Caspase Targets Research Articles

MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10.

Thrombogenic and inflammatory mediators, such as thrombin, induce NF-κB-mediated endothelial cell (EC) activation and dysfunction, which contribute to pathogenesis of arterial thrombosis. The role of anti-inflammatory microRNA-181b (miR-181b) on thrombosis remains unknown. Our previous study demonstrated that miR-181b inhibits downstream NF-κB signaling in response to TNF-α. Here, we demonstrate that miR-181b uniquely inhibits upstream NF-κB signaling in response to thrombin. Overexpression of miR-181b inhibited thrombin-induced activation of NF-κB signaling, demonstrated by reduction of phospho-IKK-β, -IκB-α, and p65 nuclear translocation in ECs. MiR-181b also reduced expression of NF-κB target genes VCAM-1, intercellular adhesion molecule-1, E-selectin, and tissue factor. Mechanistically, miR-181b targets caspase recruitment domain family member 10 (Card10), an adaptor protein that participates in activation of the IKK complex in response to signals transduced from protease-activated receptor-1. miR-181b reduced expression of Card10 mRNA and protein, but not protease-activated receptor-1. 3'-Untranslated region reporter assays, argonaute-2 microribonucleoprotein immunoprecipitation studies, and Card10 rescue studies revealed that Card10 is a bona fide direct miR-181b target. Small interfering RNA-mediated knockdown of Card10 expression phenocopied effects of miR-181b on NF-κB signaling and targets. Card10 deficiency did not affect TNF-α-induced activation of NF-κB signaling, which suggested stimulus-specific regulation of NF-κB signaling and endothelial responses by miR-181b in ECs. Finally, in response to photochemical injury-induced arterial thrombosis, systemic delivery of miR-181b reduced thrombus formation by 73% in carotid arteries and prolonged time to occlusion by 1.6-fold, effects recapitulated by Card10 small interfering RNA. These data demonstrate that miR-181b and Card10 are important regulators of thrombin-induced EC activation and arterial thrombosis. These studies highlight the relevance of microRNA-dependent targets in response to ligand-specific signaling in ECs.-Lin, J., He, S., Sun, X., Franck, G., Deng, Y., Yang, D., Haemmig, S., Wara, A. K. M., Icli, B., Li, D., Feinberg, M. W. MicroRNA-181b inhibits thrombin-mediated endothelial activation and arterial thrombosis by targeting caspase recruitment domain family member 10.

High Throughput Flow Screening Assays to Profile Cell-Mediated Killing

Abstract While enhancing T-cell mediated killing of tumor cells is emerging as a successful therapeutic approach for a variety of cancers, improvements to these therapies are actively being sought. Traditional assays for monitoring cell-mediated killing are only capable of homogenous live/dead readouts for an entire sample. As an alternative platform for cell-mediated killing studies, IntelliCyt’s iQue Screener can identify multiple cell types in suspension and report multiple cell killing readouts in streamlined no wash assay formats. We demonstrate two example high throughput assays for cell-mediated killing using NK cells and chimeric antigen receptor (CAR) T-cells. Using the NK92 cell line as an effector cell and fluorescently encoded Jurkat cells as target cells, viability and Caspase 3 activation was determined for both Jurkat and NK92 cells in the same sample, and compounds that were generally cytotoxic to both cells could be identified. Specificity of the cell-mediated killing response was demonstrated using known signal transduction inhibitors including sunitinib, U73122, pp2, and wortmannin that would attenuate the NK cell killing activity, at a fixed target to effector cell ratio. In the CAR T-cell assay, efficacy of different CARs at targeting and killing a B-cell line (NALM-6) was profiled using multiplex readouts for cell health and secreted cytokines. Multiple cytokines including inflammatory markers and Granzyme B were quantified using bead-based ELISA on the same analysis platform. These application examples highlight the robustness and flexibility of the iQue Screener for performing multiplexed screening assays with cells and beads.

