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Previous studies have investigated the inhibitory effect of BMI-1026 on cyclin-dependent kinase 1 in vitro. However, the molecular mechanisms by which BMI-1026 treatment leads to cancer cell death remain unclear. This study was conducted to investigate the anticancer mechanisms of BMI-1026 on human renal carcinoma Caki cells. BMI-1026 induced apoptosis in association with the cleavage of poly(ADP-ribose) polymerase and pro-caspase-3 and the release of apoptosis-inducing factor and cytochrome c from mitochondria in Caki cells. BMI-1026-induced apoptosis was inhibited by the pan-caspase inhibitor z-VAD-fmk. Furthermore, BMI-1026 downregulated Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP) at the transcriptional level and Mcl-1 (L) and cellular FADD-like IL-1β-converting enzyme inhibitory protein (c-FLIP (L)) at the post-transcriptional level. Interestingly, Mcl-1 (L) and c-FLIP (L), but not Bcl-2 or XIAP, played important roles in BMI-1026-induced Caki cell apoptosis. Although the constitutively active form of Akt did not attenuate BMI-1026-induced apoptosis, blockade of the PI3K/Akt pathway using a subcytotoxic concentration of the PI3K/Akt inhibitor LY294002 enhanced Caki cell apoptosis induced by BMI-1026. Electrophysiological safety was confirmed by determining the cardiotoxicity of BMI-1026 via left ventricular pressure analysis. These results suggest that BMI-1026 is a potent multitarget anticancer agent with electrophysiological safety and should be further investigated.

R428, a selective small molecule Axl inhibitor, is known to have anti-cancer effects, such as inhibition of invasion and proliferation and induction of cell death in cancer cells. The Axl receptor tyrosine kinase is highly expressed in cancer cells and the level of Axl expression is associated with survival, metastasis, and drug resistance of many cancer cells. However, the effect of Axl inhibition on overcoming anti-cancer drugs resistance is unclear. Therefore, we investigated the capability of Axl inhibition as a therapeutic agent for the induction of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) sensitivity. In this study, R428 markedly sensitized cancer cells to TRAIL-induced apoptotic cell death, but not in normal human skin fibroblast (HSF) and human umbilical vein cells (EA.hy926). Moreover, knockdown of Axl by siRNA also increased TRAIL-induced apoptosis. R428 decreased c-FLIP proteins levels via induction of miR-708 expression and survivin protein levels at the post-translational level, and we found that knockdown of Axl also decreased both c-FLIP and survivin protein expression. Overexpression of c-FLIP and survivin markedly inhibited R428 plus TRAIL-induced apoptosis. Furthermore, R428 sensitized cancer cells to multiple anti-cancer drugs-mediated cell death. Our results provide that inhibition of Axl could improve sensitivity to TRAIL through downregulation of c-FLIP and survivin expression in renal carcinoma cells. Taken together, Axl may be a tempting target to overcome TRAIL resistance.

Garcinol is a polyisoprenylated benzophenone derived from the Garcinia indica fruit that possess potential therapeutic effects such as inhibition of inflammation and tumor expansion. Here, we investigated whether garcinol induces TRAIL sensitization in renal carcinoma cells. Single treatment with garcinol or TRAIL did not effect on apoptosis. However, combined treatment with garcinol plus TRAIL significantly induced apoptosis in renal carcinoma (Caki, ACHN and A498), lung carcinoma (A549), and hepatoma (SK-Hep1) cells. In contrast, garcinol plus TRAIL did not alter cell viability in normal cells. Garcinol plus TRAIL induced up-regulation of DR5 and down-regulation of c-FLIP expression at post-translational levels. Furthermore, knock-down of DR5 by siRNA and ectopic expression of c-FLIP blocked apoptotic cell death induced by garcinol plus TRAIL. Overall, our study provides evidence that garcinol can be exploited as a potential TRAIL sensitizer.

Formaldehyde (FA) is a familiar indoor air pollutant found in everything from cosmetics to clothing, but its impact on the middle ear is unknown. This study investigated whether FA causes cytotoxicity, inflammation, or induction of apoptosis in human middle ear epithelial cells (HMEECs). Cell viability was investigated using the trypan blue assay and a cell counting kit (CCK-8) in HMEECs treated with FA for 4 or 24 h. The expression of genes encoding the inflammatory cytokine tumor necrosis factor alpha (TNF- α ) and mucin (MUC5AC) was analyzed using RT-PCR. Activation of the apoptosis pathway was determined by measuring mitochondrial membrane potential (MMP), cytochrome oxidase, caspase-9/Mch6/Apaf 3, and Caspase-Glo® 3/7 activities. The CCK-8 assay and trypan blue assay results showed a reduction in cell viability in FA-treated HMEECs. FA also increased the cellular expression of TNF- α and MUC5AC and reduced the activities of MMP and cytochrome oxidase. Caspase-9 activity increased in cells stimulated for 4 h, as well as caspase-3/7 activity in cells stimulated for 24 h. The decreased cell viability, the induction of inflammation and mucin gene expression, and the activation of the apoptosis pathway together indicate a link between environmental FA exposure and the development of otitis media.

