Mcl1 protein levels and Caspase-7 executioner protease control axial organizer cells survival.

BACKGROUND: Organizing centers are groups of specialized cells that secrete morphogens, thereby influencing development of their neighboring territories. Apoptosis is a form of programmed cell death reported to limit the size of organizers. Little is known about the identity of intracellular signals driving organizer cell death. Here we investigated in Xenopus the role of both the anti-apoptotic protein Myeloid-cell-leukemia 1 (Mcl1) and the cysteine proteases Caspase-3 and Caspase-7 in formation of the axial organizing center-the notochord-that derives from the Spemann organizer, and participates in the induction and patterning of the neuroepithelium. RESULTS: We confirm a role for apoptosis in establishing the axial organizer in early neurula. We show that the expression pattern of mcl1 is coherent with a role for this gene in early notochord development. Using loss of function approaches, we demonstrate that Mcl1 depletion decreases neuroepithelium width and increases notochord cells apoptosis, a process that relies on Caspase-7, and not on Caspase-3, activity. Our data provide evidence that Mcl1 protein levels physiologically control notochord cells' survival and that Caspase-7 is the executioner protease in this developmental process. CONCLUSIONS: Our study reveals new functions for Mcl1 and Caspase-7 in formation of the axial signalling center.

The effect of Lipopolysaccharide (LPS) pretreatment on hippocampal apoptosis in traumatic rats.

Objectives: Traumatic brain injury (TBI) is a serious medical problem that affects the quality of life. Apoptosis is a form of programmed cell death that happens after trauma. Effector caspases are responsible for initiating apoptosis.Methods: In the present study, we examined the effect of LPS preconditioning (0.1 and 0.5 mg/kg, ip; 5 days prior controlled cortical injury) on apoptosis, 4 and 12 hours after trauma. We investigated possible mechanisms on the expression of caspase3 and caspase7 in hippocampal CA1 and CA3 areas by using immunohistochemistry and Western blotting techniques and also TUNEL-positive cells.Results: Higher expression of caspase3 and caspase7 were accompanied by a higher number of dead neurons in traumatic rats 4 and 12 hours after trauma(P < 0.05). LPS preconditioning decreased caspase3 and caspase7over-expression and the number of dead neurons in the hippocampus(P < 0.05).Discussion: Our data indicate that LPS preconditioning inhibits neural damage and apoptosis induced by trauma in the hippocampus.

NADPH oxidase inhibitor VAS2870 prevents staurosporine-induced cell death in rat astrocytes.

Background Astrocytes maintain central nerve system homeostasis and are relatively resistant to cell death. Dysfunction of cell death mechanisms may underlie glioblastoma genesis and resistance to cancer therapy; therefore more detailed understanding of astrocytic death modalities is needed in order to design effective therapy. The purpose of this study was to determine the effect of VAS2870, a pan-NADPH oxidase inhibitor, on staurosporine-induced cell death in astrocytes. Materials and methods Cultured rat astrocytes were treated with staurosporine as activator of cell death. Cell viability, production of reactive oxygen species (ROS), and mitochondrial potential were examined using flow cytometric analysis, while chemiluminescence analysis was performed to assess caspase 3/7 activity and cellular ATP. Results We show here for the first time, that VAS2870 is able to prevent staurosporine-induced cell death. Staurosporine exerts its toxic effect through increased generation of ROS, while VAS2870 reduces the level of ROS. Further, VAS2870 partially restores mitochondrial inner membrane potential and level of ATP in staurosporine treated cells. Conclusions Staurosporine induces cell death in cultured rat astrocytes through oxidative stress. Generation of ROS, mitochondrial membrane potential and energy level are sensitive to VAS2870, which suggests NADPH oxidases as an important effector of cell death. Consequently, NADPH oxidases activation pathway could be an important target to modulate astrocytic death.

Methamphetamine Induces Apoptosis of Microglia via the Intrinsic Mitochondrial-Dependent Pathway.

