Proteolytic Activation Of Caspase-3 Research Articles

Nitric oxide and thioredoxin modulate the activity of caspase 9 in HepG2 cells

The interdependent and finely tuned balance between the well-established redox-based modification, S-nitrosylation, and its counteractive mechanism of S-nitrosothiol degradation, i.e., S-denitrosylation of biological protein or non-protein thiols defines the cellular fate in the context of redox homeostasis. S-nitrosylation of cysteine residues by S-nitrosoglutathione, S-nitroso-L-cysteine-like physiological and S-nitroso-L-cysteine ethyl ester-like synthetic NO donors inactivate Caspase-3, 8, and 9, thereby hindering their apoptotic activity. However, spontaneous restoration of their activity upon S-denitrosylation of S-nitrosocaspases into their reduced, free thiol active states, aided by the members of the ubiquitous cellular redoxin (thioredoxin/ thioredoxin reductase/ NADPH) and low molecular weight dithiol (lipoic acid/ lipoamide dehydrogenase/ dihydrolipoic acid/ NADPH) systems imply a direct relevance to their proteolytic activities and further downstream signaling cascades. Additionally, our previous and current findings offer crucial insight into the concept of redundancy between thioredoxin and lipoic acid systems, and the redox-modulated control of the apoptotic and proteolytic activity of caspases, triggering their cyto- and neurotoxic effects in response to nitro-oxidative stress. Thus, this might lay the foundation for the exogenous introduction of precise and efficient NO or related donor drug delivery systems that can directly participate in catering to the S-(de)-nitrosylation-mediated functional outcomes of the cysteinyl proteases in pathophysiological settings.

Anticancer Effect of Medicinal Mushroom Extract on Renal Cell Carcinoma: Alternative Therapeutic Implication

Background: To investigate if medicinal Poria mushroom extract (PE) would be a potential therapeutic agent against renal cell carcinoma (RCC) as it has been shown to have anticancer/antitumor activity with few side effects. We examined its anticancer effect on RCC as well as its potential anticancer mechanism in vitro . Methods: Anticancer effect of PE was first assessed by the dose-dependent effects (0 - 200 µg/mL) on human RCC, ACHN cells. Its anticancer mechanism was then explored focusing on specific biochemical and molecular parameters Results: PE concentrations >= 100 µg/mL led to 50-90% reduction in cell viability, accompanied by a significant decrease in hexokinase activity and cellular adenosine triphosphate (ATP) level, implying the inhibition of glycolysis. The key metabolic regulators, adenosine monophosphate (AMP)-activated protein kinase (AMPK), protein kinase B (Akt), and mammalian target of rapamycin (mTOR), were specifically modulated by PE. These results were indicative of subsequent growth cessation and cell death. Cell cycle analysis also revealed a G 1 cell cycle arrest and the upregulation of activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), and glucose-regulated protein 78 (GRP78), indicated induction of endoplasmic reticulum (ER) stress. Ultimately, such ER stress led to apoptosis, evidenced by proteolytic activation of caspase-3 (Csp-3) and poly-ADP ribose polymerase (PARP). Conclusions: PE has anticancer effect on ACHN cells, resulting in the significant reduction in cell viability. Its anticancer mechanism appears to be linked to glycolysis inhibition, modulations of metabolic pathways, and ER stress-induced apoptosis. World J Nephrol Urol. 2022;11(1):1-9 doi: https://doi.org/10.14740/wjnu433

Antioxidant activity of banana flesh and antiproliferative effect on breast and pancreatic cancer cells.

Bananas, one of the most widely consumed fruits worldwide, are a rich source of valuable phytochemicals. In this study, the antioxidant and the anticancer potential of banana flesh was investigated. Of the four kinds of banana flesh extracts, the hexane extract (HE) had the highest total polyphenol content (2.54 ± 0.60 mg GAE/g) and total flavonoid content (1.69 ± 0.34 mg RE/g), followed by the chloroform fraction, total ethanol extract, and ethanol fraction. HE was found to exert a strong radical scavenging activity on 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH•) and 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonicacid) (ABTS•) free radicals. According to the IC50 values in various cancer cell lines, HE was found to possess the greatest cell growth inhibitory potential in human pancreatic cancer PANC‐1 cells and human triple‐negative breast cancer MDA‐MB‐231 cells. HE induced apoptosis in PANC‐1 and MDA‐MB‐231 cells, as evidenced by the appearance of condensation of chromatin, proteolytic activation of caspase‐3 and 7, and increase in the level of the cleaved form of poly (ADP‐ribose) polymerase protein. Gas chromatography mass spectrometry (GC‐MS) analysis of HE identified several anticancer compounds including palmitic acid, linoleic acid, oleic acid, campesterol, stigmasterol, and γ‐sitosterol, supporting the anticancer potential of HE. Our investigation provides a rationale for the use of banana flesh to minimize the risk of cancer‐like diseases.

