Involvement Of Caspases Research Articles

Revisiting the secretion mechanism(s) of macrophage migration inhibitory factor-welcome to the "UPS club".

Revisiting the secretion mechanism(s) of macrophage migration inhibitory factor-welcome to the "UPS club".

Anticancer Effects of Viscum album Fraxini Extract on Medulloblastoma Cells in vitro

Background: Mistletoe therapy is frequently administered as a supportive treatment in diverse pediatric cancer entities including brain tumors. Medulloblastoma is the most common brain tumor in childhood. Its high risk to metastasize and its long-term sequelae caused by aggressive chemo- or radiotherapies are still challenging. Material and Methods: Effects of a lectin-rich mistletoe extract, abnobaVISCUM Fraxini, were investigated in two medulloblastoma cell lines (Daoy and ONS-76). Responsiveness of tumor cells was assessed by cell viability assays and xCELLigence real-time analyses. Moreover, impacts on proliferation, cell cycle and apoptosis were investigated. Apoptosis was studied by staining of vital mitochondria and assessing the involvement of caspases. In addition, effects on migration and invasion were analyzed. Results: Both medulloblastoma cell lines were more susceptible to treatment with the mistletoe extract than a nontumorigenic fibroblast cell line. In mistletoe-sensitive Daoy cells, reduction of proliferation and induction of caspase-mediated apoptosis were observed upon administration of 0.05 and 0.5 mg/mL abnobaVISCUM Fraxini treatment, respectively. Furthermore, mistletoe extract inhibited migration and invasion properties in Daoy and significantly impaired invasive capabilities of ONS-76 cells. Conclusion: AbnobaVISCUM Fraxini has cell line dependent antitumoral effects in medulloblastoma models. These results call for further investigations, to reveal mechanistic insights into antitumorigenic properties of mistletoe extracts.

The Venom of the Ectoparasitoid Wasp Pachycrepoideus vindemiae (Hymenoptera: Pteromalidae) Induces Apoptosis of Drosophila melanogaster Hemocytes.

The pupal ectoparasitoid Pachycrepoideus vindemiae injects venom into its fly hosts prior to oviposition. We have shown that this venom causes immune suppression in Drosophila melanogaster pupa but the mechanism involved remained unclear. Here, we show using transgenic D. melanogaster with fluorescent hemocytes that the in vivo number of plasmatocytes and lamellocytes decreases after envenomation while it has a limited effect on crystal cells. After in vitro incubation with venom, the cytoskeleton of plasmatocytes underwent rearrangement with actin aggregation around the internal vacuoles, which increased with incubation time and venom concentration. The venom also decreased the lamellocytes adhesion capacity and induced nucleus fragmentation. Electron microscopy observation revealed that the shape of the nucleus and mitochondria became irregular after in vivo incubation with venom and confirmed the increased vacuolization with the formation of autophagosomes-like structures. Almost all venom-treated hemocytes became positive for TUNEL assays, indicating massive induced apoptosis. In support, the caspase inhibitor Z-VAD-FMK attenuated the venom-induced morphological changes suggesting an involvement of caspases. Our data indicate that P. vindemiae venom inhibits D. melanogaster host immunity by inducing strong apoptosis in hemocytes. These assays will help identify the individual venom component(s) responsible and the precise mechanism(s)/pathway(s) involved.

Brainiac Caspases: Beyond the Wall of Apoptosis.

For the last two decades, caspases, a family of cysteine-aspartic proteases, have evolved from being considered solely as regulators of apoptosis or inflammation to having a wider range of functions. In this mini review, we focus on the most recent “non-apoptotic” roles of caspases in the CNS, particularly in neurons, astrocytes and oligodendrocytes. Non-apoptotic caspase functions in microglia have already been reviewed extensively elsewhere. Here we discuss the involvement of caspases in the activation of the inflammasome, autophagy, and non-apoptotic forms of cell death such as necroptosis and pyroptosis. Also, we review the involvement of caspases in synapses and the processing of aggregates key to neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s diseases. Likewise, we mention the recently described involvement of caspases in mitochondrial biogenesis, which is a function independent of the enzymatic activity. We conclude discussing the relevance that “new” functions of caspases have in the CNS and the future of this field of research.