Effect of 5-aminolevulinic acid mediated photodynamic therapy on proliferation and apoptosis of gastric cancer cell line MKN-45

Objective To explore the 5-aminolevulinic acid (5-ALA) mediated photodynamic therapy(PDT) for proliferation and apoptosis of gastric cancer cell line MKN-45. Methods The human gastric cancer cell line MKN-45 was cultured in vitro. The MTT method was used to detect fixed lighting processing (laser radiation dose fixed for 25.00 J/cm2, 5-ALA photosensitizer concentrations were 0 mmol/L, 0.25 mmol/L, 0.50 mmol/L, 1.00 mmol/L, 2.00 mmol/L), fixed photosensitizer concentration treatment (5-ALA photosensitizer concentrations fixed to 1.00 mmol/L, laser radiation dose were 0 J/cm2, 6.25 J/cm2, 12.50 J/cm2, 25.00 J/cm2, 50.00 J/cm2, 100.00 J/cm2) of the cell apoptosis rate, and then fixed 5-ALA photosensitizer 1 mmol/L and selected the control group, 5-ALA group, pure illumination group and PDT group. The promoting effect on cell apoptosis was determined by MTT, electron microscope and flow cytometry. The activity of human gastric cancer cells MKN-45 apoptosis related proteins p65 and the downstream gene regulatory protein bcl-2, bax, caspase-3 and caspase-9 were detected by using Western blot method. Results When laser radiation dose was fixed, 5-ALA photosensitizer concentrations in promoting gastric cancer cell apoptosis was positively related to the dominant (P < 0.01). When 5-ALA photosensitizer concentration was fixed, laser radiation dose effect had no longer dominant after 25.00 J/cm2 positive correlation (P = 0.613). When 5-ALA photosensitizer concentration was fixed in 1 mmol/L, compared to control group, 5-ALA group and pure illumination group, the inhibition of the MKN-45 cell proliferation in PDT group was more apparently, apoptosis rate increased obviously, signal pathways in cell apoptosis protein involved in the NF-kappa B p65 expression was reduced, the downstream gene regulation of target protein bcl-2, caspase-3 and caspase-9 expression were reduced, and bax protein expression was increased. Conclusions 5-ALA mediated photodynamic has a strong inhibitory effect on the growth of gastric cancer cell line MKN-45 in vitro. It can significantly increase the apoptosis rate of gastric cancer cells, and the effect may be carried out by activating the NF-kappa B pathway. Key words: 5-aminolevulinic acid; Photochemotherapy; Stomach neoplasms; Apoptosis

Daucosterol inhibits colon cancer growth by inducing apoptosis, inhibiting cell migration and invasion and targeting caspase signalling pathway

&lt;p class="Abstract"&gt;The aim of the present investigation was to examine and demonstrate in detail the anti-cancer and apoptotic effects of daucosterol in human colon cancer cell line HCT-116. The effects of this compound on cell migration, cell invasion, cell cycle analysis and caspase signalling pathway were also studied. Cell viability was evaluated by MTT assay using different doses of the drug. In vitro wound healing assay was used to study cell migration. Flow cytometry was involved to examine cell apoptosis as well as cell cycle phase distribution. Daucosterol induced significant, dose-dependent as well as time-dependent cytotoxic effects with IC&lt;sub&gt;50&lt;/sub&gt; values of 26.6 and 47.3 µM at 24 and 48 hours time intervals respectively. The percent of cells that migrated decreased from 99% in case of untreated control to 84.2, 45.2, 39.5 and 14.4% in groups treated with 0, 5, 50, 75 and 100 µM of daucosterol respectively. Percentage of apoptotic cells increased from 2.5% in untreated control cells to 23.6, 46.9 and 74.2% in cells treated with 5, 50 and 100 µM dose of daucosterol respectively. Daucosterol at different doses induced cell cycle arrest at sub-G1 phase of the cell cycle.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Video clip&lt;/strong&gt;&lt;/p&gt;&lt;p&gt;&lt;a href="https://youtube.com/v/CUkLDjyNUFw"&gt;Cell Migration Assay&lt;/a&gt;: 1 min&lt;/p&gt;

YAP induces cisplatin resistance through activation of autophagy in human ovarian carcinoma cells.

