by solvent extraction. The anti-proliferative activity of Khz-cp was confirmed by using Annexin-V/PI-flow cytometry analysis. Intracellular calcium increase and measurement of intracellular reactive oxygen species (ROS) were performed by using flow cytometry and inverted microscope. SNU-1 cells were treated with p38, Bcl-2 and Nox family siRNA. siRNA transfected cells was employed to investigate the expression of apoptotic, growth and survival genes in SNU-1 cells. Western blot analysis was performed to confirm the expression of the genes., Results: In the present study, Khz-cp induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz-cp was found to induce apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating P38 to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-cp-induced apoptosis was caspase dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-cp-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was shown by the translocation of the regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz-cp. Khz-cp triggered a rapid and sustained increase in [Ca2+]i that activated P38. P38 was considered to play a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation., Conclusions: In summary, these data indicate that Khz-cp preferentially induces apoptosis in cancer cells and that the signaling mechanisms involve an increase in [Ca2+]i, P38 activation, and ROS generation via NADPH oxidase and mitochondria.

The purpose of this study was to demonstrate the apoptotic activity and antitumor effect of PEGylated tumor necrosis factor-related apoptosis-inducing ligand (PEG-TRAIL) in pancreatic carcinoma Mia Paca-2 cells and in Mia Paca-2 cell-xenografted mice. PEG-TRAIL was prepared using mPEG-aldehyde (Mw 5 kDa). The apoptosis induced by PEG-TRAIL in Mia Paca-2 cells and in the tumors of Mia Paca-2 cell-xenografted mice was quantified by FACS analysis and using a TUNEL assay. Mia Paca-2 cell-xenografted BALB/c nu/nu mice were administered intratumoral injections of PEG-TRAIL (50 μg/mouse/injection) every 3 days from day 0 (~4 weeks after xenografting) to day 15. Tumor volumes were measured every 3 days from day 0 to day 27. PEG-TRAIL displayed obvious apoptotic activity in Mia Paca-2 cells; the FACS signal was shifted to the apoptotic area and the cells exhibited green fluorescence indicating apoptosis in the TUNEL assay. Furthermore, PEG-TRAIL was found to suppress tumors in Mia Paca-2 cell-xenografted mice (tumor volumes: 183.9±134.1 for PEG-TRAIL vs. 1827.3±264.5 mm(3) for saline control). In addition, in vivo TUNEL assays of tumor tissues showed that the antitumor effect of PEG-TRAIL was due apoptosis. Our findings provide clear in vivo evidence of the antitumor potential of PEG-TRAIL in a Mia Paca-2 cell-xenografted mouse model based of pancreatic cancer., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Background: Multidrug resistance is a major problem in the treatment of breast cancer, and a number of studies have attempted to find an efficient strategy with which to overcome it. In this study, we investigate the synergistic anticancer effects of resveratrol (RSV) and doxorubicin (Dox) against human breast cancer cell lines., Methods: The synergistic effects of RSV on chemosensitivity were examined in Dox-resistant breast cancer (MCF-7/adr) and MDA-MB-231 cells. In vivo experiments were performed using a nude mouse xenograft model to investigate the combined sensitization effect of RSV and Dox., Results and Conclusion: RSV markedly enhanced Dox-induced cytotoxicity in MCF-7/adr and MDA-MB-231 cells. Treatment with a combination of RSV and Dox significantly increased the cellular accumulation of Dox by down-regulating the expression levels of ATP-binding cassette (ABC) transporter genes, MDR1, and MRP1. Further in vivo experiments in the xenograft model revealed that treatment with a combination of RSV and Dox significantly inhibited tumor volume by 60%, relative to the control group., General Significance: These results suggest that treatment with a combination of RSV and Dox would be a helpful strategy for increasing the efficacy of Dox by promoting an intracellular accumulation of Dox and decreasing multi-drug resistance in human breast cancer cells., (© 2013.)

Objective: Layered double hydroxide (LDH) nanoparticles have been studied as cellular delivery carriers for anionic anticancer agents. As MTX and 5-FU are clinically utilized anticancer drugs in combination therapy, we aimed to enhance the therapeutic performance with the help of LDH nanoparticles., Method: Anticancer drugs, MTX and 5-FU, and their combination, were incorporated into LDH by reconstruction method. Simply, LDHs were thermally pretreated at 400°C, and then reacted with drug solution to simultaneously form drug-incorporated LDH. Thus prepared MTX/LDH (ML), 5-FU/LDH (FL), and (MTX + 5-FU)/LDH (MFL) nanohybrids were characterized by X-ray diffractometer, scanning electron microscopy, infrared spectroscopy, thermal analysis, zeta potential measurement, dynamic light scattering, and so forth. The nanohybrids were administrated to the human cervical adenocarcinoma, HeLa cells, in concentration-dependent manner, comparing with drug itself to verify the enhanced therapeutic efficacy., Conclusion: All the nanohybrids successfully accommodated intended drug molecules in their house-of-card-like structures during reconstruction reaction. It was found that the anticancer efficacy of MFL nanohybrid was higher than other nanohybrids, free drugs, or their mixtures, which means the multidrug-incorporated LDH nanohybrids could be potential drug delivery carriers for efficient cancer treatment via combination therapy.

