Caspase-dependent Cell Death Research Articles

Characterization and cardioprotective activity of anthocyanins from Nitraria tangutorum Bobr. by-products.

The Nitraria tangutorum Bobr. fruit is an indigenous berry of the shrub belonging to the Zygophyllaceae family which grows at an altitude of over 3000 m in the Tibetan Plateau, and has been used as a native medicinal food for treating weakness of the spleen, stomach syndrome, dyspepsia, neurasthenia, dizziness, etc. for thousands of years. Nowadays, N. tangutorum industrial juice by-products generated from health food production can be a potential low cost source of some unique bioactive ingredients. In a prior study, we established a simultaneous microwave/ultrasonic assisted enzymatic extraction method for extracting antioxidant ingredients from the industrial by-products of N. tangutorum juice. In this study, these ingredients were selectively fractionated by cation-exchange resin chromatography to obtain an anthocyanin fraction namely NJBAE. NJBAE was found to be composed of 16 anthocyanins derived from six anthocyanidins by HPLC-ESI-MS, and has an appreciable cardioprotective effect on doxorubicin-induced injured H9c2 cardiomyocytes. The cardioprotective mechanism research showed that NJBAE could directly scavenge ROS, restrict further generation of ROS, promote the activity of key antioxidase, enhance glutathione redox cycling, then affect the apoptotic signaling changes in a positive way, and finally mediate caspase-dependent cell death pathways. Therefore, NJBAE has great potential to be used for preventing and treating cardiovascular disease in the food, pharmaceutical and other emerging industries.

Doxorubicin and edelfosine lipid nanoparticles are effective acting synergistically against drug-resistant osteosarcoma cancer cells

Despite the great advances that have been made in osteosarcoma therapy during recent decades, recurrence and metastases are still the most common outcome of the primary disease. Current treatments include drugs such as doxorubicin (DOX) that produce an effective response during the initial exposure of tumor cells but sometimes induce drug resistance within a few cycles of chemotherapy. New therapeutic strategies are therefore needed to overcome this resistance. To this end, DOX was loaded into lipid nanoparticles (LN) and its efficacy was evaluated in commercial and patient-derived metastatic osteosarcoma cell lines. DOX efficacy was heavily influenced by passage number in metastatic cells, in which an overexpression of P-gp was observed. Notably, DOX-LN overcame the resistance associated with cell passage and improved DOX efficacy fivefold. Moreover, when DOX was co-administered with either free or encapsulated edelfosine (ET), a synergistic effect was observed. This higher efficacy of the combined treatment was found to be at least partially due to an increase in caspase-dependent cell death. The combination of DOX and ET is thus likely to be effective against osteosarcoma.

New orally active DNA minor groove binding small molecule CT-1 acts against breast cancer by targeting tumor DNA damage leading to p53-dependent apoptosis.

Targeting tumor DNA damage and p53 pathway is a clinically established strategy in the development of cancer chemotherapeutics. Majority of anti-cancer drugs are delivered through parenteral route for reasons like severe toxicity, lack of stability, and poor enteral absorption. Current DNA targeting drugs in clinical like anthracycline suffers from major drawbacks like cardiotoxicity. Here, we report identification of a new orally active small molecule curcumin-triazole conjugate (CT-1) with significant anti-breast cancer activity in vitro and in vivo. CT-1 selectively and significantly inhibits viability of breast cancer cell lines; retards cells cycle progression at S phase and induce mitochondrial-mediated cell apoptosis. CT-1 selectively binds to minor groove of DNA and induces DNA damage leading to increase in p53 along with decrease in its ubiquitination. Inhibition of p53 with pharmacological inhibitor as well as siRNA revealed the necessity of p53 in CT-1-mediated anti-cancer effects in breast cancer cells. Studies using several other intact p53 and deficient p53 cancer cell lines further confirmed necessity of p53 in CT-1-mediated anti-cancer response. Pharmacological inhibition of pan-caspase showed CT-1 induces caspase-dependent cell death in breast cancer cells. Most interestingly, oral administration of CT-1 induces significant inhibition of tumor growth in LA-7 syngeneic orthotropic rat mammary tumor model. CT-1 treated mammary tumor shows enhancement in DNA damage, p53 upregulation, and apoptosis. Collectively, CT-1 exhibits potent anti-cancer effect both in vitro and in vivo and could serve as a safe orally active lead for anti-cancer drug development. © 2016 Wiley Periodicals, Inc.