Auranofin Enhances Sulforaphane-Mediated Apoptosis in Hepatocellular Carcinoma Hep3B Cells through Inactivation of the PI3K/Akt Signaling Pathway.

The thioredoxin (Trx) system plays critical roles in regulating intracellular redox levels and defending organisms against oxidative stress. Recent studies indicated that Trx reductase (TrxR) was overexpressed in various types of human cancer cells indicating that the Trx-TrxR system may be a potential target for anti-cancer drug development. This study investigated the synergistic effect of auranofin, a TrxR-specific inhibitor, on sulforaphane-mediated apoptotic cell death using Hep3B cells. The results showed that sulforaphane significantly enhanced auranofin-induced apoptosis by inhibiting TrxR activity and cell proliferation compared to either single treatment. The synergistic effect of sulforaphane and auranofin on apoptosis was evidenced by an increased annexin-V-positive cells and Sub-G1 cells. The induction of apoptosis by the combined treatment caused the loss of mitochondrial membrane potential (ΔΨm) and upregulation of Bax. In addition, the proteolytic activities of caspases (-3, -8, and -9) and the degradation of poly (ADP-ribose) polymerase, a substrate protein of activated caspase-3, were also higher in the combined treatment. Moreover, combined treatment induced excessive generation of reactive oxygen species (ROS). However, treatment with N-acetyl-L-cysteine, a ROS scavenger, reduced combined treatment-induced ROS production and apoptosis. Thereby, these results deduce that ROS played a pivotal role in apoptosis induced by auranofin and sulforaphane. Furthermore, apoptosis induced by auranofin and sulforaphane was significantly increased through inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Taken together, the present study demonstrated that down-regulation of TrxR activity contributed to the synergistic effect of auranofin and sulforaphane on apoptosis through ROS production and inhibition of PI3K/Akt signaling pathway.

C2-Ceramide-Induced Rb-Dominant Senescence-Like Phenotype Leads to Human Breast Cancer MCF-7 Escape from p53-Dependent Cell Death.

Ceramide is a sphingolipid which regulates a variety of signaling pathways in eukaryotic cells. Exogenous ceramide has been shown to induce cellular apoptosis. In this study, we observed that exogenous ceramide induced two distinct morphologies of cell fate following C2-ceramide treatment between the two breast cancer cell lines MCF-7 (wild type p53) and MDA-MB-231 (mutant p53) cells. The growth assessment showed that C2-ceramide caused significant growth inhibition and apoptosis in MDA-MB-231 cells through down-regulating the expression of mutant p53 whereas up-regulating the expression of pro-apoptotic Bad, and the proteolytic activation of caspase-3. However, senescence-associated (SA)-β-galactosidase (β-gal) was regulated in MCF-7 cells after C2-ceramide treatment. The results of proliferation and apoptosis assays showed that MCF-7 cells were more resistant to C2-ceramide treatment compared to MDA-MB-231 cells. Furthermore, C2-ceramide treatment induced a time-responsive increase in Rb protein, a key regulator of senescence accompanied with the upregulation of both mRNA level and protein level of SA-genes PAI-1 and TGaseII in MCF-7 but not in MDA-MB-231 cells, suggesting that some cancer cells escape apoptosis through modulating senescence-like phenotype. The results of our present study depicted the mechanism of C2-ceramide-resistant breast cancer cells, which might benefit the strategic development of ceramide-based chemotherapeutics against cancer in the future.

Human Cytomegalovirus Immediate Early 86-kDa Protein Blocks Transcription and Induces Degradation of the Immature Interleukin-1β Protein during Virion-Mediated Activation of the AIM2 Inflammasome.