ObjectiveTo identify the role of YAP in cisplatin resistance in human ovarian cancer cells and in the regulation of autophagy in these cancer cells.Materials and methodsThe cisplatin-sensitive OV2008 parental cell line and its cisplatin-resistant variant C13K were cultured. RNA interference was used to knock down the YAP gene. Accumulation of GFP-LC3 puncta was performed by fluorescence microscopy. The formation of autophagosomes was observed by transmission electron microscopy. Drug sensitivity was examined using CCK-8 assay, while apoptosis, the level of intracellular rhodamine 123 and lysosomal acidification were analyzed by fluorescence-activated cell sorting. Acid phosphatase activity was measured using an acid phosphatase-assay kit. Real-time polymerase chain reaction, Western blotting, and immunofluorescence detection were used to detect the protein and messenger RNA expression of YAP, YAP target genes, CCND1, cleaved PARP, and caspase 3, Atg-3 and -5, and the LC3B protein.ResultsYAP signaling may regulate cisplatin resistance in ovarian cancer cells by augmenting cellular autophagic flux. After knockdown of YAP-sensitized C13K cells to cisplatin by inducing a decrease in autophagy, YAP led to an increase in autophagy via enhancement of autolysosome degradation.ConclusionYAP-mediated autophagy may play a protective role in cisplatin-resistant human ovarian cancer cells. Therefore, YAP-mediated autophagy should be explored as a new target for enhancing the efficacy of cisplatin against ovarian cancer and other types of malignancies.

MiR-155 targets Caspase-3 mRNA in activated macrophages

ABSTRACTTo secure the functionality of activated macrophages in the innate immune response, efficient life span control is required. Recognition of bacterial lipopolysaccharides (LPS) by toll-like receptor 4 (TLR4) induces downstream signaling pathways, which merge to induce the expression of cytokine genes and anti-apoptotic genes. MicroRNAs (miRNAs) have emerged as important inflammatory response modulators, but information about their functional impact on apoptosis is scarce. To identify miRNAs differentially expressed in response to LPS, cDNA libraries from untreated and LPS-activated murine macrophages were analyzed by deep sequencing and regulated miRNA expression was verified by Northern blotting and qPCR. Employing TargetScanTM we identified CASPASE-3 (CASP-3) mRNA that encodes a key player in apoptosis as potential target of LPS-induced miR-155. LPS-dependent primary macrophage activation revealed TLR4-mediated enhancement of miR-155 expression and CASP-3 mRNA reduction. Endogenous CASP-3 and cleaved CASP-3 protein declined in LPS-activated macrophages. Accumulation of miR-155 and CASP-3 mRNA in miRNA-induced silencing complexes (miRISC) was demonstrated by ARGONAUTE 2 (AGO2) immunoprecipitation. Importantly, specific antagomir transfection effectively reduced mature miR-155 and resulted in significantly elevated CASP-3 mRNA levels in activated macrophages. In vitro translation assays demonstrated that the target site in the CASP-3 mRNA 3'UTR mediates miR-155-dependent Luciferase reporter mRNA destabilization. Strikingly, Annexin V staining of macrophages transfected with antagomir-155 and stimulated with LPS prior to staurosporine (SSP) treatment implied that LPS-induced miR-155 prevents apoptosis through CASP-3 mRNA down-regulation. In conclusion, we report that miR-155-mediated CASP-3 mRNA destabilization in LPS-activated RAW 264.7 macrophages suppresses apoptosis, as a prerequisite to maintain their crucial function in inflammation.

