A non-apoptotic role for caspase-9 in muscle differentiation

Caspases, a family of cysteine proteases most often investigated for their roles in apoptosis, have also been demonstrated to have functions that are vital for the efficient execution of cell differentiation. One such role that has been described is the requirement of caspase-3 for the differentiation of skeletal myoblasts into myotubes but, as yet, the mechanism leading to caspase-3 activation in this case remains elusive. Here, we demonstrate that caspase-9, an initiator caspase in the mitochondrial death pathway, is responsible for the activation of caspase-3 in differentiating C2C12 cells. Reduction of caspase-9 levels, using an shRNA construct, prevented caspase-3 activation and inhibited myoblast fusion. Myosin-heavy-chain expression, which accompanies myoblastic differentiation, was not caspase-dependent. Overexpression of Bcl-xL, a protein that inhibits caspase-9 activation, had the same effect on muscle differentiation as knockdown of caspase-9. These data suggest that the mitochondrial pathway is required for differentiation; however, the release of cytochrome c or Smac (Diablo) could not be detected, raising the possibility of a novel mechanism of caspase-9 activation during muscle differentiation.</jats:p

Uterine Endoplasmic Reticulum Stress and Its Unfolded Protein Response May Regulate Caspase 3 Activation in the Pregnant Mouse Uterus

We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manne

Regulation of caspase-3 processing by cIAP2 controls the switch between pro-inflammatory activation and cell death in microglia.

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International Licence. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons licence, users will need to obtain permission from the licence holder to reproduce the material.The activation of microglia, resident immune cells of the central nervous system, and inflammation-mediated neurotoxicity are typical features of neurodegenerative diseases, for example, Alzheimer's and Parkinson's diseases. An unexpected role of caspase-3, commonly known to have executioner role for apoptosis, was uncovered in the microglia activation process. A central question emerging from this finding is what prevents caspase-3 during the microglia activation from killing those cells? Caspase-3 activation occurs as a two-step process, where the zymogen is first cleaved by upstream caspases, such as caspase-8, to form intermediate, yet still active, p19/p12 complex; thereafter, autocatalytic processing generates the fully mature p17/p12 form of the enzyme. Here, we show that the induction of cellular inhibitor of apoptosis protein 2 (cIAP2) expression upon microglia activation prevents the conversion of caspase-3 p19 subunit to p17 subunit and is responsible for restraining caspase-3 in terms of activity and subcellular localization. We demonstrate that counteracting the repressive effect of cIAP2 on caspase-3 activation, using small interfering RNA targeting cIAP2 or a SMAC mimetic such as the BV6 compound, reduced the pro-inflammatory activation of microglia cells and promoted their death. We propose that the different caspase-3 functions in microglia, and potentially other cell types, reside in the active caspase-3 complexes formed. These results also could indicate cIAP2 as a possible therapeutic target to modulate microglia pro-inflammatory activation and associated neurotoxicity observed in neurodegenerative disorders

Impact of L-carnitine and selenium treatment on testicular apoptosis in rats exposed to 2.45 GHz microwave energy

Objective: It has been suggested that electromagnetic radiation (EMR) by wireless devices (2.45 GHz) induces testicular apoptosis. We investigated if supplemental selenium (Se) and L-carnitine may reduce this adverse effect. Material: Twelve-week old maleWistar albino rats were used in this study. Twenty-four rats were equally divided into four groups which were named as: sham group, EMR-only, EMR+L-carnitine (1.5 mg L-carnitine/ kg/day) and EMR+Se (1.5 mg Se/kg/ every other day). Results: The level of Bcl-2, Bax, caspase-3 and -8 were compared and a significant difference was found between the sham and EMR-only groups (p < 0.05), and Bcl-2, Bax, caspase-3 and -8 expressions increased in the EMR-only group. The level of Bcl-2, Bax, tumour necrosis factor-alpha (TNF-α), caspase- 3 and -8 were compared and a significant difference was found between the sham and EMR+L-carnitine groups (p < 0.05) and Bcl-2, Bax, TNF-α, caspase-3 and -8 expressions increased in the EMR+L-carnitine group. The level of Bcl-2, Bax, TNF-α, caspase-3 and -8 were compared and a significant difference was found between the sham and EMR+Se groups (p < 0.05) and Bcl-2, Bax, TNF-α, caspase-3 and -8 expressions increased in the EMR+Se group. When the expression of caspase-8 was compared, a significant difference was found between the EMR-only and EMR+Se groups (p < 0.05). Caspase-8 expression decreased in EMR+Se group compared with EMR-only group. Conclusion: Electromagnetic radiation exposure resulted in testicular apoptosis in rats, mainly by the intrinsic pathways by down-regulated expression of caspase-8. Reduction in the activation of the intrinsic pathway of apoptosis was found higher with selenium administration compared with L-carnitine administration