Docosahexaenoic acid (DHA), a component of omega-3 fatty acids, has been reported to protect against inflammatory bone diseases such as osteoporosis and rheumatoid arthritis. However, its exact mechanism in bone resorbing cells has not been elucidated. In this study, we investigated the effects and the molecular mechanism of DHA on the proliferation, differentiation, and survival of osteoclast lineage cells using mouse bone marrow-derived macrophages (BMMs). DHA suppressed the macrophage colony-stimulating factor (M-CSF)-induced proliferation of osteoclast precursors, BMMs, in a dose-dependent manner. The attenuated proliferation of DHA-treated BMMs was related to M-CSF inhibition that selectively decreased Akt activation and downregulated cyclin D1 and cyclin D2 expression. DHA also blocked receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation from BMMs. At the molecular level, DHA inhibited JNK, ERK, and p38 MAPKs. In addition, it inhibited NF-κB signaling cascades, as demonstrated by the suppression of RANKL-mediated IκBα phosphorylation, NF-κB subunit p65 nuclear translocation, and NF-κB transcriptional activation. Accordingly, DHA attenuated the induction of c-Fos and nuclear factor of activated T cells c1 (NFATc1). Furthermore, DHA accelerated the apoptosis of mature osteoclasts by inducing Bim expression, a critical modulator of osteoclast apoptosis. Collectively, our data demonstrate that DHA exerts an anti-osteoclastogenic effect by suppressing the proliferation and differentiation of BMMs and enhancing the apoptosis of mature osteoclasts, thereby resulting in a diminished number of bone-resorptive cells., (Copyright © 2016. Published by Elsevier Inc.)

Previous studies have demonstrated the anticancer effects of the newly developed cyclin-dependent kinase inhibitor BAI in various cancer cells. However, the molecular mechanisms of the cellular effects induced by BAI have not been fully elucidated. The objective of this study was to investigate the mechanisms underlying the regulation of B cell lymphoma-2 (Bcl-2) family proteins in BAI-induced apoptosis of cancer cells. BAI induced poly(ADP-ribose) polymerase cleavage and DEVDase activation dose- and time-dependently. However, BAI-induced apoptosis was not involved in reactive oxygen species generation or mitogen-activated protein kinases pathways. On the other hand, BAI reduced the mitochondrial membrane potential (∆ψm) dose- and time-dependently, and induced the release of apoptosis-inducing factor (AIF) and cytochrome c from mitochondria in A549 and Caki cells. Furthermore, BAI-induced apoptosis was strongly associated with downregulation of B-cell lymphoma-extra large (Bcl-xL), but not Bcl-2, and BAI modulated the interactions among p53 and Bcl-2 family proteins in human cancer cells. Taken together, these results revealed that the regulations of Bcl-2 family proteins are correlated with BAI-induced apoptosis, suggesting that BAI is a potential multi-target agent of cancer.

RU486 (Mifepristone) is known as an antagonist of the progesterone receptor and glucocorticoid receptor. Here, we investigated the mechanism underlying anti-tumor activity of RU486 in renal carcinoma Caki cells. Treatment of Caki cells with RU486 was found to induce several signature ER stress markers; including ER stress-specific XBP1 splicing, and the up-regulation of glucose-regulated protein (GRP)-78 and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. RU486-induced expression of CHOP involves the putative C/EBPδ site within the CHOP promoter region. Using a combination of C/EBPδ cDNA transfection, the luciferase assay with a mutated C/EBPδ binding site and siRNA-mediated C/EBPδ knockdown, we found that the C/EBPδ site is required for RU486-mediated activation of the CHOP promoter. In addition, RU486-induced CHOP expression is down-regulated by inhibition of the p38 MAPK and JNK signaling pathways at the post-translational levels. RU486 dose-dependently induced apoptotic cell death in renal carcinoma cells. Suppression of CHOP expression by CHOP siRNA attenuated RU486-induced apoptosis. Taken together, RU486 induces pro-apoptotic ER stress through the induction of CHOP expression., (© 2015 Wiley Periodicals, Inc.)

Study Design: Animal study., Objective: To further investigate the effects of therapeutic hypothermia (TH), the present study compared autophagy and apoptosis after treatment with either therapeutic moderate systemic hypothermia or methylprednisolone sodium succinate (MP) in a rat model of acute spinal cord injury (SCI)., Summary of Background Data: The neuroprotective effects of TH have recently become an important topic in the field of SCI research., Methods: All rats were subjected to a 25-g/cm spinal cord contusion over the ninth thoracic vertebrae. After the induction of SCI, the control group did not receive any further treatment, TH group immediately received moderate systemic hypothermia for 4 hours, and MP group was administered high-dose MP. The rats were killed either 2 or 7 days after SCI, and the injured spinal cord tissues were obtained. Apoptosis and autophagy were assessed by immunohistochemical analyses and Western blot analyses. In addition, the microarchitecture of the autophagosomes was evaluated using transmission electron microscopy, and the motor activity of the rats was assessed using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale., Results: Compared with controls, there was a significant reduction in the expression levels of cleaved caspase-8, -9, and -3 in the TH- and MP-treated groups 2 days after SCI. Moreover, compared with the control group, the expression of LC3II and Beclin-1 exhibited a significant decrease on day 2 after treatment with TH. The numbers of transferase dUTP nicked-end labeling and LC3-positive cells were significantly lower on days 2 and 7. The Basso-Beattie-Bresnahan ratings were significantly higher 6 weeks after SCI in both the TH- and MP-treated groups than in the control group., Conclusion: Both TH and MP have neuroprotective effects on injured spinal cord tissues via the inhibition of apoptosis and autophagy. Thus, the application of moderate systemic hypothermia may be a useful treatment modality after acute SCI., Level of Evidence: N/A.