Secretion of interleukin-1β (IL-1β) represents a fundamental innate immune response to microbial infection that, at the molecular level, occurs following activation of proteolytic caspases that cleave the immature protein into a secretable form. Human cytomegalovirus (HCMV) is the archetypal betaherpesvirus that is invariably capable of lifelong infection through the activity of numerous virally encoded immune evasion phenotypes. Innate immune pathways responsive to cytoplasmic double-stranded DNA (dsDNA) are known to be activated in response to contact between HCMV and host cells. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) genome editing to demonstrate that the dsDNA receptor absent in melanoma 2 (AIM2) is required for secretion of IL-1β following HCMV infection. Furthermore, dsDNA-responsive innate signaling induced by HCMV infection that leads to activation of the type I interferon response is also shown, unexpectedly, to play a contributory role in IL-1β secretion. Importantly, we also show that rendering virus particles inactive by UV exposure leads to substantially increased IL-1β processing and secretion and that live HCMV can inhibit this, suggesting the virus encodes factors that confer an inhibitory effect on this response. Further examination revealed that ectopic expression of the immediate early (IE) 86-kDa protein (IE86) is actually associated with a block in transcription of the pro-IL-1β gene and, independently, diminishment of the immature protein. Overall, these results reveal two new and distinct phenotypes conferred by the HCMV IE86 protein, as well as an unusual circumstance in which a single herpesviral protein exhibits inhibitory effects on multiple molecular processes within the same innate immune response.IMPORTANCE Persistent infection with HCMV is associated with the operation of diverse evasion phenotypes directed at antiviral immunity. Obstruction of intrinsic and innate immune responses is typically conferred by viral proteins either associated with the viral particle or expressed immediately after entry. In line with this, numerous phenotypes are attributed to the HCMV IE86 protein that involve interference with innate immune processes via transcriptional and protein-directed mechanisms. We describe novel IE86-mediated phenotypes aimed at virus-induced secretion of IL-1β. Intriguingly, while many viruses target the function of the molecular scaffold required for IL-1β maturation to prevent this response, we find that HCMV and IE86 target the IL-1β protein specifically. Moreover, we show that IE86 impairs both the synthesis of the IL-1β transcript and the stability of the immature protein. This indicates an unusual phenomenon in which a single viral protein exhibits two molecularly separate evasion phenotypes directed at a single innate cytokine.

Induction of ER Stress-Mediated Apoptosis by the Major Component 5,7,4′-Trimethoxyflavone Isolated from Kaempferia parviflora Tea Infusion

Kaempferia parviflora (KP) is a famous medicinal plant from Thailand, and is a rich source of various kinds of methoxyflavones (MFs). Many kinds of food products such as tea, capsule, and liquor are manufactured from the rhizomes of KP. In this study, KP infusions were prepared with different brewing conditions, and the amounts of three major methoxylflavones, 5,7-dimethoxyflavone (DMF), 5,7,4′-trimethoxyflavone (TMF), and 3,5,7,3′,4′-pentamethoxyflavone (PMF), were analyzed. The antiproliferative activities of DMF, TMF, and PMF isolated from the brewed tea samples were evaluated. TMF was discovered to be significantly effective at inhibiting proliferation of SNU-16 human gastric cancer cells in a concentration dependent manner. TMF induced apoptosis, as evidenced by increments of sub-G1 phase, DNA fragmentation, annexin-V/PI staining, the Bax/Bcl-xL ratio, proteolytic activation of caspase-3,-7,-8, and degradation of poly (ADP-ribose) polymerase (PARP) protein. Furthermore, it was found that TMF induced apoptosis via ER stress, verified by an increase in the level of C/EBP homologous protein (CHOP), glucose regulated protein 78 (GRP78), inositol-requiring enzyme 1 α (IRE1α), activating transcription factor-4 (ATF-4), and the splice isoform of X-box-binding protein-1 (XBP-1) mRNA.

The in Vitro and in Vivo Anti-Cancer Potential of Mycobacterium Cell Wall Fraction (MCWF) Against Canine Transitional Cell Carcinoma of the Urinary Bladder