Pereskia bleo Leaves Extract Induces Cell Death via Cell Cycle Arrest and Apoptosis in Cervical Cancer Cells HeLa

Introduction: Pereskia bleo is a leafy and edible plant, locally known as “Pokok Jarum Tujuh Bilah” which has anticancer properties. This study purposed to determine the cytotoxic effects of P. bleo leaves extracts on several well-known cancer cells and elucidate its underlying mechanism in inducing cell death.Methods: Cytotoxic activity on selected cell lines was determined using MTT assay. Mechanism of cell death was investigated through cell cycle and Annexin V assay. Expression of apoptotic proteins was measured by flow cytometry method.Results: Ethyl acetate extract of P. bleo leaves (PBEA) appeared to have the strongest IC50 value (14.37 ± 8.40 μg/ml) and most active against HeLa cells was further studied for apoptosis. The cell cycle investigation by flow cytometry evidenced the increment of PBEA treated HeLa cells in G0/G1 phase and apoptotic event was detected in Annexin V assay. Analysis of apoptotic protein showed pro-apoptotic proteins (Bax, p53 and caspase 3) were triggered where as anti-apoptotic protein Bcl-2 was suppressed in treated HeLa cells.Conclusions: Our findings demonstrated that PBEA treatment induced cell death in HeLa cells by p53-mediated mechanism through arresting cell cycle at G0/G1 phase and mitochondrial-mediated pathway with involvement of pro-apoptotic proteins, anti-apoptotic protein, and caspase 3.

Alanyl-glutamine Protects Against Damage Induced by Enteroaggregative Escherichia coli Strains in Intestinal Cells.

Enteroaggregative Escherichia coli (EAEC) is an important pathogen causing enteric infections worldwide. This pathotype is linked to malnutrition in children from developing countries. Alanyl-glutamine (Ala-Gln) is an immune modulator nutrient that acts during intestinal damage and/or inflammation. This study investigated the effect of EAEC infection and Ala-Gln on cell viability, cell death, and inflammation of intestinal epithelium cells (IEC-6). Cells were infected with an EAEC prototype 042 strain, an EAEC wild-type strain isolated from a Brazilian malnourished child, and a commensal E coli HS. Gene transcription and protein levels of caspases-3, -8, and -9 and cytokine-induced neutrophil chemoattractant 1 (CINC-1/CXCL1) were evaluated using RT-qPCR, western blot analysis, and ELISA. Infections with both EAEC strains decreased cell viability and induced apoptosis and necrosis after 24 hours. Ala-Gln supplementation increased cell proliferation and reduced cell death in infected cells. Likewise, EAEC strain 042 significantly increased the transcript levels of caspases-3, -8, and -9 when compared to the control group, and Ala-Gln treatment reversed this effect. Furthermore, EAEC induced CXCL1 protein levels, which were also reduced by Ala-Gln supplementation. These findings suggest that EAEC infection promotes apoptosis, necrosis, and intestinal inflammation with involvement of caspases. Supplementation of Ala-Gln inhibits cell death, increases cell proliferation, attenuates mediators associated with cell death, and inflammatory pathways in infected cells.

Characterization of the responses of the caspase 2, 3, 6 and 8 genes to immune challenges and extracellular ATP stimulation in the Japanese flounder (Paralichthys olivaceus)

BackgroundCaspases are a family of conserved intracellular cysteine-dependent aspartate-specific cysteine proteases that play important roles in regulating cell death and inflammation. Our previous study revealed the importance of the inflammatory caspase 1 gene in extracellular ATP-mediated immune signaling in Japanese flounder, Paralichthys olivaceus. To explore the potential roles of other caspases in P. olivaceus innate immunity, we extended our study by characterizing of the responses of four additional P. olivaceus caspase genes, termed JfCaspase 2, 3, 6 and 8, to inflammatory challenge and extracellular ATP stimulation.ResultsSequence analysis revealed that the domain structures of all the Japanese flounder caspase proteins are evolutionarily conserved. Quantitative real-time PCR analysis showed that the JfCaspase 2, 3, 6 and 8 genes were expressed ubiquitously but at unequal levels in all examined Japanese flounder normal tissues. In addition, the basal gene expression levels of JfCaspase 2, 3, 6 and 8 were higher than those of JfCaspase 1 in both Japanese flounder head kidney macrophages (HKMs) and peripheral blood leukocytes (PBLs). Furthermore, immune challenge experiments showed that the inflammatory stimuli LPS and poly(I:C) significantly modulated the expression of the JfCaspase 2, 3, 6 and 8 genes in Japanese flounder immune cells. Finally, DNA fragmentation, associated with increased extracellular ATP-induced JfCaspase 2, 3, 6 and 8 gene expression and enzymatic activity, was inhibited by the caspase inhibitor Z-VAD-FMK in the HKMs.ConclusionOur findings demonstrate broad participation of multiple caspase genes in response to inflammatory stimulation in Japanese flounder immune cells and provide new evidence for the involvement of caspase(s) in extracellular ATP-induced apoptosis in fish.