6-Shogaol, a potent bioactive compound in ginger (Zingiber officinale Roscoe), has been reported for anti-inflammatory and anti-cancer activity. In this study, we investigated the effect of 6-shogaol to enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. The combined treatment with 6-shogaol and TRAIL markedly induces apoptosis in various cancer cells (renal carcinoma Caki cells, breast carcinoma MDA-MB-231 cells and glioma U118MG cells), but not in normal mesangial cells and normal mouse kidney cells. 6-Shogaol reduced the mitochondrial membrane potential (MMP) and released cytochrome c from mitochondria to cytosol via Bax activation. Furthermore, we found that 6-shogaol induced down-regulation of c-FLIP(L) expression at the post-translational levels and the overexpression of c-FLIP(L) markedly inhibited 6-shogaol plus TRAIL-induced apoptosis. Moreover, 6-shogaol increased reactive oxygen species (ROS) production in Caki cells. Pretreatment with ROS scavengers attenuated 6-shogaol plus TRAIL-induced apoptosis through inhibition of MMP reduction and down-regulation of c-FLIP(L) expression. In addition, 6-gingerol, another phenolic alkanone isolated from ginger, did not enhance TRAIL-induced apoptosis and down-regulate c-FLIP(L) expression. Taken together, our results demonstrated that 6-shogaol enhances TRAIL-mediated apoptosis in renal carcinoma Caki cells via ROS-mediated cytochrome c release and down-regulation of c-FLIP(L) expression., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Despite recent advances in therapeutic strategies for lung cancer, mortality still is increasing. In the present study, we investigated the anti-cancer effects of KMU-193, 2-(4-Ethoxy-phenyl)-N-{5-[2-fluoro-4-(4-methyl- piperazine-1-carbonyl)-phenylamino]-1H-indazol-3-yl}-acetamide in a human non-small cell lung cancer cell line A549. KMU-193 strongly inhibited the proliferation of A549 cells, but it did not have anti-proliferative effect in other types of cancer cell lines. KMU-193 further induced apoptosis in association with activation of caspase-3 and cleavage of PLC-γ1. However, KMU-193 had no apoptotic effect in untransformed cells such as TMCK-1 and BEAS-2B. Interestingly, pretreatment with z-VAD-fmk, a pan-caspase inhibitor, strongly abrogated KMU- 193-induced apoptosis. KMU-193 treatment enhanced the expression levels of p53 and PUMA. Importantly, p53 siRNA transfection attenuated KMU-193-induced apoptosis. Collectively, these results for the first time demonstrate that KMU-193 has strong apoptotic effects on A549 cells and these are largely mediated through caspase-3- and p53-dependent pathways.

Accumulative evidence suggests ginseng extract and/or its major components, ginsenosides and compound K, a metabolized ginseng saponin, have anti-cancer effects. In the present study, the effects of a ginseng butanolic extract (GBX) and an enzymatically fortified ginseng extract (FGX), with enriched ginsenosides and compound K, on the growth of KATO3 human gastric cancer cells were investigated using a cell viability assay. While treatment with GBX at 31.25-125 mg/ml for 24 h did not affect the proliferation of KATO3 cells, FGX under the same conditions inhibited cell proliferation in a concentration-dependent manner. Furthermore, Annexin V/PI-staining and flow cytometric analysis demonstrated that the population of apoptotic KATO3 cells was increased following treatment with FGX, which was greater than in the GBX-treated cells, suggesting that FGX had a stronger apoptotic effect than GBX. To investigate the underlying mechanism of the cytostatic and cytotoxic effects of the ginseng extracts, apoptosis-associated proteins were assessed using western blot analysis. The data revealed higher expression levels of B-cell lymphoma 2-associated X protein (Bax), lower expression of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) and reduced phosphorylation of mammalian target of rapamycin (mTOR) and protein kinase B (PKB) in the FGX-treated KATO3 cells than in the GBX-treated cells. Collectively, these results demonstrated for the first time, to the best of our knowledge, that FGX had stronger anti-proliferative and pro-apoptotic effects on KATO3 cells than GBX. The anti-proliferative and/or pro-apoptotic effects of FGX appeared to be mediated via the upregulation of Bax, IκBα proteolysis (activation of nuclear factor-κB) and the blocking of mTOR and PKB signals.

Introduction: Cisplatin is a widely used anticancer chemotherapeutic agent. However, it is notorious for its ototoxicity and nephrotoxicity due to induction of reactive oxygen species (ROS). Caffeic acid is a naturally occurring polyphenol present in honey that is known to reduce the generation of oxygen-derived free radicals. The objective of the present study was to evaluate the protective effects and mechanism underlying the effect of caffeic acid on cisplatin-induced ototoxicity in HEI-OC1 auditory cell lines., Methods: Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was determined by Hoechst 33258 staining and Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Cell cycle stages were analyzed by flow cytometry. The radical-scavenging activity of caffeic acid was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The expression levels of caspase-3, -8, and -9, as well as the activity of caspase-3, were evaluated., Results: Caffeic acid showed a protective effect against cisplatin-induced HEI-OC1 cell damage as demonstrated by the MTT assay. Caffeic acid decreased cell death by apoptosis and necrosis. Caffeic acid showed strong scavenging activity against the radical DPPH and decreased intracellular ROS production. Caffeic acid decreased the expression of caspase-3 and -8 and increased the activity of caspase-3., Conclusions: Caffeic acid attenuated cisplatin-induced hair cell loss in HEI-OC1 cell lines; these effects were mediated by its radical scavenging activity and inhibition of apoptosis., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The inhibition of topoisomerase can suppress the growth of cancer cells and induce apoptosis. The aim of this study was to evaluate the anticancer effects and mechanisms of action of a novel topoisomerase inhibitor, 4-(furan-2-yl)-2-(pyridin-2-yl)-5,6-dihydro-1,10-phenanthroline (FPDHP). FPDHP suppressed the growth of Caki, A549, HT29 and MDA-MB-231 cells, and induced caspase-dependent apoptosis in the Caki cells. In particular, FPDHP also induced caspase-dependent apoptosis and the downregulation of the protein expression levels of cellular FLICE-like inhibitory protein (cFLIP) and the phosphorylation of Akt in Caki cells. Notably, the overexpression of cFLIP, but not that of Akt, in part, blocked the FPDHP-mediated apoptosis in Caki cells. In addition, FPDHP was further shown to induce the caspase-independent detachment of Caki cells from the culture dish; higher populations of apoptotic cells were observed in the detached cells than in the attached cells. To the best of our knoweledge, these results collectively demonstrate for the first time that FPDHP has a killing effect on Caki cells, which is mediated through both caspase-dependent apoptosis and caspase-independent cell detachment.