Beneficial role of allicin from garlic in cervical cancer

Introduction: Cervical cancer remains a global health concern for females. Thus, in order to control cervical cancer, attempts are being made by researchers globally to somehow induce programmed cell death in the said cancerous cells. Wide spectrums of molecules are being probed for its ability to induce apoptosis in cervical cancer cells. Focus has now shifted in exploring natural compounds having antioxidant and anti-inflammatory molecules that may induce apoptosis in cancerous cells. Thus, we have employed allicin from garlic- a natural antioxidant, to probe the above in the present study.&#xd;&#xa;Objective: To probe whether or not allicin from garlic, a natural antioxidant, induces apoptosis in monocytes from patients with cervical cancer.&#xd;&#xa;Results: Allicin (500 ng/ml) reduced cell viability to 27% after 24 hours of treatment. Moreover, allicin-induced apoptosis was ascertained by measuring the activity of caspase-3, caspase-8 and caspase-9-like proteases in allicin treated and untreated monocytes from cervical cancer patients. Monocyte co-cultured with allicin for 24 hrs exhibited higher activity of caspase-3 followed by caspase-8 and caspase-9 like proteases, thereby indicating that the activation of caspase-3 like proteases was associated with reduced cell survival and apoptotic death of allicin-treated cervical cancer monocytes. This was ascertained by pre-treatment of cancer cells with cell permeable inhibitor Z-VAD-FMK (caspase-3 inhibitor), Z-IETD-FMK (caspase-8 inhibitor) and Z-LEHD-FMK (caspase-9 inhibitor) followed by allicin for 24 hrs (p&#x3c;0.001). In this case, the cell viability assay showed that the presence of Z-VAD-FMK inhibitor blocked the effect of allicin on the viability of cancer monocytes (p&#x3c;0.001).&#xd;&#xa;Conclusion: Allicin from garlic may act as an adjunct in the chemotherapy of cervical cancer.&#xd;&#xa

Metalloporphyrins inactivate caspase-3 and -8

Activation of caspases represents one of the earliest biochemical indicators for apoptotic cell death. Therefore, measurement of caspase activity is a widely used and generally accepted method to determine apoptosis in a wide range of in vivo and in vitro settings. Numerous publications characterize the role of the heme-catabolizing enzyme heme oxygenase-1 (HO-1) in regulating apoptotic processes. Different metalloporphyrins representing inducers and inhibitors of this enzyme are often used, followed by assessment of apoptotic cell death. In the present work, we found that caspase-3-like activity, as well as activity of caspase-8 measured in either Fas (CD95) ligand-treated Jurkat T-lymphocytes or by the use of recombinant caspase-3 or -8, was inhibited by different metalloporphyrins (cobalt(III) protoporphyrin IX, tin and zinc II) protoporphyrin-IX). Moreover, employing the mouse model of Fas-induced liver apoptosis these properties of porphyrins could also be demonstrated in vivo. The metalloporphyrins were shown to inhibit caspase-3-mediated PARP cleavage. Molecular modeling studies demonstrated that porphyrins can occupy the active site of caspase-3 in an energetically favorable manner and in a binding mode similar to that of known inhibitors. The data shown here introduce metalloporphyrins as direct inhibitors of caspase activity. This finding points to the need for careful employment of metalloporphyrins as modulators of HO-1

Association of B-cell Lymphoma Protein-2 and Caspase-3 Expression in Ovarian Cancer