Non-Canonical Cell Death Induced by p53.

Programmed cell death is a vital biological process for multicellular organisms to maintain cellular homeostasis, which is regulated in a complex manner. Over the past several years, apart from apoptosis, which is the principal mechanism of caspase-dependent cell death, research on non-apoptotic forms of programmed cell death has gained momentum. p53 is a well characterized tumor suppressor that controls cell proliferation and apoptosis and has also been linked to non-apoptotic, non-canonical cell death mechanisms. p53 impacts these non-canonical forms of cell death through transcriptional regulation of its downstream targets, as well as direct interactions with key players involved in these mechanisms, in a cell type- or tissue context-dependent manner. In this review article, we summarize and discuss the involvement of p53 in several non-canonical modes of cell death, including caspase-independent apoptosis (CIA), ferroptosis, necroptosis, autophagic cell death, mitotic catastrophe, paraptosis, and pyroptosis, as well as its role in efferocytosis which is the process of clearing dead or dying cells.

Induction of Apoptosis and Autophagy By Nampt Inhibition in Adult T-Cell Leukemia/Lymphoma and Leukemic Cell Lines

Introduction: Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with human T-cell leukemia virus type I (HTLV-1). Despite the recent advances in chemotherapy, allogeneic hematopoietic stem cell transplantation, and supportive care, the prognosis for patients with acute, lymphoma, or unfavorable chronic subtypes is one of the poorest among hematological malignancies. The identification of new molecular targets for ATL prevention and treatment is desired. SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent histone/protein deacetylase, plays crucial roles in various physiological processes, including aging and apoptosis. We previously reported that ATL patients had significantly higher SIRT1 protein levels and novel small-molecule SIRT1 inhibitors are highly effective against ATL cells.1,2 Nicotinamide phosphoribosyltransferase (Nampt) also known as pre-B-cell colony-enhancing factor 1 or visfatin is a rate-limiting enzyme in NAD+ biosynthesis, and it regulates intracellular ATP levels in mammalian cells. Most cancer cells (acute myeloid leukemia [AML], acute lymphoblastic leukemia [ALL], mantle cell lymphoma [MCL], chronic lymphocytic leukemia [CLL], and T-cell lymphoma) is sensitive to low concentrations of FK866, Nampt inhibitor, as measured in cytotoxicity and clonogenic assays.3Here, we assessed how Nampt is regulated in ATL cells and leukemic cell lines.Results: We observed that ATL patients had significantly higher SIRT1 and Nampt protein levels than healthy controls. FK866 induced significant growth inhibition and apoptosis (Annexin V+ cells and TUNEL) in leukemia/lymphoma cell lines (HTLV-1-related cell lines: S1T, MT-2; Jurkat and HL60). FK866 showed potent activities with GI50values of 0.63, 3.7, 1.0, and 3,4 nM for S1T, MT-2, Jurkat, and HL60 cells, respectively. FK866 also activated caspase activity (caspase-3, 8, and 9) with DNA fragmentation. However, a caspase inhibitor did not inhibit this caspase-dependent cell death. Interestingly, FK866 increased the LC3-II-enriched protein fraction, indicating autophagosome accumulation as well as autophagy. Autophagy detection was also performed using the CytoID Autophagy detection kit. Autophagy levels are increased in the presence of STF-62247 pre-treated with bafilomycin A1, a specific inhibitor of vacuolar proton ATPase, whose inhibition is known to block the fusion of autophagosomes with lysosomes for 2 h. Thus, FK866 simultaneously caused apoptosis and autophagy.Conclusion:These results suggest that Nampt inhibitor is highly effective against ATL cells in caspase-dependent or -independent manners with autophagy, and that its clinical application might improve the prognosis of patients with this fatal disease.1. Kozako T, Aikawa A, Shoji T, et al. High expression of the longevity gene product SIRT1 and apoptosis induction by sirtinol in adult T-cell leukemia cells. Int J Cancer. 2012;131:2044-2055.2. Kozako T, Suzuki T, Yoshimitsu M, et al. Novel small-molecule SIRT1 inhibitors induce cell death in adult T-cell leukaemia cells. Sci Rep. 2015;5:11345.3. Nahimana A, Attinger A, Aubry D, et al. The NAD biosynthesis inhibitor APO866 has potent antitumor activity against hematologic malignancies. Blood. 2009;113:3276-3286. DisclosuresYoshimitsu:HUYA Bioscience International: Research Funding.