Silibinin, an effective anti-cancer and chemopreventive agent, has been shown to exert multiple effects on cancer cells, including inhibition of both cell proliferation and migration. However, the molecular mechanisms responsible for these effects are not fully understood. We observed that silibinin significantly induced the expression of the non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in both p53 wild-type and p53-null cancer cell lines, suggesting that silibinin-induced NAG-1 up-regulation is p53-independent manner. Silibinin up-regulates early growth response-1 (EGR-1) expression. The ectopic expression of EGR-1 significantly increased NAG-1 promoter activity and NAG-1 protein expression in a dose-dependent manner. Furthermore, down-regulation of EGR-1 expression using siRNA markedly reduced silibinin-mediated NAG-1 expression, suggesting that the expression of EGR-1 is critical for silibinin-induced NAG-1 expression. We also observed that reactive oxygen species (ROS) are generated by silibinin; however, ROS did not affect silibinin-induced NAG-1 expression and apoptosis. In addition, we demonstrated that the mitogen-activated protein kinase (MAP kinase) signal transduction pathway is involved in silibinin-induced NAG-1 expression. Inhibitors of p38 MAP kinase (SB203580) attenuated silibinin-induced NAG-1 expression. Furthermore, we found that siRNA-mediated knockdown of NAG-1 attenuated silibinin-induced apoptosis. Collectively, the results of this study demonstrate for the first time that up-regulation of NAG-1 contributes to silibinin-induced apoptosis in cancer cells., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Anisomycin is known to inhibit protein synthesis and induce ribotoxic stress. In this study, we investigated whether anisomycin treatment could modulate TRAIL-mediated apoptosis in human renal carcinoma Caki cells. We found that anisomycin treatment (10-15 nM) alone had no effect on the level of apoptosis, but a combination treatment of anisomycin and TRAIL significantly increased the level of apoptosis in human renal carcinoma (Caki, ACHN and A498), human glioma (U251MG), and human breast carcinoma (MDA-MB-361 and MCF7) cells. Anisomycin treatment led to the down-regulation of Bcl-2 expression at the transcriptional level, and the over-expression of Bcl-2 inhibited the apoptosis induced by the combination treatment of anisomycin and TRAIL. Furthermore, anisomycin treatment resulted in the down-regulation of c-FLIP(L) and Mcl-1 at the post-transcriptional level, and the over-expression of c-FLIP(L) and Mcl-1 blocked the induction of apoptosis caused by the combination treatment of anisomycin with TRAIL. In contrast, anisomycin treatment had no effect on the levels of TRAIL-mediated apoptosis in mouse kidney cells (TMCK-1) or normal human skin fibroblasts (HSF). Cumulatively, our study demonstrates that anisomycin treatment enhances TRAIL-mediated apoptosis through the down-regulation of Bcl-2, c-FLIP(L) and Mcl-1 at the transcriptional or post-transcriptional level., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Helenalin, a sesquiterpene lactone, exhibits anti-inflammatory and anti-tumor activities. Here, we investigated whether helenalin could induce apoptosis in human renal carcinoma Caki cells. Helenalin increased apoptosis in dose dependent manner in Caki cells, and also induced apoptosis in other carcinoma cells, such as human renal carcinoma ACHN cells, human colon carcinoma HT29 and HCT116 cells. We found that helenalin markedly induced endoplasmic reticulum (ER) stress-related genes, such as regulated in development and DNA damage responses (REDD) 1, activating transcription factor-4 (ATF4) and/or the CCAAT enhancer-binding protein-homologous protein (CHOP). However, down-regulation of ATF4 and/or CHOP expression by siRNA had no effect on helenalin-induced apoptosis in Caki and HCT116 cells. Helenalin increased production of intracellular reactive oxygen species (ROS). Furthermore, ROS scavengers, N-acetylcystine (NAC), and glutathione ethyl ester (GEE), reduced helenalin-induced apoptosis. Taken together, helenalin induced apoptosis via ROS generation in human renal carcinoma Caki cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Previously, we have synthesized a novel cyclin-dependent kinase (CDK) inhibitor, 2-[1,1'biphenyl]-4-yl-N-[5-(1,1-dioxo-1λ(6) -isothiazolidin-2-yl)-1H-indazol-3-yl]acetamide (BAI) and reported its anti-cancer activity in head and neck cancer cells. In this study, we further evaluated the effect of BAI on growth of various human cancer cell lines, including A549 (nonsmall cell lung cancer), HCT116 (colon), and Caki (kidney). Profoundly, results of XTT and clonogenic assays demonstrated that BAI at nanomolar concentrations (20-60 nM) inhibited growth of A549, HCT116, and Caki cells, suggesting the anti-cancer potency. We show that BAI induced a dose-dependent apoptotic cell death in these human cancer cells, as measured by fluorescence-activated cell sorting (FACS). Interestingly, further biochemical analysis showed that treatment with BAI at 20 nM induced apoptosis in A549 cells in association with activation of caspases, cleavage of phospholipase C-γ1 (PLC-γ1), and inhibition of Akt in A549 cells. Importantly, pharmacological inhibition study revealed that pretreatment with z-VAD-fmk, a pan caspase inhibitor strongly blocked the BAI-induced apoptosis in A549 cells. Transfection analysis with Akt cDNA encoding constitutively active Akt further addressed the significance of Akt inhibition in the BAI-induced apoptosis in A549 cells. Notably, disruption of the PI3K/Akt pathway by LY294002, a PI3K/Akt inhibitor potentiated apoptosis in A549 cells by BAI at a subcytotoxic concentration. These findings collectively suggest that BAI potently inhibits growth of A549, HCT116, and Caki cells, and that the BAI-induced apoptosis in A549 cells is associated with activation of caspases, and inhibition of Akt., (Copyright © 2012 Wiley Periodicals, Inc.)