Galactomannan endowed biogenic silver nanoparticles exposed enhanced cancer cytotoxicity with excellent biocompatibility

Galactomannan isolated from the fruit rind of Punica granatum was previously reported to have excellent antioxidant, immunomodulatory and anticancer properties against both human and murine cancer cells. This polysaccharide was proved to be an anticancer agent either alone or as an adjuvant to chemotherapy. An exploration leads to the fabrication of silver nanoparticles with an average size around 30 nm and a negative surface charge of 35.2 mV using this biopolymer which acted both as reducing and capping agent and displayed good stability and biocompatibility. UV–vis spectrum of the aqueous medium containing silver nanoparticles showed an absorption peak at around 440 nm. The nanoparticles displayed an upgraded and selective cytotoxicity towards human adenocarcinoma, colorectal carcinoma and hepatocellular carcinoma cells. The induction of cancer cell toxicity was proved to be through the induction of programmed cell death pathway mediated with the active involvement of caspases. The significant anti-metastatic properties will further favour the safer in vivo application of these silver nanoparticles against neoplasia. The nontoxic nature of polysaccharide endowed the resultant silver nanoparticles with excellent biocompatibility towards red blood cells and extended the biomedical potential of this candidate. Hence, the surfactant-free green method mediated orchestration of biogenic silver nanoparticles resembled a potential theransonstic nano-construct with synergistic anticancer and immunomodulatory potential in a single platform.

Alterations in the nucleocytoplasmic transport in apoptosis: Caspases lead the way.

Apoptosis is a mode of regulated cell death that is indispensable for the morphogenesis, development and homeostasis of multicellular organisms. Caspases are cysteine-dependent aspartate-specific proteases, which function as initiators and executors of apoptosis. Caspases are cytosolic proteins that can cleave substrates located in different intracellular compartments during apoptosis. Many years ago, the involvement of caspases in the regulation of nuclear changes, a hallmark of apoptosis, was documented. Accumulated data suggest that apoptosis-associated alterations in nucleocytoplasmic transport are also linked to caspase activity. Here, we aim to discuss the current state of knowledge regarding this process. Particular attention will be focused on caspase nuclear entry and their functions in the demolition of the nucleus upon apoptotic stimuli.

Gasdermin: A new player to the inflammasome game

Pyroptosis is a lytic type of programmed cell death that was traditionally associated with the involvement of inflammatory caspases, such as caspase-1. These inflammatory caspases are activated within multi-protein complexes called inflammasomes that are assembled in response to invading pathogens and/or danger signals. Pyroptotic cell death was suggested to evolve via the formation of pores in the plasma membrane, but the exact mechanism underlying the formation of these pores remained unclear. Recently, gasdermin D, a member of the gasdermin protein family was identified as a caspase substrate and essential effector of pyroptosis, being identified as the protagonist of membrane pore formation. Gasdermins have emerged as a family of new class of cell death inducers, but many questions remain unanswered. Here, we present an overview of recent work being done in the area of programmed cell death and the latest evidence regarding the role and participation of gasdermin D as an effector of pyroptosis.

The caspase-8/RIPK3 signaling axis in antigen presenting cells controls the inflammatory arthritic response

BackgroundCaspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, but maintains functions beyond cell death that involve suppression of receptor-interacting serine-threonine kinases (RIPKs). A genome-wide association study meta-analysis revealed an SNP associated with risk of rheumatoid arthritis (RA) development within the locus containing the gene encoding for caspase-8. Innate immune cells, like macrophages and dendritic cells, are gaining momentum as facilitators of autoimmune disease pathogenesis, and, in particular, RA. Therefore, we examined the involvement of caspase-8 within these antigen-presenting cell populations in the pathogenesis of an arthritis model that resembles the RA effector phase.MethodsCreLysMCasp8flox/flox and CreCD11cCasp8flox/flox mice were bred via a cross between Casp8flox/flox and CreLysM or CreCD11c mice. RIPK3–/–CreLysMCasp8flox/flox and RIPK3–/–CreCD11cCasp8flox/flox mice were generated to assess RIPK3 contribution. Mice were subjected to K/BxN serum-transfer-induced arthritis. Luminex-based assays were used to measure cytokines/chemokines. Histological analyses were utilized to examine joint damage. Mixed bone marrow chimeras were generated to assess synovial cell survival. Flow cytometric analysis was employed to characterize cellular distribution. For arthritis, differences between the groups were assessed using two-way analysis of variance (ANOVA) for repeated measurements. All other data were compared by the Mann-Whitney test.ResultsWe show that intact caspase-8 signaling maintains opposing roles in lysozyme-M- and CD11c-expressing cells in the joint; namely, caspase-8 is crucial in CD11c-expressing cells to delay arthritis induction, while caspase-8 in lysozyme M-expressing cells hinders arthritis resolution. Caspase-8 is also implicated in the maintenance of synovial tissue-resident macrophages that can limit arthritis. Global loss of RIPK3 in both caspase-8 deletion constructs causes the response to arthritis to revert back to control levels via a mechanism potentially independent of cell death. Mixed bone marrow chimeric mice demonstrate that caspase-8 deficiency does not confer preferential expansion of synovial macrophage and dendritic cell populations, nor do caspase-8-deficient synovial populations succumb to RIPK3-mediated necroptotic death.ConclusionsThese data demonstrate that caspase-8 functions in synovial antigen-presenting cells to regulate the response to inflammatory stimuli by controlling RIPK3 action, and this delicate balance maintains homeostasis within the joint.

Involvement of caspase-8 in the differentiation of syncytial layers of the mouse placental labyrinth

Involvement of caspase-8 in the differentiation of syncytial layers of the mouse placental labyrinth

Indole-coumarin-thiadiazole hybrids: An appraisal of their MCF-7 cell growth inhibition, apoptotic, antimetastatic and computational Bcl-2 binding potential

Cancer therapeutic potential of thiadiazole hybrids incorporating pharmacologically active indole and coumarin moieties have not been explored much. In the current investigation, three new thiadiazole hybrids with spacers of varying lengths linking indole and thiadiazole units were synthesized and their structures were well-established using various spectroscopic techniques. 3-(1-(5-(3-(1H-indol-3-yl)propyl)-1,3,4-thiadiazol-2-ylimino)ethyl)-6-bromo-2H-chromen-2-one (IPTBC) exhibited dose-dependent cytotoxicity in breast adenocarcinoma (MCF-7) cells. The circumvention of apoptosis is a prominent hallmark of cancer and hence triggering apoptosis in specific cancer cells is one of the convenient and widely used approaches for the development of anticancer chemotherapeutics. The induction of apoptosis upon treatment with IPTBC was confirmed by multiple apoptosis assays like Acridine orange-ethidium bromide, Hoechst staining, TUNEL staining, and colorimetric quantification using APOPercentage™ Apoptosis assay. The apoptosis initialisation through the active involvement of caspases was confirmed by caspase profiling tests. The wound healing assay displayed an intense impairment in the motility of MCF-7 cells suggesting the anti-metastatic potential of IPTBC. The ability of IPTBC to inhibit the antiapoptotic Bcl-2 protein by acting as a small molecule BH3 mimetic was explored through docking simulation studies. Although auxiliary investigations are warranted with this promising thiadiazole hybrid IPTBC, the perspective anticancer potential through programmed cell death, anti-metastatic and probable Bcl-2 inhibitory action will enable its further exploration in oncology.

Characterization of three caspases and their pathogen-induced expression pattern in Portunus trituberculatus

Caspases are a family of proteases involved in many important biological processes including apoptosis and inflammation. In this study, we analyzed the expression patterns and effects on immune response in various tissues of the edible crab Portunus trituberculatus. PtCas 2, PtCas 3 and PtCas 4 share overall sequence identities of 55.88%–74.86%, 8.47%–46.54% and 20.11%–50.87%, respectively, with their other crustacean species. PtCas 2, PtCas 3 and PtCas 4 have the same caspase domain and catalytic site found in known caspases. The expression levels of the three caspases differed between tissues. Following bacterial and viral infection, the expression levels of the three caspases reached a maximum level at 24 h post-infection (hpi) in case of bacteria, whereas it was 48 hpi in virus. Moreover, the WSSV, Vibrio alginolyticus or V. parahaemolyticus induced the activities of PtCas 2–4 in a time-dependent manner. These results indicate an involvement of caspases in bacterial and viral induced immune response and demonstrate for the first time that PtCas 2, PtCas 3 and PtCas 4 are essential for optimal response to bacterial and virus infection in crabs.