Treatment of Hodgkin Lymphoma Xenografts with the Novel PI3K δ/γ Inhibitor RP6530 Suppresses M2 Macrophage Polarization and Results in Potent Antitumor and Antiangiogenic Effects

INTRODUCTION: Primary refractory and early-relapsed patients with Hodgkin lymphoma (HL) experience poor responses to salvage chemotherapy and dismal long-term disease control. Therefore, they represent an unmet medical need urgently requiring not only new diagnostic tools but also novel therapeutic agents to overcome the chemo-refractory phenotype.Tumor-associated macrophages (TAMs) have been implicated in the pathogenesis of HL and have been suggested to negatively impact clinical outcome. The phosphatidylinositol 3-kinase (PI3K) is a major effector pathway mediating tumor and endothelial cell survival and functions, as well as macrophage activation. As PI3Kδ and PI3Kγ are constitutively activated in several hematologic malignancies resulting in cancer development and progression, we hypothesize that the novel dual PI3K δ/γ inhibitor,RP6530, might represent a unique therapeutic opportunity for HL patients. This study aimed at investigating the activity and mechanism(s) of action of RP6530 in preclinical HL models.METHODS: The effects of RP6530 on HL cell lines (L-540, KM-H2, L-428) were investigated by assaying cell proliferation, cell death, and gene expression profiling. Upon treatment with RP6530, human macrophages co-cultured with HL cell lines were assessed by flow cytometry for cell death and expression of M1/M2 macrophage markers. The antitumor efficacy and mechanism(s) of action of RP6530 were also analyzed in NOD/SCID mice bearing HL cell line xenografts. Tumor vasculature was analyzed by in vivo biotinylation of vascular endothelial proteins.RESULTS: Exposure of HL cell lines to RP6530 (1.25 - 10 µM) induced a marked, early, and dose-dependent dephosphorylation of PI3K/Akt and MAPK pathways that was associated with inhibition of cell proliferation (L-540: 40 ± 4%, KM-H2: 33 ± 5% and L-428 30 ± 5%, mean ± SEM) and S phase cell cycle arrest (3-fold reduction, compared to vehicle-controls). Significant levels of caspase-dependent cell death were observed in L-540 (20µM: 61 ± 5 vs. 15 ± 2%), KM-H2 (20µM: 41 ± 4 vs. 12 ± 1%) and L-428 (20µM: 31± 4% vs. 12 ± 1%) cells that were associated with mitochondrial depolarization (up to 40%). Interestingly, microarray gene expression analysis of KM-H2 cell line showed that a 24-hour treatment with RP6530 decreased the amplitude of several gene signatures, including “PI3K signaling in B lymphocytes”, “Tumor cell proliferation”, “Tumor angiogenesis”, and “Macrophages activation”, while genes involved in positive regulation of cell death were upregulated. In KM-H2 co-culture, besides observing cell death of human macrophages (up to 40% following a 24-hour exposure), we found that RP6530 inhibited M2 macrophage differentiation, as suggested by decreased CD209 and CD163 expression, and increased M1 macrophage differentiation by upregulating CD40 and CD80 expression. Compared to vehicle-controls, analysis of RP6530-treated tumor xenografts showed a marked reduction of TAM (-44% and -50%, P ≤.0001 in L-540 and KM-H2, respectively), as well as tumor vessel density (average decrease: 80%), consistent with the role of PI3K signaling in the macrophage-dependent promotion of tumor growth and tumor-induced angiogenesis. This finding was paralleled by the histological detection of tumor vasculature disruption, and inhibition of phospho-Akt and -Erk on both endothelial and tumor cells. Finally, RP6530 treatment (150 mg/kg/BID/3wks) of NOD/SCID mice significantly reduced the growth of KM-H2 nodules (-60%, P ≤.0001) compared to vehicle-controls. Reduction of tumor growth was associated with a strong decrease in Ki-67 expression by tumor cells, increase in tumor apoptosis (1.2- and 12-fold in L-540 and KM-H2, respectively) and tumor necrosis (average increase: 7-fold, P≤.0001).CONCLUSIONS: These results demonstrate that the therapeutic potential of RP6530 in HL is likely due to a reduction of M2 macrophages polarization and inhibition of tumor angiogenesis. Our findings warrant further investigation in future clinical trials. DisclosuresViswanadha:Incozen Therapeutics: Employment. Vakkalanka:Rhizen Pharmaceuticals: Employment, Equity Ownership. Santoro:Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; ArQule: Membership on an entity's Board of Directors or advisory committees. Carlo-Stella:Boehringer Ingelheim: Consultancy; Rhizen Pharmaceuticals: Research Funding.