Nanoparticle-Delivered Antisense MicroRNA-21 Enhances the Effects of Temozolomide on Glioblastoma Cells.

Glioblastoma (GBM) generally exhibits high IC50 values for its standard drug treatment, temozolomide (TMZ). MicroRNA-21 (miR-21) is an oncomiR overexpressed in GBM, thus controlling important aspects of glioma biology. We hypothesized that PLGA nanoparticles carrying antisense miR-21 to glioblastoma cells might beneficially knock down endogenous miR-21 prior to TMZ treatment. PLGA nanoparticles encapsulating antisense miR-21 were effective in intracellular delivery and sustained silencing (p < 0.01) of miR-21 function in U87 MG, LN229, and T98G cells. Prior antisense miR-21 delivery significantly reduced the number of viable cells (p < 0.001), and increased (1.6-fold) cell cycle arrest at G2/M phase upon TMZ treatment in U87 MG cells. There was overexpression of the miR-21 target genes PTEN (by 67%) and caspase-3 (by 15%) upon cotreatment. This promising PLGA nanoparticle-based platform for antisense miR-21 delivery to GBM is an effective cotherapeutic strategy in cell culture, warranting the need for further studies prior to future clinical translation.

Let-7b suppresses apoptosis and autophagy of human mesenchymal stem cells transplanted into ischemia/reperfusion injured heart 7by targeting caspase-3.

IntroductionMesenchymal stem cells (MSCs) have therapeutic potential for the repair of myocardial injury. The efficacy of MSC therapy for myocardial regeneration mainly depends on the survival of cells after transplantation into the infarcted heart. In the transplanted regions, reactive oxygen species (ROS) can cause cell death, and this process depends on caspase activation and autophagosome formation.MethodsA Software TargetScan was utilized to search for microRNAs (miRNAs) that target caspase-3 mRNA. Six candidate miRNAs including let-7b were selected and transfected into human MSCs in vitro. Expression of MEK-EKR signal pathways and autophagy-related genes were detected. Using ischemia/reperfusion model (I/R), the effect of MSCs enriched with let-7b was determined after transplantation into infarcted heart area. Miller catheter was used to evaluate cardiac function.ResultsHere, we report that let-7b targets caspase-3 to regulate apoptosis and autophagy in MSCs exposed to ROS. Let-7b-transfected MSCs (let-7b-MSCs) showed high expression of survival-related proteins, including p-MEK, p-ERK and Bcl-2, leading to a decrease in Annexin V/PI- and TUNEL-positive cells under ROS-rich conditions. Moreover, autophagy-related genes, including Atg5, Atg7, Atg12 and beclin-1, were significantly downregulated in let-7b-MSCs. Using a rat model of acute myocardial infarction, we found that intramyocardial injection of let-7b-MSCs markedly enhanced left ventricular (LV) function and microvessel density, in accordance with a reduced infarct size and the expression of caspase-3.ConclusionsTaken together, these data indicate that let-7b may protect MSCs implanted into infarcted myocardium from apoptosis and autophagy by directly targeting caspase-3 signaling.

MiR-98 and its host gene Huwe1 target Caspase-3 in Silica nanoparticles-treated male germ cells.

Silica nanoparticles (NP) is one of the most commonly used nanomaterials with potential health hazards. However, the effects of Silica NP on germ cells and the underlying mechanisms are still unclear. In this study, GC-2 and TM-4, which are two different types of male germ cells were exposed to Silica NP for 24h, and then general cytotoxicity and multi-parameter cytotoxicity were evaluated. Our results showed that Silica NP could induce apoptosis in GC-2 cells. Transmission electron microscopy (TEM) results showed that Silica NP was localized in the lysosomes of GC-2 cells. High content screening (HCS) showed that Silica NP exposure could increased cell permeabilization and decreased mitochondrial membrane potential in GC-2 cells. The mRNA and protein levels of apoptosis markers (Bax, Caspase-3, Caspase-9) in GC-2 cells were significantly increased, while Bcl-2 was decreased. Accordingly, the expression level of miR-98, which can regulate Caspase-3, was significantly decreased. Huwe1, the host gene of miR-98, was positively associated with miR-98 expression after Silica NP exposure. Dual luciferase reporter assay suggested that miR-98 directly targets Caspase-3. These results suggest that Silica NP induces apoptosis via loss of mitochondrial membrane potential and Caspase-3 activation, while miR-98 plays key role in modulating this effect.