Ovarian cancer remains a major problem of women's health in the world, including Indonesia, and is associated with high rates of incidence and mortality. There are many efforts in early diagnosis on ovarian cancer, but until now there have not been found any satisfactory method. On the other hand, knowledge and research in the field of molecular biology become more advance, one of them is a mechanism to control the growth of cells in ovarian cancer through a process of programmed cell death or apoptosis. B-cell lymphoma protein 2 (Bcl-2) and caspase-3 are proteins that play a role on the mechanism of apoptosis. The purpose of this study was to determine the expression of Bcl-2 and caspase-3 and their association with ovarian cancer. Materials and method: The design of this study was a cross-sectional study. Expression of Bcl-2 and caspase-3 examined by immunohistochemistry under light microscope with 400x light power field and expression as a negative when the protein expressed in 10% or less of cells and as a positive when the protein expressed in more than 10% of cells. A number of 45 subjects were recruited in this study. Thirthy one of 45 subjects showed the expression of Bcl-2 positive (68.9%), while the positive expression of caspase-3 present in 20 subjects (44.4%). There was a significant association between the expression of Bcl-2 with the expression of caspase-3 in ovarian cancer patients (p=0.002; lambda=0.4). There was also a significant association between stage of disease with expression of Bcl-2 (p=0.002; lambda=0.3) dan expression of caspase-3 (p=0.001; lambda=0.3). Conclusion: It concluded that there is a significant association between the expression of Bcl-2 and the expression of caspase-3 in ovarian cancer

Heat-shock pretreatment inhibits sorbitol-induced apoptosis in K562, U937 and HeLa cells.

The aim of this study was to determine whether heat-shock pretreatment exerted a protective effect against sorbitol-induced apoptotic cell death in K562, U937 and HeLa cell lines and whether such protection was associated with a decreased cytochrome c release from mithocondria and a decreased activation of caspase-9 and -3. Following heat-shock pretreatment (42 6 0.3C for 1 hr), these cell lines were exposed to sorbitol for 1 hr. Apoptosis was evaluated by DNA fragmentation, whereas caspase-9,-3 activation, cytochrome c release and heat-shock protein70 (HSP70) were assayed by Western Blot. Sorbitol exposure-induced apoptosis in these different cell lines with a marked activation of caspase-9 and caspase- 3, whereas heat-shock pretreatment before sorbitol exposure, induced expression of HSP70 and inhibited sorbitol-mediated cytochrome c release and subsequent activation of caspase-9 and caspase- 3. Similarly, overexpression of HSP70 in the three cell lines studied prevented caspase-9 cleavage and activation as well as cell death. Furthermore, we showed that the mRNA expression of iNOS decreased during both the heat-shock treatment and heat-shock pretreatment before sorbitol exposure. By contrast, the expression of Cu-Zn superoxide dismutase (SOD) and Mn-SOD proteins increased during heat-shock pretreatment before sorbitol exposure. We conclude that, heat-shock pretreatment protects different cell lines against sorbitol-induced apoptosis through a mechanism that is likely to involve SOD family members

Increased S-nitrosylation and proteasomal degradation of caspase-3 during infection contribute to the persistence of adherent invasive escherichia coli (AIEC) in immune cells

Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn's disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients

Analysis Expression of ZIP1 and Caspase-3 Protein in Adenocarsinoma of the Prostate

Background: Carcinogenesis of adenocarcinoma of the prostate occurs due to dysregulation of zinc level within the cells. Intracellular zinc molecules influx is regulated by a transporter protein ZIP1, whose non-presence is predicted to inhibit apoptosis, thus leads to the development of prostate adenocarcinoma. Methods: This study was aimed to analyse the correlation of ZIP1 and Caspase-3 expression in prostate adenocarcinoma on its grading as represented by Gleason Score. This was a cross-sectional, retrospective analytical study on 31 Formalin-fixed, paraffin-embedded tissue that meets inclusion criteria. The specimen was stained using the immune-histochemistry technique for ZIP1 and Caspase-3. Protein expression of each case was counted using ImageJ analysis. Gleason score was acquired as secondary data from the cases' reports. The correlation of their expression with respect to Gleason score was analysed with Pearson's correlation using SPSS 11.5. Results: Mean expression level of ZIP1 and Caspase-3 in prostate adenocarcinoma were 35% and 33%, respectively. There was a significantly positive correlation between ZIP1 and Caspase-3 expression (r=0.379; p=0.018). However, their correlation was stronger in intermediate-grade group (r=0.73; p=0.01) and the correlation was much weaker in high-grade group (r=0.04; p=0.48). Conclusions: There was a positive correlation between ZIP1 and Caspase-3 expression in prostate adenocarcinoma.&nbsp