Exposure to ethanol during developmental stages leads to several types of neurological disorders. Apoptotic neurodegeneration due to ethanol exposure is a main feature in alcoholism. Exposure of developing animals to alcohol induces apoptotic neuronal death and causes fetal alcohol syndrome. In the present study, we observed the possible protective effect of pyruvate against ethanol-induced neurodegeneration. Exposure of developing mice to ethanol (2.5 g/kg) induces apoptotic neurodegeneration and widespread neuronal cell death in the cortex and thalamus. Co-treatment of pyruvate (500 mg/kg) protects neuronal cell against ethanol by the reduced expression of caspase-3 in these brain regions. Immunohistochemical analysis and TUNNEL at 24 h showed that apoptotic cell death induced by ethanol in the cortex and thalamus is reduced by pyruvate. Histomorphological analysis at 24 h with cresyl violet staining also proved that pyruvate reduced the number of neuronal cell loss in the cortex and thalamus. The results showed that ethanol increased the expression of caspase-3 and thus induced apoptotic neurodegeneration in the developing mice cortex and thalamus, while co-treatment of pyruvate inhibits the induction of caspase-3 and reduced the cell death in these brain regions. These findings, therefore, showed that treatment of pyruvate inhibits ethanol-induced neuronal cell loss in the postnatal seven (P7) developing mice brain and may appear as a safe neuroprotectant for treating neurodegenerative disorders in newborns and infants.

Caffeine is white crystalline xanthine alkaloid that is naturally found in some plants and can be produced synthetically. It has various biological effects, especially during pregnancy and lactation. We studied the effect of caffeine on heartbeat, survival and the expression of cell damage related genes, including oxidative stress (HSP70), mitochondrial metabolism (Cyclin G1) and apoptosis (Bax and Bcl2), at early developmental stages of zebrafish embryos. We used 100 µm concentration based on the absence of locomotor effects. Neither significant mortality nor morphological changes were detected. We monitored hatching at 48 h post-fertilization (hpf) to 96 hpf. At 60 and 72 hpf, hatching decreased significantly (P < 0.05); however, the overall hatching rate at 96 hpf was 94% in control and 93% in caffeine treatment with no significant difference (P > 0.05). Heartbeats per minute were 110, 110 and 112 in control at 48, 72 and 96 hpf, respectively. Caffeine significantly increased heartbeat - 122 and 136 at 72 and 96 hpf, respectively. Quantitative RT-PCR showed significant up-regulation after caffeine exposure in HSP70 at 72 hpf; in Cyclin G1 at 24, 48 and 72 hpf; and in Bax at 48 and 72 hpf. Significant down-regulation was found in Bcl2 at 48 and 72 hpf. The Bax/Bcl2 ratio increased significantly at 48 and 72 hpf. We conclude that increasing exposure time to caffeine stimulates oxidative stress and may trigger apoptosis via a mitochondrial-dependent pathway. Also caffeine increases heartbeat from early phases of development without affecting the morphology and survival but delays hatching. Use of caffeine during pregnancy and lactation may harm the fetus by affecting the expression of cell-damage related genes., (Copyright © 2012 John Wiley & Sons, Ltd.)

MUDENG, also known as AP5M1, was originally identified as an adaptin domain-containing gene that induced cell death in lymphoma cell lines. However, little is known of the mechanism responsible for MUDENG-mediated cell death. In this study, we investigated MUDENG changes during TRAIL-induced cell death. We found that MUDENG is rapidly processed in response to TRAIL in Jurkat and BJAB cells with time line similar to that of caspase activation. Caspase-3-mediated MUDENG cleavage was confirmed by an in vitro cleavage assay using recombinant active caspase proteins. Caspase cleavage sites (D276 and D290) were located in the adaptin domain of MUDENG, and cleaved MUDENG showed the reduced killing activity. These results suggest that the adaptin domain plays a key role in MUDENG-mediated cell death., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The Rho GTPases are the sub-group of Ras super family and identified in all eukaryotes. The Rho GTPases effect different cellular signaling pathways involved in a number of diseases such as cancer, neurological and cardiovascular disorders. Members of Rho GTPases including RhoA, RhoC and Rac1 play a major role in regulation of apoptosis in different kind of stress conditions. Here we investigated the Rho GTPase activating protein 15 (ArhGAP15) gene knock-down effect on apoptosis induced by ethanol in bovine fibroblast cells. The bovine Fibroblast cells were treated and transfected with two different concentrations (50 and 100 nM) of ArhGAP15 siRNA for 48 h respectively. Both concentrations of siRNA were effective and the results of RT-PCR revealed an efficient knock-down of ArhGAP15 mRNA in fibroblast cells. Further, the normal cells exposed to a 100 mM ethanol concentration showed a reduction in cell viability and induced the ratio of apoptosis related Bax/Bcl-2 proteins compared with ArhGAP15 siRNA transfected ethanol treated cells. Ethanol also increased caspase-3 expression in normal fibroblast cells compared with transfected cells. The ArhGAP15 knock-down cells treated with ethanol decreased Bax/Bcl-2 ratio and lower caspase-3 protein levels in ArhGAP15 knocked-down cells. Our results suggest that apoptosis induced by ethanol involves the activation of Rho GTPase activating protein 15 and silencing of the said gene protects apoptosis.

The phosphatidylinositol 3-kinase (PI3K) pathway is one of the critical signaling cascades playing important roles in the chemoresistance of human cancer cells, including ovarian cancer. In this study, we investigated the potential of targeting the PI3K p110β-isoform as a novel approach to overcome the chemoresistance in ovarian cancer. The effects on apoptosis, cell viability, proliferation and migration in chemoresistant ovarian cancer cell were determined following targeted p110β inhibition by small interfering RNA (siRNA). Seven paclitaxel (PTX)-resistant sublines (SKpacs and A2780pac) were produced from SKOV3 and A2780 ovarian cancer cell lines. We, first, evaluated the expression of PI3K p110 isoforms in chemosensitive and chemoresistant ovarian cancer cell lines and patient specimens, and found that p110β-isoform was significantly overexpressed both in a panel of ovarian cancer samples, and in PTX-resistant sublines compared with their parent cell lines. RNA interference-mediated p110β silencing augmented PTX-mediated apoptosis (31.15 ± 13.88 %) and reduced cell viability (67 %) in PTX-resistant cells, whereas targeting p110α did not show a significant change in cell viability and apoptosis. In addition, p110β silencing impaired cell proliferation (60 %) in PTX-resistant SKpac cells. We also found the combined treatment group with p110β siRNA and PTX showed a significant inhibition of tumor growth of SKpac cells compared to the PTX-only treated group in a xenograft nude mouse model. Thus, the siRNA-mediated silencing of PI3K p110β resensitizes PTX-resistant ovarian cancer cells, and may be a useful therapeutic strategy for PTX-resistant ovarian cancers.