Abstract 1255: Mifepristone causes endoplasmic reticulum stress, triggers the unfolded protein response, and increases autophagic flux in ovarian cancer cells

Abstract Mifepristone (MF) is a synthetic steroid that blocks the growth of ovarian cancer cells causing cytostasis or lethality depending on its concentration. Unbiased genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to platinum led to the identification of two key genes up-regulated by MF and that are involved in the unfolded protein response (UPR): the master chaperone of the endoplasmic reticulum (ER), glucose regulated protein (GRP) 78, and the CCAAT/enhancer binding protein homologous transcription factor (CHOP); both proteins are known for balancing ER stress in a ‘yin’ (CHOP; pro-cell death factor) ‘yang’ (GRP78; pro-survival factor) manner. GRP78 and CHOP were up-regulated in ovarian cancer cells regardless of p53 status and platinum sensitivity. Further studies revealed that MF caused expansion of the ER and triggered the UPR. The three UPR associated signaling pathways, PERK, IRE1α, and ATF6, were activated by MF. While low-dose MF caused cytostasis with compensatory UPR activation, high-dose MF induced lethality with concurrent UPR activation, up-regulation of pro-apoptotic factor Bim, down-regulation of anti-apoptotic factor Bcl-2, and cleavage of PARP, a downstream target for executer caspases. MF-induced lethality but not cytostasis was abrogated by the ER stress inhibitor salubrinal, indicating that ER stress is required when MF causes cell death but not when its action is limited to cell cycle arrest. Because ER stress involves the accumulation of misfolded/unfolded proteins that alter ER homeostasis, the UPR engages compensatory mechanisms including short-term attenuation of the rate of protein translation, and degradation of surplus proteins by the ubiquitin proteasome system (UPS) and/or autophagy. Contrary to our expectation, MF-induced UPR associated with short-term increase rather than decrease, in protein translation rate. Moreover, the accumulation of poly-ubiquitinated proteins-a surrogate marker of the activity of the UPS-was slightly decreased by MF, suggesting that ER-associated protein degradation (ERAD) may be operating at a faster pace. Finally, MF increased the accumulation of LC3-II, which results from the fusion of LC3-I and phosphatidylethanolamine along the process of autophagosome formation. MF-induced LC3-II accumulation also augmented in the presence of the lysosomal degradation inhibitor bafilomycin A1, indicating that MF increases autophagic flux. In MF-treated cells, the induction of autophagy may counterbalance and alleviate ER expansion induced by ER stress. The stimulation by MF of ER stress, UPR, and autophagy offers a therapeutic opportunity for utilizing this compound to sensitize ovarian cancer cells to aggravators of the ER and/or inhibitors of autophagy. Citation Format: Lei Zhang, Alicia Goyeneche, Rekha Srinivasan, Carlos Telleria. Mifepristone causes endoplasmic reticulum stress, triggers the unfolded protein response, and increases autophagic flux in ovarian cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1255. doi:10.1158/1538-7445.AM2015-1255