Abstract Transitional cell carcinoma (TCC), is the most common form of urinary bladder cancer in dogs and represents 2% of all reported canine cancers. Canine TCC is usually a high-grade invasive cancer and problems associated with TCC include urinary tract obstruction and distant metastases in more than 50% of affected dogs. TCC is most commonly located in the trigone region of the bladder precluding complete surgical resection. Current treatment options for TCC in dogs include medical therapy, surgery or radiation. Mycobacterium Cell Wall Fraction (MCWF) is a biological immunomodulator derived from non-pathogenic Mycobacterium phlei. MCWF possesses a potential in multiple veterinary areas such as anticancer therapy, palliative care and treatment of infectious diseases in both small and large animals. MCWF is considered a bifunctional anti-cancer agent that induces apoptosis of cancer cells and stimulates cytokine and chemokines synthesis by cells of the immune system. Here we report the results from in vitro and in vivo studies that could suggest use of MCWF as an additional treatment option for TCC in dogs. Particularly, we demonstrated that MCWF induces a concentration dependent inhibition of proliferation of K9TCC cells which was associated with the induction of apoptosis as measured by the proteolytic activation of caspase-3 and the degradation of PARP. Furthermore, we demonstrated the safety and potential for in vivo MCWF treatment efficacy in dogs bearing stage T2 TCC by reducing clinical signs, and improving the quality of life in dogs with TCC.

Abstract 2330: BI5: a novel SMAC mimetic that triggers tumor cell death and potentiates PD-1 mediated cancer therapy

Abstract Background: Inhibitors of apoptosis proteins (IAPs) regulate cellular apoptosis by interfering with the proteolytic activities of caspases. IAP inhibitors (SMAC mimetics) have been developed to restore the defective apoptosis that characterizes many tumour cells. Emerging evidence demonstrates that IAPs are critical components of immune-modulatory pathways that control innate and adaptive immunity. Accordingly, SMAC mimetics hold the promise of both inducing tumour cell killing and stimulating the immune system to recognize and eliminate dying tumour cells. Here we show that BI5 primes immune components and synergises with PD-1 checkpoint inhibitors to promote eradication of syngeneic tumors. Methods: Here we report the efficacy and modulation of the immune response by a potent and selective SMAC mimetic, BI5. We characterised the effect of BI5 on tumor growth inhibition as a single agent and in combination with an anti-PD-1 antibody in syngeneic mouse tumor models. A detailed 17-colour multi-color flow cytometry analysis was used to investigate the mechanisms by which the SMAC mimetic interacts with anti-PD-1 therapy in vivo. Results: Treatment of the syngeneic mouse tumor models MBT-2 and EMT-6 with the SMAC mimetic in combination with an anti-PD-1 antibody results in remarkable tumor regressions in vivo. Importantly, the combined effect of the SMAC mimetic and anti-PD-1 on tumor growth was dependent on the adaptive immune system in vivo. Mechanistic studies show that degradation of IAP triggers tumor cell death, which leads to a potent activation of dendritic cells in the draining lymph nodes and a subsequent influx of T and NK cells into the tumor microenvironment. Interestingly, in the presence of the SMAC mimetic alone, an induction of PD-1 expression on tumor-infiltrating CD8+ T cells was observed, which in turn resulted in the exhaustion of these cells and tumor outgrowth. In the presence of the anti-PD-1 antibody, T cells are reactivated leading to potent and long term tumor eradication. Conclusion: We show that our SMAC mimetic leads to a potent induction of immunogenic cell death and sets up a “virtuous cycle” by potentiating dendritic cell and T cell mediated immune responses that further promote induction of cell death. These effects are potentiated by checkpoint inhibitors, leading to long term tumor control. Tumours with minimal T-cell infiltration are poorly responsive to PD-1 monotherapy. These studies indicate that SMAC mimetics, such as BI5, represent promising and tolerated combination partners for checkpoint inhibitors in patients that lack a strong immune inflammatory signature. Citation Format: Markus Reschke, Maria Antonietta Impagnatiello, Ulrich Reiser, Dirk Scharn, Walter Spevak, Alexander Savchenko, Andreas Wernitznig, Martina Sykora, Rebecca Langlois, Elisabeth Zier, Daniel Zach, Sabine Kallenda, Pilar Garin-Chesa, Jens Quant, Mark Pearson, Darryl McConnell, Norbert Kraut, Juergen Moll. BI5: a novel SMAC mimetic that triggers tumor cell death and potentiates PD-1 mediated cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2330. doi:10.1158/1538-7445.AM2017-2330

Induction of apoptosis by Moutan Cortex Radicis in human gastric cancer cells through the activation of caspases and the AMPK signaling pathway