Smad7 has been known as a negative regulator for the transforming growth factor-β (TGF-β) signaling pathway through feedback regulation. However, Smad7 has been suspected to have other biological roles through the regulation of gene transcription. By screening differentially regulated genes, we found that the caspase 8 gene was highly up-regulated in Smad7-expressing cells. Smad7 was able to activate the caspase 8 promoter through recruitment of the interferon regulatory factor 1 (IRF1) transcription factor to the interferon-stimulated response element (ISRE) site. Interaction of Smad7 on the caspase 8 promoter was confirmed with electrophoretic mobility shift assay and chromatin immunoprecipitation experiment. Interestingly, Smad7 did not directly interact with the ISRE site, but it increased the binding activity of IRF1 with ISRE. These results support that Smad7 recruits IRF1 protein on the caspase 8 promoter and functions as a transcriptional coactivator. To confirm the biological significance of caspase 8 up-regulation, we tested tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cell death assay in breast cancer cells. Smad7 in apoptosis-resistant MCF7 cells markedly sensitized the cells to TRAIL-induced cell death by restoring the caspase cascade. Furthermore, restoration of caspase 8-mediated apoptosis pathway repressed the tumor growth in the xenograft model. In conclusion, we suggest a novel role for Smad7 as a transcriptional coactivator for caspase 8 through the interaction with IRF1 in regulation of the cell death pathway.

Acquired resistance to tamoxifen (Tam) is a critical problem in breast cancer therapy. Therefore, new potential strategies for Tam-resistant breast cancer are needed recently. In this study, we synthesized a novel histone deacetylase (HDAC) inhibitor, MHY218, for the development of potent inhibitors of HDAC and evaluated its biological activities by monitoring the anticancer effects in Tam-resistant MCF-7 (TAMR/MCF-7) cells via in vitro and in vivo studies. MHY218 significantly inhibited the proliferation of TAMR/MCF-7 cells in a dose-dependent manner. The total HDAC enzyme activity was significantly inhibited, corresponding with inhibition of acetylated H3 and H4 expression in TAMR/MCF-7 cells. HDAC1, 4, and 6 expression levels were decreased in response to MHY218 treatment. Cell cycle analysis indicated that MHY218 induced G2/M phase cell cycle arrest. As expected, apoptotic cell death was observed in response to MHY218 treatment. Interestingly, levels of beclin-1 and LC3-II, the markers of autophagy, were increased in TAMR/MCF-7 cells treated with MHY218. The efficacy of MHY218 was also compared with that of SAHA in vivo in a xenograft model of nude mice bearing a TAMR/MCF-7 cells. MHY218 (10 mg/kg, twice a week for 21 days) completely inhibited tumor growth and MHY218 markedly inhibited the expression of proliferative cell nuclear antigen (PCNA) in tumor tissue. These results indicate that MHY218 can induce caspase-independent autophagic cell death rather than apoptotic cell death. The MHY218-induced autophagic cell death could be a new strategy in the treatment of Tam-resistant human breast cancer.

Sirtuins (SIRTs), NAD+-dependent class III histone deacetylases (HDACs), play an important role in the regulation of cell division, survival and senescence. Although a number of effective SIRT inhibitors have been developed, little is known about the specific mechanisms of their anticancer activity. In this study, we investigated the anticancer effects of sirtinol, a SIRT inhibitor, on MCF-7 human breast cancer cells. Apoptotic and autophagic cell death were measured. Sirtinol significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner. The IC50 values of sirtinol were 48.6 µM (24 h) and 43.5 µM (48 h) in MCF-7 cells. As expected, sirtinol significantly increased the acetylation of p53, which has been reported to be a target of SIRT1/2. Flow cyto-metry analysis revealed that sirtinol significantly increased the G1 phase of the cell cycle. The upregulation of Bax, downregulation of Bcl-2 and cytochrome c release into the cytoplasm, which are considered as mechanisms of apoptotic cell death, were observed in the MCF-7 cells treated with sirtinol. The annexin V-FITC assay was used to confirm sirtinol-induced apoptotic cell death. Furthermore, the expression of LC3-II, an autophagy-related molecule, was significantly increased in MCF-7 cells after sirtinol treatment. Autophagic cell death was confirmed by acridine orange and monodansylcadaverine (MDC) staining. Of note, pre-treatment with 3-methyladenine (3-MA) increased the sirtinol-induced MCF-7 cell cytotoxicity, which is associated with blocking autophagic cell death and increasing apoptotic cell death. Based on our results, the downregulation of SIRT1/2 expression may play an important role in the regulation of breast cancer cell death; thus, SIRT1/2 may be a novel molecular target for cancer therapy and these findings may provide a molecular basis for targeting SIRT1/2 in future cancer therapy.