Methamphetamine (METH) is a drug of abuse, the acute and chronic use of which induces neurotoxic responses in the human brain, ultimately leading to neurocognitive disorders. Our goals were to understand the impact of METH on microglial mitochondrial respiration and to determine whether METH induces the activation of the mitochondrial-dependent intrinsic apoptosis pathway in microglia. We assessed the expression of pro- apoptosis genes using qPCR of RNA extracted from a human microglial cell line (HTHU). We examined the apoptosis-inducing effects of METH on microglial cells using digital holographic microscopy (DHM) to quantify real-time apoptotic volume decrease (AVD) in microglia in a noninvasive manner. METH treatment significantly increased AVD, activated Caspase 3/7, increased the gene expression levels of the pro- apoptosis proteins, APAF-1 and BAX, and decreased mitochondrial DNA content. Using immunofluorescence analysis, we found that METH increased the expression of the mitochondrial proteins cytochrome c and MCL-1, supporting the activation of mitochondrion-dependent (intrinsic) apoptosis pathway. Cellular bio-energetic flux analysis by Agilent Seahorse XF Analyzer revealed that METH treatment increased both oxidative and glycolytic respiration after 3 h, which was sustained for at least 24 h. Several events, such as oxidative stress, neuro-inflammatory responses, and mitochondrial dysfunction, may converge to mediate METH-induced apoptosis of microglia that may contribute to neurotoxicity of the CNS. Our study has important implications for therapeutic strategies aimed at preserving mitochondrial function in METH abusing patients.

Chlorinated cobalt alkyne complexes derived from acetylsalicylic acid as new specific antitumor agents.

[(Prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl (Co-ASS), an organometallic derivative of the irreversible cyclooxygenase-1/2 (COX-1/2) inhibitor acetylsalicylic acid (ASS), demonstrated high growth-inhibitory potential against various tumor cell lines and inhibition of both COX isoenzymes. With the objective of increasing the selectivity for COX-2, we introduced a chlorine substituent in position 3, 4, 5, or 6 of the ASS moiety, respectively. Increased COX-2 selectivity is desirable as this isoenzyme is predominantly related to the development of cancer and abnormal tissue growth. The new compounds were investigated in comprehensive cellular biological assays to identify the impact of the chlorine substitution at the complex on COX-1/2 inhibition, antiproliferative activity, apoptosis, metabolic activity, cell-based COX inhibition, and cellular uptake. Chlorination distinctly reduced the effects at isolated COX-1 (about 25% inhibition at 10 µM; Co-ASS: 82.7%), while those at COX-2 remained almost unchanged (about 65% inhibition at 10 µM; Co-ASS: 78.5%). In cellular systems, with exception of the 6-Cl derivative, all compounds showed notable antitumor activity in COX-1/2 expressing tumor cells (HT-29 (IC50 = 1.5-2.7 µM), MDA-MB-231 (IC50 = 5.2-8.0 µM)), but were distinctly less active in the COX-1/2-negative MCF-7 breast cancer cell line (IC50 = 15.2-22.9 µM). All complexes possess high selectivity for tumor cells, because they did not influence the growth of the non-tumorigenic, human bone marrow stromal cell line HS-5. These findings clearly demonstrate that the interference with the COX-1/2 cascade contributes to the mode of anticancer action of the cobalt alkyne complexes.

Plasma Derived From Human Umbilical Cord Blood Modulates Mitogen-Induced Proliferation of Mononuclear Cells Isolated From the Peripheral Blood of ALS Patients.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by degeneration of motor neurons in the spinal cord and brain. This disease clinically manifests as gradual muscular weakness and atrophy leading to paralysis and death by respiratory failure. While multiple interdependent factors may contribute to the pathogenesis of ALS, increasing evidence shows the possible presence of autoimmune mechanisms that promote disease progression. The potential use of plasma derived from human umbilical cord blood (hUCB) as a therapeutic tool is currently in its infancy. The hUCB plasma is rich in cytokines and growth factors that are required for growth and survival of cells during hematopoiesis. In this study, we investigated the effects of hUCB plasma on the mitogen-induced proliferation of mononuclear cells (MNCs) isolated from the peripheral blood of ALS patients and apoptotic activity by detection of caspase 3/7 expression of the isolated MNCs in vitro. Three distinct responses to phytohemagglutinin (PHA)-induced proliferation of MNCs were observed, which were independent of age, disease duration, and the ALS rating scale: Group I responded normally to PHA, Group II showed no response to PHA, while Group III showed a hyperactive response to PHA. hUCB plasma attenuated the hyperactive response (Group III) and potentiated the normal response in Group I ALS patients, but did not alter that of the nonresponders to PHA (Group II). The elevated activity of caspase 3/7 observed in the MNCs from ALS patients was significantly reduced by hUCB plasma treatment. Thus, study results showing different cell responses to mitogen suggest alteration in lymphocyte functionality in ALS patients that may be a sign of immune deficiency in the nonresponders and autoimmunity alterations in the hyperactive responders. The ability of hUCB plasma to modulate the mitogen cell response and reduce caspase activity suggests that the use of hUCB plasma alone, or with stem cells, may prove useful as a therapeutic in ALS patients.