cFLIP inhibits caspase 8 recruitment and processing at the death-inducing signaling complex (DISC), which is known to inhibits apoptosis mediated by death receptors such as Fas and death receptor 5 (DR5) as well as apoptosis mediated by anticancer therapeutic drugs. We observed that oxaliplatin induced apoptosis, the activation of DEVDase activity, DNA fragmentation, and cleavage of PLC-gamma1 and degradation of XIAP protein in dose-dependent manners, which was prevented by pretreatment with z-VAD or NAC, suggesting that oxaliplatin-induced apoptosis was mediated by caspase- or reactive oxygen species (ROS)-dependent pathways. Furthermore, ectopic expression of cFLIPs potently attenuated oxaliplatin-induced apoptosis, whereas cFLIP(L) had less effect. Interestingly, we found that the protein level of XIAP was sustained in oxaliplatin-treated cFLIPs overexpressing cell, which was caused by the increased XIAP protein stability and that the phospho-Akt level was high compared to vector-transfected cell. The increased XIAP protein stability was lessened by PI3K inhibitor LY294002 treatment in cFLIPs overexpressing cells. Thus, our findings imply that the anti-apoptotic functions of cFLIPs may be attributed to inhibit oxaliplatin-induced apoptosis through the sustained XIAP protein level and Akt activation.

Sanguinarine is a benzophenanthridine alkaloid derived from the root of Sanguinaria canadensis and other poppy-fumaria species, possessing potent antibacterial, antifungal, and anti-inflammatory activities. In this study, we investigated the underling mechanisms by which sanguinarine induce apoptosis in human breast cancer MDA-231 cells. Treatment of MDA-231 cells with sanguinarine induced remarkable apoptosis accompanying the generation of ROS. Consistently, sanguinarine-induced apoptosis was mediated by the increased reproductive cell death. Pretreatment with NAC or GSH attenuated sanguinarine-induced apoptosis, suggesting the involvement of ROS in this cell death. During sanguinarin-induced apoptosis, protein levels of pro-caspase-3, Bcl-2, cIAP2, XIAP, and c-FLIPs were reduced. Sanguinarine-mediated apoptosis was substantially blocked by ectopic expression of Bcl-2 and cFLIPs. Additionally, we found that sub-lethal doses of sanguinarine remarkably sensitized breast cancer cells to TRAIL-mediated apoptosis, but the cell death induced by sanguinarine and TRAIL in combination was not blocked by overexpression of Bcl-2 or Akt. Therefore, combinatory treatment of sanguinarine and TRAIL may overcome the resistance of breast cancer cells due to overexpression of Akt or Bcl-2.

Mithramycin A is a DNA-binding antitumor agent, which has been clinically used in the therapies of several types of cancer and Paget's disease. In this study, we investigated the combined effect of mithramycin A and tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) on apoptosis of cancer cells. In Caki renal cancer cells, which are resistant to TRAIL, cotreatment with subtoxic doses of mithramycin A and TRAIL resulted in a marked increase in apoptosis. This combined treatment was also cytotoxic to Caki cells overexpressing Bcl-2 but not to normal mesengial cells. Moreover, apoptosis by the combined treatment with mithramycin A and TRAIL was dramatically induced in various cancer cell types, thus offering an attractive strategy for safely treating malignant tumors. Mithramycin A-stimulated TRAIL-induced apoptosis was blocked by pretreatment with the broad caspase inhibitor zVAD-fmk or Crm-A overexpression, showing its dependence on caspases. We found that mithramycin A selectively down-regulated XIAP protein levels in various cancer cells. Luciferase reporter assay and the chromatin immunoprecipitation assay using the XIAP promoter constructs show that mithramycin A down-regulates the transcription of XIAP gene through inhibition of Sp1 binding to its promoter. Although XIAP overexpression significantly attenuated apoptosis induced by mithramycin A plus TRAIL, suppression of XIAP expression by transfection with its small interfering RNA prominently enhanced TRAIL-induced apoptosis. We present here for the first time that mithramycin A-induced suppression of XIAP transcription plays a critical role in the recovery of TRAIL sensitivity in various cancer cells.

