APT131 Sigma-AldrichCaspase 3 Colorimetric Activity Assay Kit, DEVD

Enoxacin has been identified as a small molecule inhibitor of binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments. Consistent with this hypothesis, enoxacin dose-dependently reduced the number of multinuclear cells expressing tartrate-resistant acid phosphatase (TRAP) activity produced by RANK-L-stimulated osteoclast precursors. Enoxacin (50 μM) did not induce apoptosis as measured by TUNEL and caspase-3 assays. V-ATPases containing the a3-subunit, but not the housekeeping a1-subunit, were isolated bound to actin. Treatment with enoxacin reduced the association of V-ATPase subunits with the detergent-insoluble cytoskeleton. Quantitative PCR revealed that enoxacin triggered significant reductions in several osteoclast-selective mRNAs, but levels of various osteoclast proteins were not reduced, as determined by quantitative immunoblots, even when their mRNA levels were reduced. Immunoblots demonstrated that proteolytic processing of TRAP5b and the cytoskeletal protein L-plastin was altered in cells treated with 50 μM enoxacin. Flow cytometry revealed that enoxacin treatment favored the expression of high levels of DC-STAMP on the surface of osteoclasts. Our data show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel mechanism that involves changes in posttranslational processing and trafficking of several proteins with known roles in osteoclast function. We propose that these effects are downstream to blocking the binding interaction between a3-containing V-ATPases and microfilaments.

The purpose of this study was to investigate the anticancer properties of an anthocyanin-pyruvic acid adduct extract, which is being developed aiming to be further applied in the food industry. An anthocyanin extract from blueberry (extract I) and an anthocyanin-pyruvic acid adduct extract (extract II) were tested on two breast cancer cell lines (MDA-MB-231 and MCF7). Proliferation was assessed by SRB assay and ³H-thymidine incorporation. Caspase-3 activity was determined in the presence of both extracts. Their capacity as chemoattractants and their invasive potential were also assayed. In both cell lines, extracts I and II significantly reduced cell proliferation at 250 μg/mL, after 24 h of cell incubation. Caspase-3 activity was not altered by the extracts (250 μg/mL) in either cell line, with the exception of extract II in MCF-7, which increased its activity, probably explaining its effects on cell proliferation. Both extracts (250 μg/mL) demonstrated significant antiinvasive potential in both cell lines. Furthermore, they did not demonstrate any capacity for chemotaxis. In conclusion, blueberry anthocyanins and the respective anthocyanin-pyruvic acid adducts demonstrated anticancer properties by inhibiting cancer cell proliferation and by acting as cell antiinvasive factors and chemoinhibitors. The anthocyanin-pyruvic acid adduct extract showed a more pronounced effect in MDA-MB-231, suggesting an effect independent of estrogen receptors.

Previous studies suggest that Transforming growth factor-1beta (TGF-1beta) administration in human fibroblasts exposed to oxidative stress is able to modulate hyaluronan synthases (HASs). HAS modulation in turn increases high molecular weight (Hyaluronan) HA concentration. Nuclear factor kB (NF-kB) is a response transcription factor involved in inflammation and acts by enabling the expression of certain detrimental molecules. Caspases are specific proteases responsible for regulating and programming cell death. HA at medium molecular weight together with chondroitin-4-sulphate proved to be effective on NF-kB and caspases. We investigated whether the protective effect afforded by the high molecular weight HA produced by TGF-1beta treatment has any effect on NF-kB and apoptosis activation in fibroblast cultures exposed to oxidative stress. Generation of free radicals gives rise to cell death, increases lipid peroxidation, activates NF-kB, reduces its cytoplasmic inhibitor IkBalpha, augments caspase-3 and caspase-7 gene expression and their relative protein activity, and depletes catalase (CAT) and glutathione peroxidase (GPx). Treatment of fibroblasts with TGF-1beta 12 h before inducing oxidative stress greatly increased HA levels, ameliorated cell survival, inhibited lipid peroxidation, blunted NF-kB translocation, normalized IkBalpha protein, reduced caspase gene expression and protein levels, and restored the endogenous antioxidants CAT and GPx. Since it was previously reported that antioxidants can work as inhibitors of NF-kB and apoptosis induction we can hypothesize that endogenous HA, by inhibiting lipid peroxidation, may block a step whereby free radical activity converges in the signal transduction pathway leading to NF-kB and caspase activation.