Curcumin induces differential expression of cytoprotective enzymes but similar apoptotic responses in fibroblasts and myofibroblasts.

Excessive extracellular matrix (ECM) deposition and tissue contraction after injury can lead to esthetic and functional problems. Fibroblasts and myofibroblasts activated by transforming growth factor (TGF)-ß1 play a key role in these processes. The persistence of (myo)fibroblasts and their excessive ECM production and continuous wound contraction have been linked to pathological scarring. The identification of compounds reducing myofibroblast survival and function may thus offer promising therapeutic strategies to optimize impaired wound healing. The plant-derived polyphenol curcumin has shown promising results as a wound healing therapeutic in vivo; however, the exact mechanism is still unclear. In vitro, curcumin induces apoptosis in various cell types via a reactive oxygen species (ROS)-dependent mechanism. Here we treated human dermal fibroblasts with TGF-ß1 to induce myofibroblast differentiation, and compared the responses of fibroblasts and myofibroblasts to 25 µM curcumin. Curcumin induced caspase-independent apoptosis in both fibroblasts and myofibroblasts in a ROS-dependent manner. Oxidative stress leads to the induction of several antioxidant systems to regain cellular homeostasis. We detected stress-induced induction of heme oxygenase (HO)-1 in fibroblasts but not in myofibroblasts following curcumin exposure. Instead, myofibroblasts expressed higher levels of heat shock protein (HSP)72 compared to fibroblasts in response to curcumin, suggesting that TGF-ß1 treatment alters the stress-responses of the cells. However, we did not detect any differences in curcumin toxicity between the two populations. The differential stress responses in fibroblasts and myofibroblasts may open new therapeutic approaches to reduce myofibroblasts and scarring.

Induction of apoptosis by eicosapentaenoic acid in esophageal squamous cell carcinoma.

BACKGROUND: Eicosapentaenoic acid (EPA) suppresses the proliferation of cell lines derived from colon, pancreatic, breast and other cancers. Few reports have described the effect of EPA on esophageal cancer cell lines. MATERIALS AND METHODS: We investigated the effect of EPA on the proliferation of the esophageal squamous cell carcinoma cell lines TE11 and KYSE180 with a WST-1 assay. Apoptosis was evaluated with a DNA fragmentation assay. Levels of apoptosis-related proteins (caspase-3, -7, -9 and poly (ADP-ribose) polymerase (PARP)) and cleaved caspase-3, -7, -9 and PARP were evaluated by western blot analysis. RESULTS: After exposure to EPA for 24 h, KYSE180 and TE11 cell proliferation was suppressed in a dose-dependent manner (p<0.05). In addition, caspase -3, -7, -9 and PARP were activated. EPA (0.1 µM, 1 µM, 10 µM) induced apoptosis in a dose-dependent manner, as detected by the DNA fragmentation assay. CONCLUSION: EPA shows potential as a new treatment for esophageal cancer.

Andrographolide induces apoptosis of C6 glioma cells via the ERK-p53-caspase 7-PARP pathway.

BACKGROUND: Glioma is the most malignant tumor of the central nervous system. Efforts on the development of new chemotherapy are mandatory. Andrographolide (AND), a diterpenoid lactone isolated from the Andrographis paniculata, has been shown to have antitumor activities in several types of cancer cells. Whether AND can exert its antitumor activity in glioblastoma cells remains unknown. This study examined the anticancer effects of AND, both in vitro and in vivo. METHODS: Cell apoptosis was assayed by flow cytometry and nuclear staining. The signaling pathway for AND was determined by western blotting. The effects of AND on tumor growth was evaluated in a mouse model. RESULTS AND CONCLUSION: In vitro, with application of specific inhibitors and siRNA, AND-induced apoptosis was proven through ROS-ERK-P53-caspase 7-PARP signaling pathway. In vivo, AND significantly retarded tumor growth and caused regression of well-formed tumors in vivo. Furthermore, AND did not induce apoptosis or activate ERK and p53 in primary cultured astrocyte cells, and it may serve as a potential therapeutic candidate for the treatment of glioma.