오미자 종자 정유에 의한 인체백혈병 U937 세포의 apoptosis 유도

Schisandrae fructus [Schizandra chinensis (Turcz.) Baillon] is a medicinal herb widely used for treating various inflammatory and immune diseases in East Asian countries. The Schisandrae Semen essential oil (SSeo) from this plant has pharmacological activities, including antioxidant, antimicrobial, and antitumoral activities. Nevertheless, the biological activities and underlying molecular mechanisms of the potential anti-cancer effects of this oil remain unclear. In the present study, we investigated the potential inhibition of apoptosis signaling pathways by SSeo in human leukemia U937 cells and evaluated the underlying molecular mechanism. Exposure to SSeo resulted in a concentration-dependent growth inhibition due to apoptosis, which was verified by DNA fragmentation, the presence of apoptotic bodies, and an increase in the sub-G1 ratio. Induction of apoptotic cell death by SSeo was correlated with the down-regulation of members of the inhibitor of apoptosis protein (IAP) family (including X-linked inhibitor of apoptosis protein (XIAP), cIAP-1, and surviving) and anti-apoptotic Bcl-2, and with up-regulation of death receptor (DR) 4 and DR5, depending on dosage. SSeo treatment also induced Bid truncation, mitochondrial dysfunction, proteolytic activation of caspase-3, -8 and -9, and concomitant degradation of activated caspase-3 target proteins such as poly (ADP-ribose) polymerase. Taken together, these findings suggest that SSeo may be a potential chemotherapeutic agent for use in the control of human leukemia cells. Further studies are needed to identify its active compounds.

Interactions between mitochondria and the transcription factor myocyte enhancer factor 2 (MEF2) regulate neuronal structural and functional plasticity and metaplasticity.

The classical view of mitochondria as housekeeping organelles acting in the background to simply maintain cellular energy demands has been challenged by mounting evidence of their direct and active participation in synaptic plasticity in neurons. Time-lapse imaging has revealed that mitochondria are motile in dendrites, with their localization and fusion and fission events regulated by synaptic activity. The positioning of mitochondria directly influences function of nearby synapses through multiple pathways including control over local concentrations of ATP, Ca(2+) and reactive oxygen species. Recent studies have also shown that mitochondrial protein cascades, classically associated with apoptosis, are involved in neural plasticity in healthy cells. These findings link mitochondria to the plasticity- and metaplasticity-associated activity-dependent transcription factor myocyte enhancer factor 2 (MEF2), further repositioning mitochondria as potential command centres for regulation of synaptic plasticity. Intriguingly, MEF2 and mitochondrial functions appear to be intricately intertwined, as MEF2 is a target of mitochondrial apoptotic caspases and, in turn, MEF2 regulates mitochondrial genome transcription essential for production of superoxidase and hydrogen peroxidase. Here, we review evidence supporting mitochondria as central organelles controlling the spatiotemporal expression of neuronal plasticity, and attempt to disentangle the MEF2-mitochondria relationship mediating these functions.

Reversible inhibition of lysine specific demethylase 1 is a novel anti-tumor strategy for poorly differentiated endometrial carcinoma.

BackgroundEndometrial cancer is the most common gynecologic malignancy. Type II endometrial carcinoma is often poorly differentiated and patients diagnosed with Type II disease (~11%) are disproportionately represented in annual endometrial cancer deaths (48%). Recent genomic studies highlight mutations in chromatin regulators as drivers in Type II endometrial carcinoma tumorigenesis, suggesting the use of epigenetic targeted therapies could provide clinical benefit to these patients. We investigated the anti-tumor efficacy of the LSD1 inhibitor HCI2509 in two poorly differentiated Type II endometrial cancer cell lines AN3CA and KLE.MethodsThe effects of HCI2509 on viability, proliferation, anchorage-independent growth, global histone methylation, LSD1 target gene induction, cell cycle, caspase activation and TUNEL were assayed. KLE cells were used in an orthotopic xenograft model to assess the anti-tumor activity of HCI2509.ResultsBoth AN3CA and KLE cells were sensitive to HCI2509 treatment with IC50s near 500 nM for cell viability. Inhibition of LSD1 with HCI2509 caused decreased proliferation and anchorage independent growth in soft agar, elevated global histone methylation, and perturbed the cell cycle in both cell lines. These effects were largely dose-dependent. HCI2509 treatment also caused apoptotic cell death. Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen. Tumor burden was distributed log-normally. Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p = 0.034).ConclusionsOur findings demonstrate the anti-cancer properties of the LSD1 inhibitor HCI2509 on poorly differentiated endometrial carcinoma cell lines, AN3CA and KLE. HCI2509 showed single-agent efficacy in orthotopic xenograft studies. Continued studies are needed to preclinically validate LSD1 inhibition as a therapeutic strategy for endometrial carcinoma.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-752) contains supplementary material, which is available to authorized users.