Evodiamine is one of the major bioactive compounds that have been isolated and purified from the fruit of Evodiae fructus. Evodiamine exhibits antitumor activities against the human tumor cells, including multidrug-resistant tumor cells. However, the molecular mechanism involved in cell death induced by evodiamine treatment remains poorly understood. In the present study, we showed that evodiamine activated the caspase-dependent apoptotic pathway. This apoptosis was only partially inhibited by a pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, which suggested that evodiamine-induced apoptosis in leukemic U937 cells is partially caspase independent. We observed the nuclear translocation of apoptosis-inducing factor in evodiamine-induced apoptosis of U937 cells, which may be responsible for the caspase-independent apoptotic execution. We next showed that evodiamine induced the substantial amount of apoptosis both in Bcl-2- and Akt-overexpressing U937 cells but not in human peripheral blood mononuclear cells. Although benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone inhibited caspase activity in Bcl-2-overexpressing U937 cells, it completely prevented neither the induction of apoptosis or the nuclear translocation of apoptosis-inducing factor, which suggests that evodiamine is, at least in part, able to bypass the resistance of leukemia cells via caspase-independent apoptotic pathways. Thus, therapeutic strategy using evodiamine may warrant further evaluation.

Quercetin, a natural product derived from grapes, has been shown to prevent carcinogenesis in murine models. We report here that quercetin induces anti-proliferation and arrests G2/M phase in U937 cells. The G2/M phase accumulation was accompanied by an increase in the level of the cyclin B. In contrast, the level of the cyclin D, cyclin E, E2F1, and E2F2 was marked decreased in quercetin-treated U937 cells. Removal of quercetin from the culture medium stimulates U937 cells to synchronously re-enter the cell cycle, decrease expression level of cyclin B, and increased the expression level of cyclin D and cyclin E. These data demonstrate that quercetin causes reversible G2/M phase arrest, which was related with dramatic changes in the level of cyclin B, cyclin D, and cyclin E. Quercetin-induced down-regulation of cyclin D and cyclin E was associated with suppression of transcriptional levels but not protein stability. In addition, quercetin-treated U937 cells showed DNA fragmentation, increased sub-G1 population, and generated a 60kDa cleavage product of PLC-gamma1 in a dose-dependent manner, which were significantly inhibited by z-VAD-fmk. These data clearly indicate that quercetin-induced apoptosis is associated with caspase activation. In summary, the growth inhibition of the quercetin is highly related to cell cycle arrest at the G2/M phase and induction of caspase-dependent apoptosis in human promonocytic U937 cells.

The effects of forced treadmill exercise on cell proliferation and apoptosis in the hippocampal dentate gyrus in Sprague-Dawley rats were investigated. The animals were classified into three groups: the control group, the easy exercise group, and the moderate exercise group. In the control group, rats were left on the treadmill without running for 30 min per day, while rats in the exercise groups were made to run on the treadmill for the same duration. All rats were injected intraperitoneally with 5-bromo-2'-deoxyuridine (BrdU) one-hour prior to exercise once a day for 7 consecutive days beginning at the start of the exercise regimens. Each of the rats was sacrificed 2 h after the last exercise. Both the easy and moderate exercise groups revealed increased number of BrdU-positive cells in the dentate gyrus compared to the control group. The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay revealed very few apoptotic cells, with no statistically significant differences among the groups. These results showed that treadmill exercise increases cell proliferation without altering of apoptosis in the dentate gyrus of the hippocampus.

Abstract: Preclinical Research & Development Angelicin is a furocoumarin derived from Psoralea corylifolia L. fruit that has anti‐inflammatory and anti‐tumor activity. In the present study, the effect of angelicin in enhancing tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐induced apoptotic cell death was studied in Caki (renal carcinoma) cells. Angelicin alone and TRAIL alone had no effect on apoptosis, but in combination these compounds markedly induced apoptosis in the cancer cell lines while not inducing apoptosis in normal cells. The combination treatment induced accumulation of the sub‐G1 population, DNA fragmentation, and activated caspase 3 activity in Caki cells, induced down‐regulation of c‐FLIP expression post‐translationally, and over‐expression of c‐FLIP markedly blocked apoptosis induced by combined treatment with angelicin plus TRAIL. This study provides evidence that angelicin might be a TRAIL sensitizer. [ABSTRACT FROM AUTHOR]

G protein-coupled receptor 120 (GPR120) plays an important role in the regulation of inflammation and lipid metabolism. In this study, we investigated the role of GPR120 in osteoclast development and found that GPR120 regulates osteoclast differentiation, survival and function. We observed that GPR120 was highly expressed in osteoclasts compared to their precursors, bone marrow-derived macrophages (BMMs). Activation of GPR120 by its ligand GW9508 suppressed receptor activator of NF-kB ligand (RANKL)-induced osteoclast differentiation and the expression of nuclear factor of activated T cells c1 (NFATc1), a key modulator of osteoclastogenesis. GPR120 activation further inhibited the RANKL-stimulated phosphorylation of IκBα and JNK. In addition to osteoclast differentiation, GPR120 activation increased the apoptosis of mature osteoclasts by inducing caspase-3 and Bim expression. Activation of GPR120 also interfered with cell spreading and actin cytoskeletal organization mediated by M-CSF but not by RANKL. Coincident with the impaired cytoskeletal organization, GPR120 activation blocked osteoclast bone resorbing activity. Furthermore, knockdown of GPR120 using small hairpin RNA abrogated all these inhibitory effects on osteoclast differentiation, survival, and function. Together, our findings identify GPR120 as a negative modulator of osteoclast development that may be an attractive therapeutic target for bone-destructive diseases. [ABSTRACT FROM AUTHOR]