Characterization and sorting of flow cytometric populations in human semen.

Human semen is a complex biological matrix. It contains mature spermatozoa, immature germ cells, residual apoptotic bodies and, in some cases, epithelial cells and leucocytes. Hence, one of the challenges in applying flow cytometry in spermatology is the correct recognition of spermatozoa and their separation from signals of other semen cells/elements. In this study, we show that semen spermatozoa are included in a well-defined, flame-shaped FSC/SSC region (FR), by demonstrating that the count of the spermatozoa contained in such region overlaps that obtained by microscopy in the same samples. In FR, nuclear staining of semen samples reveals three different populations: unstained, brighter and dimmer. Unstained elements were previously characterized as apoptotic bodies of testis origin and the brighter elements represent the majority of semen spermatozoa, whereas the composition and the origin of the population with a lower nuclear staining is less clear, albeit we have previously shown that all the elements constituting it are positive for TUNEL. In this study, we sorted all the elements contained in FR region and demonstrated that the dimmer elements are spermatozoa. To further characterize dimmer spermatozoa, we evaluated apoptotic caspases and chromatin immaturity, the latter detected by aniline blue (AB) and chromomycin A (CMA3) staining. We found that caspases were much more expressed in the dimmer spermatozoa (71.4 ± 18.8%) than in the brighter (46.7 ± 15.1%), whereas similar amounts of spermatozoa with chromatin immaturity were found in both populations (brighter, AB: 48.2 ± 19.5%; CMA3: 48.5 ± 20.4% and dimmer, AB: 43.4 ± 19.8%; CMA3: 36.1 ± 18.0%). Hence, the role of apoptosis in generating dimmer spermatozoa and their DNA fragmentation appears clear, whereas the involvement of defects during the chromatin packaging remains elusive.

The Pla protease of Yersinia pestis degrades fas ligand to manipulate host cell death and inflammation.

Pneumonic plague is a deadly respiratory disease caused by Yersinia pestis. The bacterial protease Pla contributes to disease progression and manipulation of host immunity, but the mechanisms by which this occurs are largely unknown. Here we show that Pla degrades the apoptotic signaling molecule Fas ligand (FasL) to prevent host cell apoptosis and inflammation. Wild-type Y. pestis, but not a Pla mutant (Δpla), degrades FasL, which results in decreased downstream caspase-3/7 activation and reduced apoptosis. Similarly, lungs of mice challenged with wild-type Y. pestis show reduced levels of FasL and activated caspase-3/7 compared to Δpla infection. Consistent with a role for FasL in regulating immune responses, Δpla infection results in aberrant proinflammatory cytokine levels. The loss of FasL or inhibition of caspase activity alters host inflammatory responses and enables enhanced Y. pestis outgrowth in the lungs. Thus, by degrading FasL, Y. pestis manipulates host cell death pathways to facilitate infection.

Enhanced antitumor efficacy with combined administration of astragalus and pterostilbene for melanoma.

Astragalus, a commonly used traditional Chinese medicine, has exhibited antitumor actions in patients. In this study, in vitro and in vivo antitumor effects of astragalus and synergistic antitumor efficacy in combination with pterostilbene were investigated. Melanoma cells were treated with pterostilbene (Pt), graduated doses of astragalus injection (AI), or these in combination. Cell viability was measured using a MTT assay. Released nucleosomes and caspase activity were measured using enzyme-linked immunosorbent assay. Growth inhibition in vitro and in vivo was also assessed. Analysis of variance and t tests were used for statistical analysis. Significant reduction (p<0.05) in cellular proliferation were observed with AI and AI-Pt in a time- and concentration-dependent manner. Apoptosis and caspase-3/7 activity were significantly increased by AI and AI-Pt treatment (p<0.05). In vivo, AI inhibited melanoma tumor growth, with inhibition rates ranging from 36.5 to 62.3%, by inducing apoptosis via up-regulation Bax expression and the Bax/Bcl-2 ratio and down-regulating Bcl-2 expression. AI significantly inhibits the growth of melanoma in vitro and in vivo by inducing apoptosis. These data suggest that combined treatment of astragalus with pterostilbene enhances antitumor efficacy.