Synthesis and bio-molecular study of (+)-N-Acetyl-α-amino acid dehydroabietylamine derivative for the selective therapy of hepatocellular carcinoma.

BackgroundThe purpose of present work is to synthesize novel (+)-Dehydroabietylamine derivatives (DAAD) using N-acetyl-α-amino acid conjugates and determine its cytotoxic effects on hepatocellular carcinoma cells.MethodsAn analytical study was conducted to explore cytotoxic activity of DAAD on hepatocellular carcinoma cell lines. The cytotoxicity effect was recorded using sulforhodamine B technique. Cell cycle analysis was performed using Propidium Iodide (PI) staining. Based on cell morphology, anti growth activity and microarray findings of DAAD2 treatment, Comet assay, Annexin V/PI staining, Immunoperoxidase assay and western blots were performed accoringly.ResultsHep3B cells were found to be the most sensitive with IC50 of 2.00 ± 0.4 μM against (+)-N-(N-Acetyl-L-Cysteine)-dehydroabietylamine as DAAD2. In compliance to time dependent morphological changes of low cellular confluence, detachment and rounding of DAAD2 treated cells; noticeable changes in G2/M phase were recorded may be leading to cell cycle cessation. Up-regulation (5folds) of TUBA1A gene in Hep3B cells was determined in microarray experiments. Apoptotic mode of cell death was evaluated using standardized staining procedures including comet assay and annexin V/PI staining, Immuno-peroxidase assay. Using western blotting technique, caspase dependant apoptotic mode of cell death was recorded against Hep3B cell line.ConclusionIt is concluded that a novel DAAD2 with IC50 values less than 8 μM can induce massive cell attenuation following caspase dependent apoptotic cell death in Hep3B cells. Moreover, the corelation study indicated that DAAD2 may have vital influence on cell prolifration properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2942-5) contains supplementary material, which is available to authorized users.