Regulation of axon arborization pattern in the developing chick ciliary ganglion: Possible involvement of caspase 3.

During a certain critical period in the development of the central and peripheral nervous systems, axonal branches and synapses are massively reorganized to form mature connections. In this process, neurons search their appropriate targets, expanding and/or retracting their axons. Recent work suggested that the caspase superfamily regulates the axon morphology. Here, we tested the hypothesis that caspase 3, which is one of the major executioners in apoptotic cell death, is involved in regulating the axon arborization. The embryonic chicken ciliary ganglion was used as a model system of synapse reorganization. A dominant negative mutant of caspase-3 precursor (C3DN) was made and overexpressed in presynaptic neurons in the midbrain to interfere with the intrinsic caspase-3 activity using an in ovo electroporation method. The axon arborization pattern was 3-dimensionally and quantitatively analyzed in the ciliary ganglion. The overexpression of C3DN significantly reduced the number of branching points, the branch order and the complexity index, whereas it significantly elongated the terminal branches at E6. It also increased the internodal distance significantly at E8. But, these effects were negligible at E10 or later. During E6-8, there appeared to be a dynamic balance in the axon arborization pattern between the "targeting" mode, which is accompanied by elongation of terminal branches and the pruning of collateral branches, and the "pathfinding" mode, which is accompanied by the retraction of terminal branches and the sprouting of new collateral branches. The local and transient activation of caspase 3 could direct the balance towards the pathfinding mode.

P08.57 Involvement of both the extrinsic and intrinsic apoptotic pathways with clomipramine treatment of human glioblastoma cells under normoxic and hypoxic conditions

AbstractIntroduction:The tricyclic antidepressant clomipramine, used clinically since the 1960’s, has been shown to have selective cytotoxicity against glioma cells in vitro and has also been shown to cross the blood brain barrier. While reports suggest clomipramine acts via the mitochondria, the mechanism of action is not fully understood.Materials and Methods:In this study, 8 human biopsy-derived glioblastoma early passage cell lines were treated with 0–200µM clomipramine over 72hrs. Cell viability was measured using the MTS assay, cellular ATP levels using the Cell Titer Glo assay and capsase activation by Caspase-Glo 3/7, 8 and 9 assays. Immunocytochemistry using Mitotracker Red and cytochrome C antibodies was performed. The JC-1 dye was used to evaluate mitochondrial transmembrane potential and DAPI was used to characterise the cell cycle.Results:Clomipramine reduced cell viability in a dose and time dependent manner with IC50 values in the range of 22–60µM at 24hrs. Compared to controls, following 24 hr treatment with 75µM clomipramine, there was a significant reduction of cellular ATP levels (by approximately 100%, p<0.0001), depolarisation of the mitochondrial transmembrane potential and release of cytochrome C from the mitochondria. The extrinsic apoptotic pathway was activated at 12 hours whereas the intrinsic (mitochondrial-mediated) pathway was activated at 24 hours as shown by elevation of caspase 8, 9 and 3/7 levels. The reduction in cell viability in response to clomipramine was attenuated by the pan-caspase inhibitor Z-VAD-FMK, indicating involvement of caspases in the mechanism of action. With the most significant effects observed with SEBTA-023; 24 hours at 75µM clomipramine cell viability increased by 50% and 67% under hypoxic and normoxic conditions respectively (p<0.0001). The effects on the cell cycle were evaluated and clomipramine treatment led to cell cycle arrest in G2/M phase. Two other tricyclic antidepressants, imipramine and amitriptyline were also examined and clomipramine was deemed to be the most effective inducer of apoptosis.Conclusions:This work provides further insights to the mechanism of action of this class of repurposed drug as a potential adjuvant treatment for GBM.

Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines.

Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma.