Oxidative stress, inflammation and apoptosis play a critical role in the onset and progression of cellular damage. It was previously reported that hyaluronan (HA) and chondroitin-4-sulphate (C4S) were able to protect human skin fibroblasts from oxidative stress. This antioxidant activity is due to the chelation of transition metal ions. Nuclear factor kB (NF-kB), complexed with the inhibitory protein IkB alpha, is an ubiquitous response transcription factor involved in inflammatory reactions and acts by inducing cytokine expression, chemokines and cell adhesion molecules. Caspases are specific proteases responsible for the regulation and the execution of apoptotic cell death. The damage caused by free radicals may be amplified greatly by the activation of these factors. The study investigated whether the ability of these glycosaminoglycans (GAGs) to reduce oxidative damage in fibroblast cultures involves NF-kB and caspases modulation.The treatment of fibroblasts with both HA and C4S limited the cell damage induced by FeSO(4) plus ascorbate. An interesting aspect of this treatment was that these GAGs significantly inhibited NF-kB DNA binding, as confirmed by the normalization of IkB alpha protein, and reduced caspase activation at both mRNA and protein level. A possible explanation for these results, since lipid peroxidation intermediates may induce NF-kB and caspase activation, is that HA and C4S indirectly blocked NF-kB DNA binding and apoptosis by inhibiting reactive oxygen species (ROS) production.These data suggest that, during oxidative stress, HA and C4S may reduce cell damage by inhibiting NF-kB and apoptosis activation as well as protecting cells from free radical attack. According to these finding the use of HA and C4S could be positive both as tool to clarify the exact mechanism of GAGs/ROS interaction, and also as drug therapy to reduce oxidative stress during inflammation.

OBJECTIVE: The purpose of this study is to explore whether or not the levels of caspase-3 (Casp3), neuron-specific enolase (NSE), and high-sensitivity C-reactive protein (hsCRP) were elevated in cerebrospinal fluid (CSF) and serum of patients after aneurysmal subarachnoid hemorrhage (SAH). METHODS: This prospective clinical study consisted of 20 patients who experienced recent aneurysmal SAH and 15 control patients who experienced hydrocephalus without any other central nervous system disease. CSF and serum samples obtained within the first 3 days, and on the fifth and seventh days of SAH were assayed for Casp3, NSE, and hsCRP by using enzyme-linked immunosorbent assay. RESULTS: Levels of Casp3, NSE, and hsCRP in the CSF (P = 0.00001, P = 0.00001, and P 0.003, respectively) and in the serum (P = 0.00001, P 0.01, and P = 0.00001, respectively) of SAH patients were found to be elevated when compared with controls with normal pressure hydrocephalus. CONCLUSION: The authors have demonstrated the synchronized elevation of Casp3, NSE, and hsCRP in both CSF and serum of patients with aneurysmal SAH. Further studies with a large number of patients are recommended to more accurately determine the roles of these molecules in aneurysmal SAH.

OBJECTIVE: To determine whether sFas, caspase-3, proteins which propagate apoptosis, and bcl-2, a protein which inhibits apoptosis, would be increased in cerebrospinal fluid (CSF) in patients with severe traumatic brain injury (TBI) and to examine the correlation of sFas, caspase-3, and bcl-2 with each other and with clinical variables. METHODS: sFas, caspase-3, and bcl-2 were measured in CSF of 14 patients with severe TBI on days 1, 2, 3, 5, 7, and 10 post-trauma. The results were compared with CSF samples from control patients who had no brain and spinal pathology and had undergone spinal anesthesia for some other reason. Soluble Fas and bcl-2 were measured by ELISA while caspase-3 was measured enzymatically. RESULTS: No sFas, caspase-3, and bcl-2 activities were found in CSF of controls, but activities significantly increased in CSF of patients at all time points post-trauma (p 0.01). Caspase-3 significantly correlated to intracranial pressure (p = 0.01) and cerebral perfusion pressure (p = 0.04). Soluble Fas and caspase-3 peaks coincided on day 5 post-trauma and there was significant association between sFas and caspase-3 increase (p = 0.01). CONCLUSION: This study indicates a prolonged activation of pro-apoptotic (sFas, caspase-3) and anti-apoptotic (bcl-2) proteins after severe TBI in humans. The degree of activation of particularly caspase-3 may be related to the severity of the injury. Parallel increases of these three molecules may indicate a pivotal role of apoptosis in the pathophysiology of post-traumatic brain oedema, secondary cell destruction and chronic cell loss following severe TBI and may open new targets for post-traumatic therapeutic interventions.