Glioblastoma multiforme (GBM), the most common and aggressive primary brain malignancy, is incurable despite the best combination of current cancer therapies. For the development of more effective therapies, discovery of novel candidate tumor drivers is urgently needed. Here, we report that peroxiredoxin 4 (PRDX4) is a putative tumor driver. PRDX4 levels were highly increased in a majority of human GBMs as well as in a mouse model of GBM. Reducing PRDX4 expression significantly decreased GBM cell growth and radiation resistance in vitro with increased levels of ROS, DNA damage, and apoptosis. In a syngenic orthotopic transplantation model, Prdx4 knockdown limited GBM infiltration and significantly prolonged mouse survival. These data suggest that PRDX4 can be a novel target for GBM therapies in the future.

Background: Silibinin, a natural polyphenolic flavonoid, has been reported to induce cell death in various cancer cell types. However, the molecular mechanism is not clearly defined. Our previous study showed that silibinin induces glioma cell death and its effect was effectively prevented by calpain inhibitor. The present study was therefore undertaken to examine the role of calpain in the silibinin-induced glioma cell death., Methods: U87MG cells were grown on well tissue culture plates and cell viability was measured by MTT assay. ROS generation and △ψm were estimated using the fluorescence dyes. PKC activation and Bax expression were measured by Western blot analysis. AIF nuclear translocation was determined by Western blot and immunocytochemistry., Results: Silibinin induced activation of calpain, which was blocked by EGTA and the calpain inhibitor Z-Leu-Leu-CHO. Silibinin caused ROS generation and its effect was inhibited by calpain inhibitor, the general PKC inhibitor GF 109203X, the specific PKCδ inhibitor rottlerin, and catalase. Silibinin-induce cell death was blocked by calpain inhibitor and PKC inhibitors. Silibinin-induced PKCδ activation and disruption of △ψm were prevented by the calpain inhibitor. Silibinin induced AIF nuclear translocation and its effect was prevented by calpain inhibitor. Transfection of vector expressing microRNA of AIF prevented the silibinin-induced cell death., Conclusions: Silibinin induces apoptotic cell death through a calpain-dependent mechanism involving PKC, ROS, and AIF nuclear translocation in U87MG human glioma cells., (© 2011 Jeong et al; licensee BioMed Central Ltd.)

Background: Hepatocyte nuclear factor-3alpha (HNF-3alpha) has been known to act as a repressor in the pathogenesis of many cancers. Herein, we investigated the effect of HNF-3alpha overexpression in prostate cancer cells., Methods: HNF-3alpha was overexpressed in prostate cancer cells using an adenovirus recombinant expressing wild-type HNF-3alpha. The apoptosis of prostate cancer cells was determined by TUNEL, FACS, and caspase activity analyses., Results: Adenovirus-mediated overexpression of HNF-3alpha caused cell death in prostate cancer cells as assessed by changes in cellular and nuclear morphology, TUNEL analysis, and caspase activations. Furthermore, FACS analysis showed an increased sub-G1 phase of cell cycle as well as the G2/M phase with a corresponding decrease in S phases. HNF-3alpha overexpression caused the upregulation of p53 protein and its accumulation, together with HNF-3alpha, in the cytoplasm. It also causes Bax protein to localize to the mitochondria-enriched fraction. These findings suggest that multiple apoptotic pathways seem to be involved in the HNF-3alpha-induced cell death: pathways involving the accumulation of p53 protein in the cytoplasm and subsequent cytochrome c release, and other pathways involving death receptor signaling and caspase-8 activation., Conclusions: The results of the current study suggest a novel function of HNF-3alpha as a killer of malignant prostate cancer cells, which reveals HNF-3alpha as a promising therapeutic molecule for prostate cancers., (Prostate 70: 353-361, 2010. (c) 2009 Wiley-Liss, Inc.)

Background: MicroRNAs (miRNAs) have emerged as novel genetic regulators of cell functions such as proliferation, apoptosis and cancer., Aims: The aim of this study was to evaluate the role of a specific miRNA in modulating hepatic cell functions., Methods: C57Bl/6 mice were administered anti-fas receptor antibodies to induce liver cell apoptosis. miRNAs were purified from the liver tissue and evaluated using an miRNA microarray. The role of miRNA-491&#95;5p, which was overexpressed in the model, in modulating hepatic cell functions was evaluated. miRNA-491&#95;5p was overexpressed in Hep G2 cells, followed by the addition of tumour necrosis factor (TNF)-alpha, and induction of apoptosis as well as genes involved in apoptosis pathways were evaluated. The effect of miRNA-491&#95;5p target genes on apoptosis was also analysed by inhibiting their expression by siRNA-induced gene silencing., Results: Upregulation of miRNA-491&#95;5p was found in a high-dose anti-fas receptor antibody group. Overexpression of microRNA-491&#95;5p sensitized Hep G2 cells for TNF-alpha-induced apoptosis, and also caused an inhibition of alpha-fetoprotein, (AFP), heat shock protein-90 (hsp-90) and nuclear factor-kappaB (NF-kappaB). Overexpression of miRNA-491&#95;5p or inhibition of AFP and hsp-90 resulted in an increased apoptosis in TNF-alpha-treated Hep G2 cells., Conclusions: One of the miRNAs that is associated with the acute liver injury mouse model, miRNA-491&#95;5p, sensitizes Hep G2 cells for TNF-alpha-induced apoptosis, at least in part, by inhibiting AFP, hsp-90 and NF-kappaB.