Reactive oxygen species at the crossroads of inflammasome and inflammation.

Inflammasomes form a crucial part of the innate immune system. These are multi-protein oligomer platforms that are composed of intracellular sensors which are coupled with caspase and interleukin activating systems. Nod-like receptor protein (NLRP) 3, and 6 and NLRC4 and AIM2 are the prominent members of the inflammasome family. Inflammasome activation leads to pyroptosis, a process of programmed cell death distinct from apoptosis through activation of Caspase and further downstream targets such as IL-1β and IL-18 leading to activation of inflammatory cascade. Reactive oxygen species (ROS) serves as important inflammasome activating signals. ROS activates inflammasome through mitogen-activated protein kinases (MAPK) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Dysregulation of inflammasome plays a significant role in various pathological processes. Viral infections such as Dengue and Respiratory syncytial virus activate inflammasomes. Crystal compounds in silicosis and gout also activate ROS. In diabetes, inhibition of autophagy with resultant accumulation of dysfunctional mitochondria leads to enhanced ROS production activating inflammasomes. Activation of inflammasomes can be dampened by antioxidants such as SIRT-1. Inflammasome and related cascade could serve as future therapeutic targets for various pathological conditions.

MiR-221: A Critical Player in Apoptosis As A Target of Caspase-3

Caspases are a family of proteases that function as the primary mediators of apoptosis. miR-221 is an oncogenic miRNA that may have both anti-angiogenic and angiogenic effect. The aim of this work was to compare the expression levels of miR-221 and its target caspase-3 in different cancer cell lines and to find out a relationship between these two. We also analyzed binding efficiency of miR-221 to 3’UTR region of CASP3 gene computationally. Our results indicate that expression of caspase-3 is quite lower as compared to miR-221 expression in all of the selected cancer cell lines. Also, our computational results showed that miR-221-3p, of which binding site has two SNPs (rs369703524, C/T; rs189187745, T/A) on CASP3 3’UTR region. As a result, we conclude that miR-221 may have a crucial role in repressing the expression of caspase-3 which may contribute to a lower apoptotic rate, thus supporting the selection of more aggressive cancer cells.

HSP70 sequestration by free α-globin promotes ineffective erythropoiesis in β-thalassaemia.

β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM.

Verification of a putative caspase substrate.

Proteomic approaches have been adopted to survey the degradome of caspases during apoptosis. These approaches provide a comprehensive list of substrates and give clues to which pathways are altered during apoptosis by activated caspases. However, substrates identified by large-scale proteomic screening need to be validated as bona fide caspase targets. This ensures that conclusions derived from the screen are based on real substrates and not on artifacts of the proteomic screen. The validation method described in this protocol uses radiolabeled versions of the putative substrates synthesized using in vitro transcription/translation methods. These are incubated with purified caspases to determine whether they are genuine caspase substrates.

Expression patterns of miR-221 and its target Caspase-3 in different cancer cell lines.