Induction of apoptosis by diphenyldifluoroketone in osteogenic sarcoma cells is associated with activation of caspases.

The aim of the present study was to investigate and compare the effects of diferuloylmethane (curcumin) and diphenyldifluoroketone (EF-24) on cell growth and apoptosis induction in human osteogenic sarcoma cells. This was examined by MTT assay, nuclear DAPI staining, caspase-activation assay, flow cytometry analysis and immunoblotting in Saos2 human osteogenic sarcoma cells. Curcumin and EF-24 inhibited the growth of Saos2 cells in a dose-dependent manner. The apparent potency of EF-24 was more than 3-fold higher that of curcumin. Treatment with curcumin or EF-24 resulted in nuclear condensation and fragmentation in the cells. The caspase-3/-7 activities were detected in living cells treated with curcumin or EF-24. Flow cytometry showed that the rate of apoptosis was increased by curcumin and EF-24 compared to the control. Curcumin and EF-24 promoted the proteolytic cleavages of procaspase-3/-7/-8/-9 with increases in the amount of cleaved caspase-3/-7/-8/-9. The curcumin- or EF-24-induced apoptosis in the Saos2 cells was mediated by the expression of Fas and activation of caspase-8, caspase-3 and poly(ADP-ribose) polymerase. Immunoblotting revealed the Bid and Bcl-2 proteins to be downregulated, and truncated-Bid, Bax and p53 proteins to be upregulated by curcumin and EF-24. Curcumin and EF-24 increased the Bax/Bcl-2 ratio significantly. These results suggest that the curcumin and EF-24 inhibit cell proliferation and induce apoptotic cell death in Saos2 human osteogenic sarcoma cells via both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway, and may have potential properties for anti-osteosarcoma drug discovery.

Obestatin changes proliferation, differentiation and apoptosis of porcine preadipocytes.

Obestatin, originally identified and purified from rat stomach extracts, was reported to bind to orphan G protein-coupled receptor, GPR39, and inhibit appetite and gastric motility. This study was conducted to investigate the effects of porcine obestatin on proliferation, differentiation and apoptosis of porcine preadipocytes isolated from subcutaneous fat of piglets. At indicated times of culture, morphology of preadipocytes and accumulated lipid droplets within the cells were identified by invert microscope. After treating with obestatin (0, 0.1, 1, 10 and 100nM), cell proliferation was measured by MTT method and protein expression of CCAAT/enhancer binding protein-α (C/EBPα), peroxisome proliferator-activated receptor-γ (PPARγ), Caspase-7 and Caspase-9 was determined by Western Blot, mRNA expression of GPR39 and Caspase-3 was analyzed by RT-PCR, and the activity of Caspase-3 was measured by spectrophotometric method. The results showed that obestatin had no effect on GPR39 expression, while promotes the optical density (OD) value of cells, enhanced protein expression of PPARγ and C/EBPa, decreased mRNA expression and activity of Caspase-3, and inhibited protein expression of Caspase-7 and Caspase-9 in a dose-dependent manner. These results suggested that obestatin enhances proliferation and differentiation of preadipocytes promoting PPARγ and C/EBPa expression, and inhibiting preadipocyte apoptosis by decreasing expression of Caspase-3, Caspase-7 and Caspase-9.

Loss of caspase 7 expression is associated with poor prognosis in renal cell carcinoma clear cell subtype.