Mulberry has been reported to contain wide range of polyphenols and have chemopreventive activity. However, little has been known regarding the effect of mulberry fruit extracts on cell viability in vitro in human glioma cells and the anticancer efficacy in vivo. This study was undertaken to examine the effect of mulberry fruit (Moris fructus; MF) extracts on cell viability in vitro and anticancer efficacy in vivo. Cell viability and cell death were estimated by MTT assay and trypan blue exclusion assay, respectively. Reactive oxygen species (ROS) generation was measured using the fluorescence probe DCFH-DA. The mitochondrial transmembrane potential was measured with DiOC(6)(3). Bax expression and cytochrome c release were measured by Western blot analysis. Caspase activity was estimated using colorimetric kit. Cell migration was estimated using the scratched wound model. In vivo anticancer efficacy of MF extracts was evaluated using a subcutaneously injected mouse tumor model. Changes in proliferation and apoptosis were estimated by immunohistochemistric analysis. MF extracts resulted in apoptotic cell death in a dose- and time-dependent manner. MF extracts increased ROS generation, and the MF extract-induced cell death was also prevented by antioxidants, suggesting that ROS generation plays a critical role in the MF extract-induced cell death. Western blot analysis showed that treatment of MF extracts caused an increase in Bax expression, which was inhibited by the antioxidant N-acetylcysteine (NAC). MF extracts induced depolarization of mitochondrial membrane potential, and its effect was inhibited by the antioxidants NAC and catalase. MF extracts induced cytochrome c release, which was inhibited by NAC. Caspase activity was stimulated by MF extracts, and caspase inhibitors prevented the MF extract-induced cell death. Treatment of MF extracts inhibited cell migration. Oral MF extracts administration in animals with subcutaneous U87MG glioma cells reduced tumor volume. Subsequent tumor tissue analysis showed a decrease in PCNA-positive cells, an increase in TUNEL-positive cells, and caspase activation. From these data, we concluded that MF extracts reduce glioma tumor growth through inhibition of cell proliferation resulting from induction of apoptosis. These findings suggest that MF extracts result in human glioma cell death in vitro through ROS-dependent mitochondrial pathway and glioma tumor growth in vivo via reduction of tumor cell proliferation and induction of apoptosis.

DNA damage stabilizes the p53 tumor suppressor protein that determines the cell fate by either cell cycle arrest or cell death induction. Noxa, the BH3-only Bcl-2 family protein, was shown to be a key player in p53-induced cell death through the mitochondrial dysfunction; however, the molecular mechanism by which Noxa induces the mitochondrial dysfunction to cause cell death in response to genotoxic agents is largely unknown. Here, we show that the mitochondrial-targeting domain (MTD) of Noxa is a prodeath domain. Peptide containing MTD causes massive necrosis in vitro through cytosolic calcium increase; it is released from the mitochondria by opening the mitochondrial permeability transition pore. MTD peptide-induced cell death can be inhibited by calcium chelator BAPTA-AM. Moreover, MTD peptide shows the potent tumor-killing activities in mice by joining with tumor-homing motifs.

In the present study, we identified a missense mutation (G199V) in KAT-18 cell line established from primary cultures of anaplastic thyroid cancer (ATC). Notably, knockdown of this mutant (mt) p53 reduced cell viability and exerted antitumor activity equivalent to high doses of several chemotherapeutic agents. We showed that p53 knockdown had an antitumor effect via the induction of apoptosis. We further examined the underlying mechanism by which mt p53 (G199V) gains antiapoptotic function in KAT-18 cells. Microarray analysis revealed that p53 knockdown modified the expression of numerous apoptosis-related genes. Importantly, p53 knockdown led to downregulation of signal transducer and activator of transcription-3 (STAT3) gene expression. We further observed that p53 knockdown induced the downregulation of STAT3 protein. We also observed that a STAT3 inhibitor augmented the reduction of cell viability induced by p53 knockdown, whereas interleukin-6 treatment alleviated this effect. In addition, overexpression of STAT3 protected ATC cells against cell death induced by p53 knockdown. Taken together, these data show that mt p53 (G199V) gains antiapoptotic function mediated by STAT3 in ATC cells. Inhibition of the function of mt p53 (G199V) could be a novel and useful therapeutic strategy for decreasing the extent and severity of toxicity due to chemotherapeutic agents.

Bcl-2 protects tumor cells from the apoptotic effects of various antineoplastic agents. Increased expression of Bcl-2 has been associated with poor response to chemotherapy in various malignancies, including leukemia. Therefore, bypassing the resistance conferred by anti-apoptotic factors such as Bcl-2 represents an attractive therapeutic strategy against cancer cells, including leukemic cells. We undertook this study to examine whether SAHA (suberoylanilide hydroxamic acid) overcomes the resistance by Bcl-2 in human leukemic cells, with a specific focus on the involvement of PML-NBs. Experiments were conducted with Bcl-2-overexpressing human leukemic U937 cells. Since we previously demonstrated that overexpression of Bcl-2 attenuates resveratrol-induced apoptosis in human leukemic U937 cells, resveratrol-treated U937 cells were used as a negative control. The present study indicates that SAHA at 1-7 microM, the dose range known to induce apoptosis in various cancer cells, overcomes the anti-apoptotic effects of Bcl-2 in Bcl-2-overexpressing human leukemic U937 cells. Notably, we observed that SAHA-induced formation of mature promyelocytic leukemia (PML) nuclear bodies (NBs) correlates with overcoming the anti-apoptotic effects of Bcl-2 in human leukemic U937 cells. Thus, PML protein and the formation of mature PML-NBs could be considered as therapeutic targets that could help bypass the resistance to apoptosis conferred by Bcl-2. Elucidating exactly how PML regulates Bcl-2 will require further work.

Downstream of Bid (DOBI) known as Pus10, has been identified as a modulator of TRAIL-induced cell death using RNAi library screening. The crystal structure of DOBI has revealed that it is a crescent-shaped protein containing the pseudouridine synthase catalytic domain and a THUMP-containing domain. Here, we demonstrated that DOBI is expressed in various tissues such as heart and lung, and is also expressed in various tumor cells such as HeLa and A549. Although ectopic expression of DOBI does not promote TRAIL death signaling in HeLa cells, knock-down of DOBI expression using shRNA inhibited TRAIL death signaling. DOBI is cleaved into a 54 kD cleaved DOBI during cell death, and the recombinant DOBI protein can be directly cleaved by caspases-3, or -8 in vitro. Together, these data suggest that the cleaved DOBI may acquire a new function, possibly by cooperating with tBid in the mitochondrial event of cell death caused by TRAIL.