Caspases are important initiators and most well-known finishers of apoptosis. By changing the death propagation homeostatic equilibrium, their different expression patterns might trigger the progression of hazardous diseases like cancer. miR-221 is an oncogenic miRNA. It is known to have both anti-angiogenic and angiogenic effect. The aim of this work was to compare the expression levels of miR-221 and its target caspase-3 in different cancer cell lines and to find out a relationship between these two. We also tried to establish a prominent relationship between miR-221 and its role in apoptosis by studying their expression levels. Our results indicate that expression of caspase-3 is quite lower as compared to miR-221 expression in all of the selected cancer cell lines. As a result, we conclude that miR-221 may have a crucial role in repressing the expression of caspase-3 which may contribute to a lower apoptotic rate, thus supporting the selection of more aggressive cancer cells. To our knowledge, this is the first study related to the expression levels of caspase-3 and miR-221 in different cell lines at the same time. We expect that our study might pave the way for better understanding the role of miR-221 in apoptotic regulation of caspase-3.

Synthesis of novel anticancer agents through opening of spiroacetal ring of diosgenin

Diosgenin has been modified to furostane derivatives after opening the F-spiroacetal ring. The aldehyde group at C26 in derivative 8 was unexpectedly transformed to the ketone 9. The structure of ketone 9 was confirmed by spectroscopy and finally by X-ray crystallography. Five of the diosgenin derivatives showed significant anticancer activity against human cancer cell lines. The most potent molecule of this series i.e. compound 7, inhibited cellular growth by arresting the population at G0/G1 phase of cell division cycle. Cells undergo apoptosis after exposure to the derivative 7 which was evident by increase in sub G0 population in cell cycle analysis. Docking experiments showed caspase-3 and caspase-9 as possible molecular targets for these compounds. This was further validated by cleavage of PARP, a caspase target in apoptotic pathway. Compound 7 was found non-toxic up to 1000mg/kg dose in acute oral toxicity in Swiss albino mice.

Adenoviral-Mediated Glial Cell Line–Derived Neurotrophic Factor Gene Transfer Has a Protective Effect on Sciatic Nerve Following Constriction-Induced Spinal Cord Injury

Neuropathic pain due to peripheral nerve injury may be associated with abnormal central nerve activity. Glial cell-line-derived neurotrophic factor (GDNF) can help attenuate neuropathic pain in different animal models of nerve injury. However, whether GDNF can ameliorate neuropathic pain in the spinal cord dorsal horn (SCDH) in constriction-induced peripheral nerve injury remains unknown. We investigated the therapeutic effects of adenoviral-mediated GDNF on neuropathic pain behaviors, microglial activation, pro-inflammatory cytokine expression and programmed cell death in a chronic constriction injury (CCI) nerve injury animal model. In this study, neuropathic pain was produced by CCI on the ipsilateral SCDH. Mechanical allodynia was examined with von Frey filaments and thermal sensitivity was tested using a plantar test apparatus post-operatively. Target proteins GDNF-1, GDNFRa-1, MMP2, MMP9, p38, phospho-p38, ED1, IL6, IL1β, AIF, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, cleaved PARP, SPECTRIN, cleaved SPECTRIN, Beclin-1, PKCσ, PKCγ, iNOS, eNOS and nNOS were detected. Microglial activity was measured by observing changes in immunoreactivity with OX-42. NeuN and TUNEL staining were used to reveal whether apoptosis was attenuated by GDNF. Results showed that administrating GDNF began to attenuate both allodynia and thermal hyperalgesia at day 7. CCI-rats were found to have lower GDNF and GDNFRa-1 expression compared to controls, and GDNF re-activated their expression. Also, GDNF significantly down-regulated CCI-induced protein expression except for MMP2, eNOS and nNOS, indicating that the protective action of GDNF might be associated with anti-inflammation and prohibition of microglia activation. Immunocytochemistry staining showed that GDNF reduced CCI-induced neuronal apoptosis. In sum, GDNF enhanced the neurotrophic effect by inhibiting microglia activation and cytokine production via p38 and PKC signaling. GDNF could be a good therapeutic tool to attenuate programmed cell death, including apoptosis and autophagy, consequent to CCI-induced peripheral nerve injury.