Cytotoxicity of 11-epi-Sinulariolide Acetate Isolated from Cultured Soft Corals on HA22T Cells through the Endoplasmic Reticulum Stress Pathway and Mitochondrial Dysfunction

Natural compounds from soft corals have been increasingly used for their antitumor therapeutic properties. This study examined 11-epi-sinulariolide acetate (11-epi-SA), an active compound isolated from the cultured soft coral Sinularia flexibilis, to determine its potential antitumor effect on four hepatocellular carcinoma cell lines. Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the results demonstrated that 11-epi-SA treatment showed more cytotoxic effect toward HA22T cells. Protein profiling of the 11-epi-SA-treated HA22T cells revealed substantial protein alterations associated with stress response and protein synthesis and folding, suggesting that the mitochondria and endoplasmic reticulum (ER) play roles in 11-epi-SA-initiated apoptosis. Moreover, 11-epi-SA activated caspase-dependent apoptotic cell death, suggesting that mitochondria-related apoptosis genes were involved in programmed cell death. The unfolded protein response signaling pathway-related proteins were also activated on 11-epi-SA treatment, and these changes were accompanied by the upregulated expression of growth arrest and DNA damage-inducible protein (GADD153) and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), the genes encoding transcription factors associated with growth arrest and apoptosis under prolonged ER stress. Two inhibitors, namely salubrinal (Sal) and SP600125, partially abrogated 11-epi-SA-related cell death, implying that the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)–activating transcription factor (ATF) 6–CHOP or the inositol-requiring enzyme 1 alpha (IRE1α)–c-Jun N-terminal kinase (JNK)–cJun signal pathway was activated after 11-epi-SA treatment. In general, these results suggest that 11-epi-SA exerts cytotoxic effects on HA22T cells through mitochondrial dysfunction and ER stress cell death pathways.

Inhibiting c-Jun N-terminal kinase partially attenuates caffeine-dependent cell death without alleviating the caffeine-induced reduction in mitochondrial respiration in C2C12 skeletal myotubes

Caffeine is a widely consumed stimulant that has previously been shown to promote cytotoxic stress and even cell death in numerous mammalian cell lines. Thus far there is little information available regarding the toxicity of caffeine in skeletal muscle cells. Our preliminary data revealed that treating C2C12 myotubes with 5 mM caffeine for 6 h increased nuclear fragmentation and reduced basal and maximal oxygen consumption rate (OCR) in skeletal myotubes. The purpose of this study was to further elucidate the pathways by which caffeine increased cell death and reduced mitochondrial respiration. We specifically examined the role of c-Jun N-terminal kinase (JNK), which has previously been shown to simultaneously increase caspase-dependent cell death and reduce mitochondrial respiration in other mammalian cell lines. We found that caffeine promoted a dose-dependent increase in cell death in multinucleated myotubes but did not in mononucleated myoblasts. The addition of 10 μM Z-DEVD-FMK, a specific inhibitor of executioner caspases, completely inhibited caffeine-dependent cell death. Further, the addition of 400 μM dantrolene, a specific ryanodine receptor (RYR) inhibitor, prevented the caffeine-dependent increase in cell death and the reduction in basal and maximal OCR. We also discovered that caffeine treatment significantly increased the phosphorylation of JNK and that the addition of 30 μM SP600125 (JNKi), a specific JNK inhibitor, partially attenuated caffeine-induced cell death without preventing the caffeine-dependent reduction in basal and maximal OCR. Our results suggest that JNK partially mediates the increase in caspase-dependent cell death but does not contribute to reduced mitochondrial respiration in caffeine-treated skeletal muscle cells. We conclude that caffeine increased cell death and reduced mitochondrial respiration in a calcium-dependent manner by activating the RYR and promoting reticular calcium release.

Biological Potential of Halfsandwich Ruthenium(II) and Iridium (III) Complexes.

In vitro studies with the ruthenium(II) and analogous iridium(III) complexes [Ru(η6- p-cymene)Cl2{Ph2PCH2CH2CH2S(O)xPh-κP}], [Ru(η6-p-cymene)Cl{Ph2PCH2CH2CH2S(O)xPh- κP,κS}][PF6] (1-4), [Ir(η5-C5Me5)Cl2{Ph2PCH2CH2CH2S(O)xPh-κP}] and [Ir(η5-C5Me5)Cl{Ph2 PCH2CH2CH2S(O)xPh-κP,κS}][PF6] (5-8; x = 0, 1) revealed the high selectivity toward the 8505C, A253, MCF-7, SW480 and 518A2 cancer cell lines. Thus, the cationic ruthenium complex 4 proved to be the most selective one. In case of the neutral and cationic ruthenium complexes 1-4 the caspase-dependent apoptotic cell death was proven as the main cause of the drug's tumoricidal action on 8505C cell line.