OBJECTIVE: To investigate the expression of CASP7 protein in renal cell carcinoma clear cell subtype (ccRCC) and its value to predict cancer-specific survival (CSS). METHODS: A tissue microarray containing 120 samples of ccRCC, 45 non-ccRCC, and 66 nontumor paired samples from patients who underwent partial or radical nephrectomy was hybridized with anti-CASP7 antibody. Tissue sections were scored according to intensity and the percentage of stained cells. CASP7 immunostaining scores were used to estimate the association with clinicopathologic parameters and calculate Kaplan-Meier survival curves. RESULTS: Reduced CASP7 expression was observed in ccRCC and non-ccRCC subtypes in comparison with nontumor renal tissues (P <.0001). CASP7 immunostaining was associated (P <.05) with clinicopathologic parameters (size, incidental tumor, clinical stage, renal vein invasion, and tumor necrosis) and correlated with CSS (P = .032) and global survival (P = .046) of patients with ccRCC. In addition, CASP7 expression was able to substratify patients with ccRCC with favorable prognosis according to low clinical stage, in which negative CASP7 staining was associated with patients with lower CSS (P = .045). Finally, CASP7 staining was able to provide significant stratification according to CSS (P = .018) among patients with ccRCC with disease relapse. CONCLUSION: Our results implicate the loss of CASP7 expression in the aggressiveness of ccRCC and indicate its potential use as a clinical prognostic marker of the disease.

Engineered soluble monomeric IgG1 CH3 domain: generation, mechanisms of function, and implications for design of biological therapeutics.

Most of the therapeutic antibodies approved for clinical use are full-size IgG1 molecules. The interaction of the IgG1 Fc with the neonatal Fc receptor (FcRn) plays a critical role in maintaining their long half-life. We have hypothesized that isolated Fc domains could be engineered to functionally mimic full-size IgG1 (nanoantibodies) but with decreased (10-fold) size. Here, we report for the first time the successful generation of a soluble, monomeric CH3 domain (mCH3). In contrast to the wild-type dimeric CH3, the mCH3 exhibited pH-dependent binding to FcRn similar to that of Fc. The binding free energy of mCH3 to FcRn was higher than that of isolated CH2 but lower than that of Fc. Therefore, CH3 may contribute a larger portion of the free energy of binding to FcRn than CH2. A fusion protein of mCH3 with an engineered antibody domain (m36.4) also bound to FcRn in a pH-dependent fashion and exhibited significantly higher neutralizing activity against HIV-1 than m36.4-Fc fusion proteins. The m36.4-mCH3 fusion protein was monomeric, stable, soluble, and expressed at a high level in Escherichia coli. We also found that engineering an additional disulfide bond in mCH3 remarkably increased its thermal stability, whereas the FcRn binding was not affected. These data suggest that mCH3 could not only help in the exploration of the dual mechanisms of the CH3 contribution to Fc functions (dimerization and FcRn interactions) but could also be used for the development of candidate therapeutics with optimized half-life, enhanced tissue penetration, access to sterically restricted binding sites, and increased therapeutic efficacy.

Stable inhibition of mmu-miR-466h-5p improves apoptosis resistance and protein production in CHO cells.

MiRNAs have been shown to be involved in regulation of multiple cellular processes including apoptosis. Since a single miRNA can affect the expression of several genes, the utilization of miRNAs for apoptosis engineering in mammalian cells can be more efficient than the conventional approach of manipulating a single gene. Mmu-miR-466h-5p was previously shown to have a pro-apoptotic role in CHO cells by reducing the expression of several anti-apoptotic genes and its transient inhibition delayed both the activation of Caspase-3/7 and the loss of cell viability. The present study evaluates the effect of stable inhibition of mmu-miR-466h-5p in CHO cells on their ability to resist apoptosis onset and their production properties. The expression of mmu-miR-466h-5p in the engineered anti-miR-466h CHO cell line was significantly lower than in the negative control and the parental CHO cells. These engineered cells reached higher maximum viable cell density and extended viability compared with negative control and parental CHO cells in batch cell cultures which resulted in the 53.8% and 41.6% increase of integral viable cells. The extended viability of anti-miR-466h CHO cells was the result of delayed Caspase-3/7 activation by more than 35h, and the increased levels of its anti-apoptotic gene targets (smo, stat5a, dad1, birc6, and bcl2l2) to between 2.1- and 12.5-fold compared with the negative control CHO in apoptotic conditions. The expression of secreted alkaline phosphatase (SEAP) increased 43% and the cell-specific productivity increased 11% in the stable pools of anti-miR-466h CHO compared with the stable pools of negative control CHO cells. The above results demonstrate the potential of this novel approach to create more productive cell lines through stable manipulation of specific miRNA expression.