Purpose: Terminal myelocystocele (TMC) is thought to be caused by a misstep during secondary neurulation. However, due to the paucity of data on secondary neurulation and the rarity of TMC, proofs of this pathogenetic mechanism are unavailable. Based on a previous observation that TMC resembles a step of secondary neurulation in chick, a closer look was taken at secondary neurulation of chick embryos focusing on the cerebrospinal fluid-filled distal neural tube (terminal balloon). Methods: Chick embryos at Hamburger and Hamilton (H-H) stages of 28, 30, 33, 35, 37, and 40 were harvested. Hematoxying-eosin staining, additional immunohistochemistry (laminin, cytokeratin, nestin), and scanning electron microscopy were performed. Results: In H-H stages 28 to 30, after merging of the lumina of the primary and secondary neural tubes, the caudal end of the confluent tube dilates into a balloon-like structure (terminal balloon). As the proximal tube progressively becomes narrower, the terminal balloon dilates even further, and its wall fuses with the surface ectoderm (H-H stage 33). Later in H-H stages 35 to 40, the terminal balloon shrinks and becomes detached from the surface ectoderm and ultimately disappears, as the proximal lumen of the secondary neural tube continues to collapse. Conclusion: A dilated balloon doubtlessly exists in the terminal secondary neural tube in chick embryos, and its subsequent disappearance occurs in a variable time course and sequence. Arrest of apoptosis resulting in failure of detachment of the terminal balloon from the surface ectoderm may well be the basis for human TMC. [ABSTRACT FROM AUTHOR]

We report here that p21WAF1/CIP1, an inhibitor of cyclin kinases, underwent proteolytic processing into a smaller fragment, p14, in the early stage of apoptosis in SK-HEP-1 cells. Apoptosis was induced by either staurosporine or ginsenoside Rh2, a ginseng saponin with a dammarane skeleton. Proteolytic processing was the result of caspase-3 activity, which accompanied the early changes in cell morphology and DNA fragmentation. p21WAF1/CIP1 translated in vitro was cleaved into a p14 fragment when incubated with cell extracts obtained from either ginsenoside Rh2-treated or staurosporine-treated cells. Cleavage was equally inhibited in both cases by adding Ac-DEVD-CHO, a specific caspase-3 inhibitor, but not by Ac-YVAD-CHO, a specific caspase-1 inhibitor. Similarly, p21WAF1/CIP1 was efficiently cleaved by recombinant caspase-3, overexpressed in Escherichia coli. Moreover, the endogenous p21WAF1/CIP1 of untreated cell extracts was also cleaved by recombinant caspase 3, as measured by immunoblotting. Mutation analysis allowed identification of two caspase-3 cleavage sites, DHVD112/L and SMTD149/F, which are located within or near the interaction domains for cyclins, Cdks, and proliferating cell nuclear antigen (PCNA). Taken together, these results show that ginsenoside Rh2 and staurosporine increase caspase-3 activity, which in turn directly cleaves p21WAF1/CIP1 during the early stages of apoptosis. We propose that proteolytic cleavage of p21WAF1/CIP1 is a functionally relevant event that allows release of the cyclin/Cdk complex from the p21WAF1/CIP1 inhibitor, resulting in the elevated levels of cyclin/Cdk kinase activity seen in the earlier stage of apoptosis. [ABSTRACT FROM AUTHOR]

In ginsenoside Rh2-treated rat glioma C6Bu-1 cells, apoptotic morphological changes, such as cell shrinkage, chromatin condensation and pyknosis were confirmed by means of electron microscopy. To evaluate whether induction of apoptosis by ginsenoside Rh2 is mediated by the members of Bcl-2 family, we first established C6Bu-1 cells overexpressing Bcl-2. It was demonstrated that the expression of Bcl-2, Bcl-xL and Bax was not altered in ginsenoside Rh2-treated C6Bu-1 cells. Bcl-2 overexpressing C6Bu-1 cells failed to prevent from ginsenoside Rh2-induced cell death. These results suggest the existence of other apoptotic pathway that requires induction of apoptosis by ginsenoside Rh2 rather than the pathway through Bcl-2, Bcl-xL or Bax in C6Bu-1 cells. [ABSTRACT FROM AUTHOR]