Molecular characterization reveals involvement of four caspases in the antibacterial immunity of tongue sole (Cynoglossus semilaevis)

Caspases are a family of proteases involved in many important biological processes including apoptosis and inflammation. In this study, we analyzed in a comparative manner the expression patterns and immune effects of four different types of caspases, i.e. caspase-1 (CsCas1), caspase-2 (CsCas2), caspase-3 (CsCas3), and caspase-9 (CsCas9), from the teleost fish tongue sole (Cynoglossus semilaevis). CsCas1, CsCas2, CsCas3, and CsCas9 share 35.4%–58.5%, 61.2%–75.3%, 52.3%–65.6%, and 63.0%–76.2% overall sequence identities, respectively, with their counterparts in teleost species. CsCas1, CsCas2, CsCas3, and CsCas9 possess the caspase domain and catalytic site conserved in known caspases. The expressions of the four caspases were detected in a wide range of tissues, however, the expression levels varied between different tissues and caspases. Following bacterial infection, the expressions of the four caspases were upregulated or downregulated to significant extents in a time- and tissue-dependent manner. In vivo analysis showed that overexpression of each of the caspases in tongue sole significantly enhanced the ability of the fish to resist bacterial dissemination in and colonization of tissues. These results indicate an involvement of fish caspases in bacteria-induced immune response and demonstrate for the first time that caspase-1, 2, 3, and 9 are essential to the optimal defense against bacterial infection in fish.

Molecular Mechanism of Crocin Induced Caspase Mediated MCF-7 Cell Death: In Vivo Toxicity Profiling and Ex Vivo Macrophage Activation.

Crocus sativus and its major constituent crocin are well established to have anti-cancer properties in breast cancer cells (MCF-7). However the role of C. sativus extract (CSE) and crocin on caspase signaling mediated MCF-7 cell death at molecular level is remains unclear. In this study, we tried to unravel role of CSE and crocin on caspase mediated MCF-7 cells death and their in vivo preclinical toxicity profiling and immune stimulatory effect. CSE extract was fractionated by HPLC and crocin was isolated and characterized by NMR, IR, and MS. MCF-7 cells were treated with both CSE and crocin and expression of Bcl-2 and Bax was assessed after 24 and 36 hours. Furthermore, caspase 3, caspase 8 and caspase 9 expression was determined by Western blotting after 24 hours of treatment. DNA fragmentation analysis was performed for genotoxicity of CSE and crocin in MCF-7 cells. The in vivo toxicity profile of CSE (300 mg/kg of b.wt) was investigated in normal Swiss albino mice. In addition, peritoneal macrophages were collected from crocin (1, 1.5 and 2 mg/kg body weight) treated mice and analyzed for ex vivo yeast phagocytosis. Immunoblot analysis revealed that there was time dependent decline in anti-apoptotic Bcl-2 with simultaneous upregulation of Bax in CSE and crocin treated MCF-7 cells. Further CSE and crocin treatment downregulated caspase 8 and 9 and cleaved the caspase 3 after 24 hours. Both CSE and crocin elicited considerable DNA damage in MCF-7 cells at each concentration tested. In vivo toxicity profile by histological studies revealed no observable histopathologic differences in the liver, kidney, spleen, lungs and heart in CSE treated and untreated groups. Crocin treatment elicited significant dose and time dependent ex vivo yeast phagocytosis by peritoneal macrophages. Our study delineated involvement of pro-apoptotic and caspase mediated MCF-7 cell death by CSE and crocin at the molecular level accompanied with extensive DNA damage. Further we found that normal swiss albino mice can tolerate the maximum dose of CSE. Crocin enhanced ex vivo macrophage yeast phagocytic ability.

Glucose regulated protein 78 (GRP78) inhibits apoptosis and attentinutes chemosensitivity of gemcitabine in breast cancer cell via AKT/mitochondrial apoptotic pathway

The underlying mechanism of gemcitabine resistance during breast cancer treatment remains unclear. Glucose regulated protein 78 (GRP78) frequently triggered by anticancer agents, was substantially elevated in gemcitabine resistant sublines. Ectopic expression of GRP78 changes gemcitabine chemosensitivity and apoptosis levels in breast cancer cells. Further experiments showed an involvement of caspase 9, not caspase 8, in gemcitabine resistance and GRP78-mediated chemosensitivity, suggesting that mitochondria apoptotic pathway was activated by GRP78. This finding was further supported by the observations of AKT activation, Bcl-2 increase, Bax and Bim decrease. Conclusively, GRP78 plays a vital role in gemcitabine resistance and clinical strategy to improve gemcitabine efficacy in breast cancer by manipulating GRP78 should be explored.