T cell depletion protects against alveolar destruction due to chronic cigarette smoke exposure in mice.

The role of T cells in chronic obstructive pulmonary disease (COPD) is not well understood. We have previously demonstrated that chronic cigarette smoke exposure can lead to the accumulation of CD4(+) and CD8(+) T cells in the alveolar airspaces in a mouse model of COPD, implicating these cells in disease pathogenesis. However, whether specific inhibition of T cell responses represents a therapeutic strategy has not been fully investigated. In this study inhibition of T cell responses through specific depleting antibodies, or the T cell immunosuppressant drug cyclosporin A, prevented airspace enlargement and neutrophil infiltration in a mouse model of chronic cigarette smoke exposure. Furthermore, individual inhibition of either CD4(+) T helper or CD8(+) T cytotoxic cells prevented airspace enlargement to a similar degree, implicating both T cell subsets as critical mediators of the adaptive immune response induced by cigarette smoke exposure. Importantly, T cell depletion resulted in significantly decreased levels of the Th17-associated cytokine IL-17A, and of caspase 3 and caspase 7 gene expression and activity, induced by cigarette smoke exposure. Finally, inhibition of T cell responses in a therapeutic manner also inhibited cigarette smoke-induced airspace enlargement, IL-17A expression, and neutrophil influx in mice. Together these data demonstrate for the first time that therapeutic inhibition of T cell responses may be efficacious in the treatment of COPD. Given that broad immunosuppression may be undesirable in COPD patients, this study provides proof-of-concept for more targeted approaches to inhibiting the role of T cells in emphysema development.

Oncolytic virotherapy for human bone and soft tissue sarcomas using live attenuated poliovirus.

The poliovirus receptor CD155, is essential for poliovirus to infect and induce death in neural cells. Recently, CD155 has been shown to be selectively expressed on certain types of tumor cells originating from the neural crest, including malignant glioma and neuroblastoma. However, the expression pattern of CD155 in soft tissue sarcoma has not been examined. Therefore, we first examined CD155 expression in sarcoma cell lines, and found the expression of both CD155 mRNA and protein in 12 soft and bone tissue sarcoma cell lines. Furthermore, we examined the effect of live attenuated poliovirus (LAPV) on 6 bone and soft tissue sarcoma cell lines in vitro, and found that LAPV induced apoptosis by activating caspases 7 and 3 in all of these cell lines. Furthermore, in BALB/c nu/nu mice xenotransplanted with HT1080 fibrosarcoma cells, administration of live attenuated poliovirus caused growth suppression of the tumors. These results suggest that oncolytic therapy using a LAPV may represent a new option for the treatment of bone and soft tissue sarcomas.

Targeted suppression of µ-calpain and caspase 9 expression and its effect on caspase 3 and caspase 7 in satellite cells of Korean Hanwoo cattle.

The calpains play an important role in cell death and cell signalling. Caspases catalyse wholesale destruction of cellular proteins which is a major cause of cellular death. The current study looks at the function of µ-calpain and caspase 9, using RNAi (RNA interference)-mediated silencing, and to observe the mRNA expression level of caspase genes during satellite cell growth. The satellite cells were treated with siRNA (small interfering RNA) of µ-calpain and caspase 9 separately. There was reduction of 16 and 24% in CAPN1 (calpain1)-siRNA2 and CAPN1-siRNA3 transfected cells respectively, whereas it was 60 and 56% in CAPN1-siRNA1 and CAPN1-siRNA4 transfected cells respectively. CAPN1-siRNA4 and CAPN1-siRNA1 treated cells showed more reduction in caspase 3 and 7 gene expression. CARD9 (caspase recruitment domain 9)-siRNA1 and CARD9-siRNA2-treated cells showed reduction of 40 and 49% respectively. CARD9-siRNA1 and CARD9-siRNA2 showed an increase in caspase 3 gene expression, whereas CARD9-siRNA2 showed reduction in caspase 7 gene expression. These results suggest a strong cross-talk between µ-calpain and the caspase enzyme systems. Suppression of target genes, such as µ-calpain and caspase 9, might have genuine potential in the treatment of skeletal muscle atrophy.