The Lysosomal Trafficking Transmembrane Protein 106B Is Linked to Cell Death

A common genetic variation in the transmembrane protein 106B (TMEM106B) gene has been suggested to be a risk factor for frontotemporal lobar degeneration (FTLD) with inclusions of transactive response DNA-binding protein-43 (TDP-43) (FTLD-TDP), the most common pathological subtype in FTLD. Furthermore, previous studies have shown that TMEM106B levels are up-regulated in the brains of FTLD-TDP patients, although the significance of this finding remains unknown. In this study, we show that the overexpression of TMEM106B and its N-terminal fragments induces cell death, enhances oxidative stress-induced cytotoxicity, and causes the cleavage of TDP-43, which represents TDP-43 pathology, using cell-based models. TMEM106B-induced death is mediated by the caspase-dependent mitochondrial cell death pathways and possibly by the lysosomal cell death pathway. These findings suggest that the up-regulation of TMEM106B may increase the risk of FTLD by directly causing neurotoxicity and a pathological phenotype linked to FTLD-TDP.

Mitochondrial localization of mutant superoxide dismutase 1 triggers caspase-dependent cell death in a cellular model of familial amyotrophic lateral sclerosis.

The mutations in superoxide dismutase 1 (SOD1) cause approximately 20% of familial amyotrophic lateral sclerosis cases. A toxic gain of function has been considered to be the cause of the disease, but its molecular mechanism remains uncertain. To determine whether the subcellular localization of mutant SOD1 is crucial to mutant SOD1-mediated cell death, we produced neuronal cell models with accumulation of SOD1 in each subcellular fraction/organelle, such as the cytosol, nucleus, endoplasmic reticulum, and mitochondria. We showed that the localization of mutant SOD1 in the mitochondria triggered the release of mitochondrial cytochrome c followed by the activation of caspase cascade and induced neuronal cell death without cytoplasmic mutant SOD1 aggregate formation. Nuclear and endoplasmic reticulum localization of mutant SOD1 did not induce cell death. These results suggest that the localization of mutant SOD1 in the mitochondria is critical in the pathogenesis of mutant SOD1-associated familial amyotrophic lateral sclerosis.

Molecular mechanisms of X‐linked retinitis pigmentosa

PurposeRetinitis Pigmentosa is a class of inherited retinal degeneration that causes progressive vision impairment even blindness. The mutations of Retinitis Pigmentosa GTPase Regulator (RPGR) gene account for up to 20% of RP cases. RPGR protein is highly expressed at the connecting cilium of vertebrates’ photoreceptors. RPGR protein has the function of regulating protein trafficking from basal body to axoneme. The trafficking is controlled by the protein complex formed by RPGR, RPGRIP1 and IFT proteins, although its precise function yet to be identified. RPGR−/− mice model was employed for investigating disease mechanisms during retinal degeneration.MethodsCell death pathway and protein trafficking were detected by immunohistochemistry.ResultsThe retina of RPGR−/− mice started to undergo degeneration from 3M old. The caspase dependent cell death pathway was found to be activated during degeneration. The Apoptosis Inducing Factor existed in outer nuclear layer indicated that caspase‐independent apoptosis also contributed to cell death. The activated microglia, showed migration from outer plexiform layer to ONL in the retina of RPGR−/− mice and the downstream inflammation pathway were activated in RPGR−/− retina. Prior to the onset of retinal degeneration, the key elements involved in the phototransduction such as rhodopsin already mislocalized and accumulated in inner segment and outer nuclear layer. The mislocalization may result from the abnormal formation of the regulator of membrane trafficking Rab8 in RPGR−/− mice that controls the fusion of rhodopsin transport carrier and directs the cargo delivery across the CC.ConclusionsIn Conclusion, multi‐pathways were involved in the photoreceptor cell death and the deficiency of RPGR led to abnormal localization of the crucial proteins that participate in photoreceptor maintenance and phototransduction.