TRAIL is an apoptotic cell death-inducing ligand that belongs to a TNF superfamily. To identify the regulators that govern the susceptibility to TRAIL, TRAIL-resistant HeLa (TR) cells were established by repeatedly treating HeLa cells with TRAIL. Here we showed that scaffolding protein Homer1 plays a decisive role in regulating the apoptotic susceptibility to TRAIL. TR cells showing the normal susceptibility to FasL and chemotherapeutic agent etoposide expressed the lower protein levels of Homer1 than parental HeLa cells. They showed the delayed activation of caspases-8, Bid cleavage and Bax translocation to mitochondria in response to TRAIL. Reconstitution of Homer1 expression in TR cells significantly restored the susceptibility to TRAIL. In addition, knock-down of Homer1 using interfering shRNA in parental HeLa cells lost the susceptibility to TRAIL. Together, our data indicate that Homer1 plays a critical role in determining the apoptotic susceptibility to TRAIL.

We have previously demonstrated that the synthetic peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist ciglitazone induces apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) and nuclear translocation of apoptosis inducing factor (AIF) in opossum kidney (OK) renal epithelial cells. However, the precise mechanism by which ciglitazone induces activation of p38 MAPK and the role of AIF in the induction of the apoptosis are not defined. This study was therefore undertaken to determine whether the roles of reactive oxygen species (ROS) generation and intracellular Ca(2+) in the ciglitazone-induced activation of p38 MAPK and whether AIF nuclear translocation is responsible for the ciglitazone-induced apoptosis in OK renal epithelial cells. Ciglitazone caused generation of ROS and an increase in intracellular Ca(2+). Ciglitazone-induced cell death was reduced by the antioxidant Trolox, the Ca(2+) chelator EGTA, and the store-operated Ca(2+) channels (SOCC) blocker lanthanum chloride (La(3+)), indicating involvement of ROS and Ca(2+) in the ciglitazone-induced cell death. Ciglitazone-induced intracellular Ca(2+) increase was decreased by Trolox, while ROS generation was not affected by EGTA and La(3+), suggesting that ROS generation promote the increase of intracellular Ca(2+). Transfection of small interfering RNA (siRNA) of p38 MAPK or vector expressing microRNA (miRNA) of AIF prevented the ciglitazone-induced cell death. Activation of p38 MAPK, mitochondrial membrane depolarization, and AIF nuclear translocation induced by ciglitazone were inhibited by Trolox, EGTA and La(3+). Taken together, these results suggest that ROS-dependent intracellular Ca(2+) increase is responsible for activation of p38 MAPK and nuclear translocation of AIF by ciglitazone.

One of the pathways by which alcohol induces hepatocyte apoptosis is via oxidative stress. We screened several chemically-synthesized small molecules and found LAS-0811, which inhibits oxidative stress. In this study, we elucidated its role in inhibiting alcohol-induced apoptosis in hepatocyte-like VL-17A cells. VL-17A cells were pre-incubated with LAS-0811, followed by ethanol incubation. Ethanol-induced reactive oxygen species and apoptosis were significantly inhibited in LAS-0811 pre-treated cells. VL-17A cells were transfected with a reporter (ARE/TK-GFP) plasmid containing green fluorescent protein (GFP) as a reporter gene and the anti-oxidant response element as the promoter. LAS-0811 pre-treatment significantly induced the GFP expression compared to the cells treated with ethanol alone. LAS-0811 induced the activation of nrf2 and enhanced the expression and activity of glutathione peroxidase, one of the downstream targets of nrf2. The results indicate that LAS-0811 protects VL-17A cells against ethanol-induced oxidative stress and apoptosis at least in part via nrf2 activation.

Purpose: To better understand the mechanism underlying the anti-apoptotic activity of alphaB-crystallin in RPE cells., Methods: Cells of the human retinal pigment epithelial line ARPE-19 were treated with a histone deacetylase inhibitor (HDACI), suberoylanilide hydroxamic acid (SAHA), with or without alphaB-crystallin siRNA. To examine the mechanism underlying the cell death induced in ARPE-19 cells, nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, Western blot analysis, confocal microscopy, and coimmunoprecipitation assay were undertaken., Results: The present study demonstrated that an HDACI, SAHA, at the usual doses or the silencing of alphaB-crystallin by siRNA alone did not effectively induce apoptosis in ARPE-19 cells. Silencing of alphaB-crystallin likely abolishes the anti-apoptotic activity of alphaB-crystallin. The data indicated that silencing of alphaB-crystallin sensitizes ARPE19 cells to SAHA-induced apoptosis and leads them to stage 1 apoptosis. alphaB-Crystallin associates with HDAC1 on SC35 speckles, and silencing of alphaB-crystallin abolishes this association, resulting in the induction of apoptosis. The data indicated that the association between alphaB-crystallin and HDAC1 on SC35 speckles plays a pivotal role in anti-apoptotic activity., Conclusions: Knockout of alphaB-crystallin may be a promising new approach to enhance therapeutic potency for proliferative vitreoretinopathy without compromising efficacy.