Simple Summary: Dexamethasone (DEX) is commonly used as immunosuppressive and chemotherapeutic agent. The effects of DEX on cell death is different, depending on cell types and stimuli. Here, we found that DEX inhibited tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death in cancer cells. Upregulation of c-FLIP(L) and downregulation of death receptor 5 (DR5) play a critical role in anti-apoptotic effects of DEX in TRAIL-induced apoptosis. DEX upregulated c-FLIP(L) expression at the transcriptional levels through the GSK-3β signaling pathway. Furthermore, DEX also modulated protein stability of DR5 via the GSK-3β/Cbl axis-mediated ubiquitin–proteasome system. Therefore, DEX-induced GSK3β activation plays a critical role in the modulation of c-FLIP(L) and DR5. This finding suggests that DEX reduced effects of anti-cancer drugs in cancer cells. Dexamethasone (DEX), a synthetic glucocorticoid, is commonly used as immunosuppressive and chemotherapeutic agent. This study was undertaken to investigate the effects of DEX on the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in cancer cells. We found that upregulation of c-FLIP(L) and downregulation of death receptor 5 (DR5; receptor for TRAIL ligand) contribute to the anti-apoptotic effect of DEX on TRAIL-induced apoptosis. DEX increased c-FLIP(L) expression at the transcriptional levels through the GSK-3β signaling pathway. The pharmacological inhibitor and catalytic mutant of GSK-3β suppressed DEX-induced upregulation of c-FLIP(L) expression. Furthermore, GSK-3β specific inhibitor markedly abolished DEX-mediated reduction of TRAIL-induced apoptosis in human renal cancer cells (Caki-1 and A498), human lung cancer cells (A549), and human breast cancer cells (MDA-MB361). In addition, DEX decreased protein stability of DR5 via GSK-3β-mediated upregulation of Cbl, an E3 ligase of DR5. Knockdown of Cbl by siRNA markedly inhibited DEX-induced DR5 downregulation. Taken together, these results suggest that DEX inhibits TRAIL-mediated apoptosis via GSK-3β-mediated DR5 downregulation and c-FLIP(L) upregulation in cancer cells. [ABSTRACT FROM AUTHOR]

Maritoclax, an active constituent isolated from marine bacteria, has been known to induce Mcl-1 downregulation through proteasomal degradation. In this study, we investigated the sensitizing effect of maritoclax on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human renal carcinoma cells. We found that combined treatment with maritoclax and TRAIL markedly induced apoptosis in renal carcinoma (Caki, ACHN and A498), lung cancer (A549) and hepatocellular carcinoma (SK-Hep1) cells. The upregulation of death receptor 5 (DR5) and downregulation of cellular FLICE-inhibitory protein (cFLIP) were involved in maritoclax plus TRAIL-induced apoptosis. Maritoclax-induced DR5 upregulation was regulated by induction of C/EBP homologous protein (CHOP) expression. Interestingly, maritoclax induced cFLIP downregulation through the increased expression of miR-708. Ectopic expression of cFLIP prevented combined maritoclax and TRAIL-induced apoptosis. Taken together, maritoclax sensitized TRAIL-induced apoptosis through CHOP-mediated DR5 upregulation and miR-708-mediated cFLIP downregulation. [ABSTRACT FROM AUTHOR]

Withaferin A, a withanolide derived from the medicinal plant Withania somnifera , has been reported to exhibit anti-tumorigenic activity against various cancer cells. In this study, we show that withaferin A inhibits the constitutive and recombinant human growth-arrest-specific protein 6 (rhGas6)-induced phosphorylation of Axl and STAT3. In addition, withaferin A also induces the down-regulation of Axl protein expression in a lysosome-dependent manner and inhibits rhGas6-induced wound healing and cell migration. Furthermore, the overexpression of Axl attenuates withaferin A-induced apoptosis. Taken together, the data from the present study indicate that the withaferin A-mediated down-regulation of the Gas6/Axl signaling pathway mediates the inhibition of cell migration and the induction of apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: Osteoclasts are polykaryons that have the unique capacity to degrade bone. Modulation of osteoclast formation and function is a promising strategy for the treatment of bone-destructive diseases. Here, we report that obovatol, a natural compound isolated from Magnolia obovata, inhibits receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL)-induced osteoclast differentiation in vitro and inflammatory bone loss in vivo. We found that obovatol strongly inhibited osteoclast formation from bone marrow-derived macrophages in a dose-dependent manner without cytotoxicity. Obovatol significantly suppressed RANKL-induced activation of NF-κB, c-Jun-N-terminal kinase, and extracellular signal-regulated kinase signaling pathways. Obovatol also inhibited RANKL-induced expression of the genes c-Fos and nuclear factor of activated T cells c1, which are transcription factors important for osteoclastogenesis. In addition to osteoclast differentiation, obovatol blocked cytoskeletal organization and abrogated the bone resorbing activity of mature osteoclast. Obovatol also accelerated osteoclast apoptosis through the induction of caspase-3 activation. Consistent with its in vitro anti-resorptive effect, obovatol prevented bone loss induced by lipopolysaccharide in vivo. Together, our data suggest that obovatol may be a useful therapeutic agent for the treatment of pathological bone disorders characterized by excessive osteoclastic bone resorption. [Copyright &y& Elsevier]

Abstract: Here, we first demonstrated the neuroprotective effect of acupuncture after SCI. Acupuncture applied at two specific acupoints, Shuigou (GV26) and Yanglingquan (GB34) significantly alleviated apoptotic cell death of neurons and oligodendrocytes, thereby leading to improved functional recovery after SCI. Acupuncture also inhibited caspase-3 activation and reduced the size of lesion cavity and extent of loss of axons. We also found that the activation of both p38 mitogen-activated protein kinase and resident microglia after injury are significantly attenuated by acupuncture. In addition, acupuncture significantly reduced the expression or activation of pro-nerve growth factor, proinflammatory factors such as tumor necrosis factor-α, interleukin-1β, interleukin-6, nitric oxide synthase, cycloxygenase-2, and matrix metalloprotease-9 after SCI. Thus, our results suggest that the neuroprotection by acupuncture may be partly mediated via inhibition of inflammation and microglial activation after SCI and acupuncture can be used as a potential therapeutic tool for treating acute spinal injury in human. [Copyright &y& Elsevier]