Natriuretic peptide receptor A inhibition suppresses gastric cancer development through reactive oxygen species-mediated G2/M cell cycle arrest and cell death

Natriuretic peptide receptor A (NPRA), the major receptor for atrial natriuretic peptide (ANP), has been implicated in tumorigenesis; however, the role of ANP-NPRA signaling in the development of gastric cancer remains unclear. Immunohistochemical analyses indicated that NPRA expression was positively associated with gastric tumor size and cancer stage. NPRA inhibition by shRNA induced G2/M cell cycle arrest, cell death, and autophagy in gastric cancer cells, due to accumulation of reactive oxygen species (ROS). Either genetic or pharmacologic inhibition of autophagy led to caspase-dependent cell death. Therefore, autophagy induced by NPRA silencing may represent a cytoprotective mechanism. ROS accumulation activated c-Jun N-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). ROS-mediated activation of JNK inhibited cell proliferation by disturbing cell cycle and decreased cell viability. In addition, AMPK activation promoted autophagy in NPRA-downregulated cancer cells. Overall, our results indicate that the inhibition of NPRA suppresses gastric cancer development and targeting NPRA may represent a promising strategy for the treatment of gastric cancer.

Loss of heterogeneous nuclear ribonucleoprotein L (HNRNP L) leads to mitochondrial dysfunction, DNA damage response and caspase-dependent cell death in hematopoietic stem cells

Loss of heterogeneous nuclear ribonucleoprotein L (HNRNP L) leads to mitochondrial dysfunction, DNA damage response and caspase-dependent cell death in hematopoietic stem cells

β-escin selectively targets the glioblastoma-initiating cell population and reduces cell viability.

Glioblastoma multiforme (GBM) is a highly aggressive tumour of the central nervous system and is associated with an extremely poor prognosis. Within GBM exists a subpopulation of cells, glioblastoma-initiating cells (GIC), which possess the characteristics of progenitor cells, have the ability to initiate tumour growth and resist to current treatment strategies. We aimed at identifying novel specific inhibitors of GIC expansion through use of a large-scale chemical screen of approved small molecules. Here, we report the identification of the natural compound β-escin as a selective inhibitor of GIC viability. Indeed, β-escin was significantly cytotoxic in nine patient-derived GIC, whilst exhibiting no substantial effect on the other human cancer or control cell lines tested. In addition, β-escin was more effective at reducing GIC growth than current clinically used cytotoxic agents. We further show that β-escin triggers caspase-dependent cell death combined with a loss of stemness properties. However, blocking apoptosis could not rescue the β-escin-induced reduction in sphere formation or stemness marker activity, indicating that β-escin directly modifies the stem identity of GIC, independent of the induction of cell death. Thus, this study has repositioned β-escin as a promising potential candidate to selectively target the aggressive population of initiating cells within GBM.

PMCA2 silencing potentiates MDA-MB-231 breast cancer cell death initiated with the Bcl-2 inhibitor ABT-263

PMCA2 overexpression in some breast cancers suggests that this calcium pump isoform may play a role in breast pathophysiology. To investigate PMCA2 as a potential drug target for breast cancer therapy, we assessed the functional consequence of PMCA2 silencing on cell death pathways and calcium signals in the basal-like MDA-MB-231 breast cancer cell line. Silencing PMCA2 expression alone has no effect on MDA-MB-231 cell viability, however, PMCA2 silencing promotes calcium-induced cell death initiated with the calcium ionophore ionomycin. Assessment of cytoplasmic calcium responses generated with various agents including ionomycin demonstrates that in MDA-MB-231 cells, PMCA2 does not play a major role in shaping global calcium signals. We also examined the ability of PMCA2 silencing to modulate caspase-dependent cell death triggered by a Bcl-2 inhibitor that is in clinical development for the treatment of various cancers, ABT-263 (Navitoclax). Despite the lack of effect on global calcium responses, PMCA2 silencing augmented Bcl-2 inhibitor (ABT-263)-mediated MDA-MB-231 breast cancer cell death. These studies provide evidence that PMCA2 inhibitors could sensitize PMCA2-positive breast cancers to cell death initiators that work through mechanisms involving the Bcl-2 survival pathway.