This study examined the mechanism for the anti-cancer effects of histone deacetylase (HDAC) inhibitor trichostatin A (TsA) in H-ras-transformed human breast epithelial (MCF10A-ras) cells. The effects of TsA on anti-cancer effects of MCF10A-ras cells were determined by measuring the level of cell cycle regulator expression and apoptotic cell death using Western blotting and flow cytometry analysis, respectively. TsA induced morphological changes, apoptotic cell death and modulation of the cell cycle regulatory proteins in the MCF10A-ras cells. TsA increased the levels of acetylated histone H3 and H4 in MCF10A-ras cells. In addition, TsA markedly down-regulated the expression of cyclin D1 and CDK4, up-regulated the expression of p21WAF1 and p53 and induced cell cycle arrest at the G1 phase in MCF10A-ras cells. The levels of hyperphosphorylation of the Rb protein were lower in MCF10A-ras cells after the TsA treatment. Furthermore, the up-regulation of p53 promoted Bax expression, which led to the activation of pro-caspase-3 and eventually to apoptosis in MCF10A-ras cells. TsA significantly increased the levels of ERK1/2 phosphorylation in MCF10A-ras cells. Overall, the TsA-activated ERK pathway plays an important role in cell cycle arrest and apoptosis through the ERK-dependent induction of p21 in Ras-related human cancer cells.

Tumor hypoxia, which is caused by the rapid proliferation of tumor cells and aberrant vasculature in tumors, results in inadequate supplies of oxygen and nutrients to tumor cells. Paradoxically, these unfavorable growth conditions benefit tumor cell survival, although the mechanism is poorly understood. We have demonstrated for the first time that hypoxia inhibits TRAIL-induced apoptosis by blocking translocation of Bax from cytosol to the mitochondria in tumor cells. However, it is largely unknown how hypoxia-inhibited Bax translocation attenuates TRAIL-induced apoptosis. Here, we demonstrate that despite its inhibitory activity in TRAIL-induced apoptosis, hypoxia does not affect TRAIL-triggered proximal apoptotic signaling events, including caspase-8 activation and Bid cleavage. Instead, hypoxia inhibited processing of caspase-3, leading to incomplete activation of the caspase. Importantly, hypoxia-blocked translocation of Bax to the mitochondria significantly inhibited releasing the mitochondrial factors, such as cytochrome c and Smac/DIABLO, to the cytosol in response to TRAIL. It is well-known that complete processing/activation of caspase-3 requires Smac/DIABLO released from mitochondria. Therefore, our data indicate that an engagement of the apoptotic mitochondrial events leading to caspase-3 activation is blocked by hypoxia. Our data shed new light on understanding of the apoptotic signal transduction and targets regulated by tumor hypoxia.

Even though there is no direct evidence to prove the cellular and molecular changes induced by radiofrequency (RF) radiation itself, we cannot completely exclude the possibility of any biological effect of mobile phone frequency radiation. We established a carousel-type exposure chamber for 849 MHz or 1763 MHz of mobile phone RF radiation to expose RF to the heads of C57BL mice. In this chamber, animals were irradiated intermittently at 7.8 W/kg for a maximum of 12 months. During this period, the body weights of 3 groups-sham, 849 MHz RF, and 1763 MHz RF-did not show any differences between groups. The brain tissues were obtained from 3 groups at 6 months and 12 months to examine the differences in histology and cell proliferation between control and RF exposure groups, but we could not find any change upon RF radiation. Likewise, we could not find changes in the expression and distribution of NeuN and GFAP in hippocampus and cerebellum, or in cell death by TUNEL assay in RF exposure groups. From these data, we conclude that the chronic exposure to 849 MHz and 1763 MHz RF radiation at a 7.8 W/kg specific absorption rate (SAR) could not induce cellular alterations such as proliferation, death, and reactive gliosis.

A screening system comprised of a randomized hybrid-ribozyme library has previously been used to identify pro-death genes in Fas-mediated apoptosis, and short sequence information of candidate genes from this system was previously reported by Kawasaki and Taira [H. Kawasaki, K. Taira, A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries, Nucleic Acids Res. 30 (2002) 3609-3614]. In this study, we have cloned the full-length of the candidate's open reading frames and found that one of the candidates, referred to as MUDENG (Mu-2 related death-inducing gene), which is composed of 490 amino acids that contain the adaptin domain found in the mu2 subunit of APs related to clathrin-mediated endocytosis, is able to induce cell death by itself. Ectopic expression of MUDENG induced cell death in Jurkat T cells and HeLa cells. In addition, when MUDENG expression was evaluated by immnuohistochemical staining, it was found in most tissues, including the intestine and testis. Furthermore, MUDENG appears to be evolutionary conserved from mammals to amphibians, suggesting that it may have a common role in cell death. Taken together, these results suggest that MUDENG is likely to play an important role in cell death in various tissues.

Acetylcholinesterase (AChE) is emerging as an important component in leading to apoptosis. Our previous study demonstrated that silencing of the AChE gene blocked the interaction between cytochrome c and apoptotic protease-activating factor-1 (Apaf-1) in etoposide-induced apoptosis of HT-29 cells. We undertook this study to further dissect the molecular role of AChE in apoptosome formation. The present study elicited that small interfering RNA (siRNA) to cytochrome c gene blocked the interaction of AChE with Apaf-1, whereas siRNA to Apaf-1 gene did not block the interaction of AChE with cytochrome c, indicating that the interaction of AChE with cytochrome c is required for the interaction between cytochrome c and protease-activating factor-1. We further observed that AChE is localized to caveolae via interacting with caveolin-1 during apoptosis and that the disruption of caveolae prevented apoptosome formation. These data indicate that the interactions of AChE with caveolin-1 and subsequently with cytochrome c appear to be indispensable for apoptosome formation.