The proprotein convertase furin is required to maintain viability of alveolar rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Success of current therapies is still limited and outcome is particularly poor for metastatic alveolar rhabdomyosarcoma (aRMS). We previously identified the proprotein convertase furin as potential target for specific drug delivery with RMS-homing peptides. Furin is a protease that converts inactive precursor proteins into bioactive proteins and peptides. In this study, we investigate the biological role of furin in aRMS progression in vitro and in vivo. Furin expression was confirmed in over 86% RMS biopsies in a tissue microarray (n=89). Inducible furin silencing in vitro led to significant impairment of cell viability and proliferation in all investigated aRMS cell lines, but not in MRC5 fibroblasts. Furthermore, the aRMS cell lines Rh3 and Rh4 revealed to be very sensitive to furin silencing, undergoing caspase-dependent cell death. Notably, furin silencing in vivo led to complete remission of established Rh4 tumors and to delayed growth in Rh30 tumors. Taken together, these findings identify furin as an important factor for aRMS progression and survival. Thus, we propose furin as a novel therapeutic target for treatment of aRMS.

The double benefit of Spalax p53: surviving underground hypoxia while defying lung cancer cells in vitro via autophagy and caspase-dependent cell death.

The blind subterranean mole rat, Spalax ehrenbergi, is a model organism for hypoxia tolerance. This superspecies have adapted to severe environment by altering an array of hypoxia-mediated genes, among which an alteration in the p53 DNA binding domain (corresponding to R174K in humans) that hinders its transcriptional activity towards apoptotic genes. It is well accepted that apoptosis is not the only form of programmed cell death and that mechanisms that depend on autophagy are also involved. In the current work we have extended our research and investigated the possibility that Spalax p53 can activate autophagy. Using two complementary assays, we have established that over-expression of the Spalax p53 in p53-null cells (human lung cancer cells, H1299), potently induces autophagy. As Spalax is considered highly resistant to cancer, we further studied the relative contribution of autophagy on the outcome of H1299 cells, following transfection with Spalax p53. Results indicate that Spalax p53 acts as a tumor suppressor in lung cancer cells, inducing cell death that involves autophagy and caspases and inhibiting cell number, which is exclusively caspase-dependent. To conclude, the Spalax p53 protein was evolutionary adapted to survive severe underground hypoxia while retaining the ability to defy lung cancer.

GSK621 Targets Glioma Cells via Activating AMP-Activated Protein Kinase Signalings.

Here, we studied the anti-glioma cell activity by a novel AMP-activated protein kinase (AMPK) activator GSK621. We showed that GSK621 was cytotoxic to human glioma cells (U87MG and U251MG lines), possibly via provoking caspase-dependent apoptotic cell death. Its cytotoxicity was alleviated by caspase inhibitors. GSK621 activated AMPK to inhibit mammalian target of rapamycin (mTOR) and downregulate Tetraspanin 8 (Tspan8) in glioma cells. AMPK inhibition, through shRNA knockdown of AMPKα or introduction of a dominant negative (T172A) AMPKα, almost reversed GSK621-induced AMPK activation, mTOR inhibition and Tspan8 degradation. Consequently, GSK621’s cytotoxicity in glioma cells was also significantly attenuated by AMPKα knockdown or mutation. Further studies showed that GSK621, at a relatively low concentration, significantly potentiated temozolomide (TMZ)’s sensitivity and lethality against glioma cells. We summarized that GSK621 inhibits human glioma cells possibly via activating AMPK signaling. This novel AMPK activator could be a novel and promising anti-glioma cell agent.