Purpose: This study was undertaken to develop a new treatment modality that would be able to minimize fibrosis and provide better outcome with glaucoma filtration surgery (GFS)., Methods: We examined whether co-treatment with mitomycin-C (MMC) and histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) efficiently induces apoptosis on rabbit Tenon's capsule fibroblasts (TCF) in vitro. We further examined the effect of co-treatment with SAHA and MMC on the alteration of IOP and the bleb survival in rabbits following GFS., Results: Co-treatment of MMC and SAHA efficiently induces apoptosis in TCFs via the up-regulation of p53 and increased phosphorylation of p53 on serine 15 and 392. Also, co-treatment of SAHA and low-dose MMC decreases IOP, prolongs bleb survival, and induces apoptosis of cells under the bleb area following GFS., Conclusion: This study shows that a co-treatment of SAHA and MMC could improve the outcome of GFS.

Apoptosis is considered to be a programmed and controlled mode of cell death, whereas necrosis has long been described as uncontrolled and accidental cell death resulting from extremely harsh conditions. In the following review, we will discuss the features and physiological meanings as well as recent advances in the elucidation of the signaling pathways of both apoptotic cell death and programmed necrotic cell death.

Although momilactone B has been studied as an allelochemical of rice (Oryza sativa L.), to date we have no report showing the effect of momilactone B on mammalian cells. This study was undertaken to examine whether this allelochemical has anticancer activity on cancer cells. We show here that momilactone B at micromolar doses has antitumor efficacy by inducing apoptosis in several blood cancer cells including human leukemic T cells. In addition, our study elucidated that anticancer activity of momilactone B on human leukemic T cells resulted from the induction of apoptosis via caspase and mitochondria. From these results, momilactone B can be considered as a novel therapeutic strategy for human leukemic T cells from its direct apoptosis-inducing activity.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family and a potent inducer of apoptosis. Gemcitabine, chemotherapeutic agent is well known to be involved in synergistic cytotoxic effect with TRAIL. But the mechanism of this synergistic effect is still unclear. To examine the effects and synergistic mechanism of gemcitabine on TRAIL-induced apoptosis, A549, HCT116 and SNU638 cells were pretreated with gemcitabine and treated with TRAIL protein. Gemcitabine significantly enhanced A549 cell death induced by TRAIL, but it was inhibited by the pan-caspase inhibitor, Z-VAD-fmk. The combined treatment of both induced the activation of caspase-8 and reduced the mitochondrial transmembrane potential (MTP) and also translocated Bax protein and released the cytochrome-c in A549 cells when compared with that of negative control. In addition, the gemcitabine pretreatment up-regulated DR-5 and p53 protein expression in a time-dependent manner, which suggests the possible involvement of the p53 protein as a transcriptional factor for DR-5 up-regulation. Thus, we report our findings that gemcitabine enhanced the TRAIL-induced apoptosis and the apoptotic signals are mediated by DR-5-dependent pathway and mitochondrial pathway. Taken together, gemcitabine enhanced TRAIL-induced apoptosis via DR-5 up-regulation and lowering MTP, and suggest that gemcitabine may be used as a successful chemotherapeutic agent for ligand type tumor therapy combined with TRAIL.

Melanoma is an intractable tumor that has shown very impressive and promising response to local administration of high dose recombinant TNF-alpha in combination with IFN-gamma in clinical studies. In this study, we investigated the effect of IL-6/sIL-6R on TNF-alpha-resistant B16/F10.9 melanoma cells. A low dose of TNF-alpha or IL-6/sIL-6R had minimal affect on the cell growth. However, the highly active fusion protein of sIL-6R and IL-6 (IL6RIL6), covalently linked by a flexible peptide, sensitized TNF-alpha-resistant F10.9 melanoma cells to TNF-alpha-induced apoptosis. Stimulation of the cells with IL6RIL6 plus TNF-alpha resulted in both the activation of caspase-3 and the reduction of bcl-2 expression. Flow cytometry analysis showed that IL6RIL6-upregulated TNF-R55 and TNF-R75 expression, suggesting an increase in TNF-alpha responsiveness by IL6RIL6 resulting from the induction of TNF receptors. Moreover, exposure of F10.9 cells to neutralizing antibody to TNF-R55 significantly inhibited IL6RIL6/TNF-alpha-induced cytotoxicity. These results suggest that the IL6/sIL6R/gp130 system, which sensitizes TNF-alpha-resistant melanoma cells to TNF-alpha-induced apoptosis, may provide a new target for immunotherapy.

In a preliminary study, we found that benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD- fmk), unlike Boc-aspartyl(OMe)-fluoromethylketone (BocD-fmk), at usual dosage could not prevent genistein-induced apoptosis of p815 mastocytoma cells. This study was undertaken to reveal the mechanism underlying the incapability of zVAD-fmk in preventing this type of apoptosis. We observed that 14-3-3 protein level was reduced in genistein-treated cells and that BocD-fmk but not zVAD-fmk prevented the reduction of 14-3-3 protein level and the release of Bad from 14-3-3. We also demonstrated that truncated Bad to Bcl-xL interaction in genistein- treated cells was prevented by BocD-fmk but not by zVAD-fmk treatment. Our data indicate that BocD- fmk, compared to zVAD-fmk, has a certain preference for inhibiting 14-3-3/Bad signalling pathway. We also elucidated that this differential efficacy of BocD-fmk and zVAD-fmk resulted from the different effect in inhibiting caspase-6 and that co-treatment of zVAD-fmk and caspase-6 specific inhibitor substantially prevented genistein-induced apoptosis. Our data shows that caspase-6 plays a role on Bad/14-3-3 pathway in genistein-induced apoptosis of p815 cells, and that the usual dose of zVAD-fmk, in contrast to BocD-fmk, did not prevent caspase-6 acting on 14-3-3/Bad-mediated event.