Moutan Cortex Radicis, the root bark of Paeonia×suffruticosa Andrews, Paeoniaceae, has been widely used in traditional medicine therapy. Although it has been shown to possess many pharmacological activities, the molecular mechanisms of its anti-cancer activity have not been clearly elucidated. In the present study, we investigated the pro-apoptotic effects of the ethanol extract of Moutan Cortex Radicis in human gastric cancer AGS cells. Moutan Cortex Radicis treatment inhibited the cell viability of AGS cells in a concentration-dependent manner, which was associated with apoptotic cell death. Moutan Cortex Radicis's induction of apoptosis was connected with the upregulation of death receptor 4, death receptor 5, tumor necrosis factor-related apoptosis-inducing ligand, Fas ligand, and Bax, and the downregulation of Bcl-2 and Bid. Moutan Cortex Radicis treatment also induced the loss of mitochondrial membrane potential (Δψm), the proteolytic activation of caspases (-3, -8, and -9), and the degradation of poly(ADP-ribose) polymerase, an activated caspase-3 substrate protein. However, the pre-treatment of a caspase-3 inhibitor significantly attenuated Moutan Cortex Radicis-induced apoptosis and cell viability reduction. In addition, Moutan Cortex Radicis treatment effectively activated the adenosine monophosphate-activated protein kinase signaling pathway; however, a specific inhibitor of AMPK significantly reduced Moutan Cortex Radicis-induced apoptosis. Overall, the results suggest that the apoptotic activity of Moutan Cortex Radicis may be associated with a caspase-dependent cascade through the activation of both extrinsic and intrinsic signaling pathways connected with adenosine monophosphate-activated protein kinase activation, and Moutan Cortex Radicis as an activator of adenosine monophosphate-activated protein kinase could be a prospective application to treat human cancers.

Analysis of porcine granulosa cell death signaling pathways induced by vinclozolin

Recent studies suggest that disturbing androgen-signaling pathways in porcine ovarian follicles may cause granulosa cell (GC) death. For this reason, we investigated which apoptotic pathway is initiated after GC exposure to an environmental antiandrogen, vinclozolin (Vnz), in vitro. Immunocytochemistry, Western blots, and fluorometric assays were used to quantify caspase-3 and -9 expression and activity. To elucidate the specific mechanism of Vnz action and toxicity, GCs were assessed for viability, cytotoxicity, and apoptotic activity using the ApoTox-Glo Triplex Assay. To further determine the mechanism of GC death induced by Vnz, we used the Apoptosis Antibody Array Kit. In response to Vnz stimulus, we found an increased level of caspase-3 protein expression (P ≤ 0.001) and an increase in caspase-3 proteolytic activity (P ≤ 0.001), confirming that Vnz is a potent proapoptotic factor. The strong immunoreaction of caspase-9 after Vnz treatment (P ≤ 0.001) suggests that intrinsic mitochondrial apoptosis pathway was activated during GC death. On the other hand, caspase-8, being a part of the extrinsic receptor pathway, was also activated (P ≤ 0.001). Therefore, it is possible that Vnz induces porcine granulosal apoptosis also through a parallel pathway. Activation of these two pathways was confirmed by the Apoptosis Antibody Array Kit. In conclusion, it is possible that the intrinsic signaling pathway may not act as an initial trigger for GC apoptosis but might contribute to the amplification and propagation of apoptotic cell death in the granulosa layer after treatment with this antiandrogen. Moreover, Vnz disturbs the physiological process of programmed cell death. Consequently, this could explain why atretic follicles are rapidly removed and suggests that normal function of the ovarian follicle may be destroyed.

Thyroid hormone and anti-apoptosis in tumor cells.

The principal secretory product of the thyroid gland, L-thyroxine (T4), is anti-apoptotic at physiological concentrations in a number of cancer cell lines. Among the mechanisms of anti-apoptosis activated by the hormone are interference with the Ser-15 phosphorylation (activation) of p53 and with TNFα/Fas-induced apoptosis. The hormone also decreases cellular abundance and activation of proteolytic caspases and of BAX and causes increased expression of X-linked inhibitor of apoptosis (XIAP). The anti-apoptotic effects of thyroid hormone largely are initiated at a cell surface thyroid hormone receptor on the extracellular domain of integrin αvβ3 that is amply expressed and activated in cancer cells. Tetraiodothyroacetic acid (tetrac) is a T4 derivative that, in a model of resveratrol-induced p53-dependent apoptosis in glioma cells, blocks the anti-apoptotic action of thyroid hormone, permitting specific serine phosphorylation of p53 and apoptosis to proceed. In a nanoparticulate formulation limiting its action to αvβ3, tetrac modulates integrin-dependent effects on gene expression in human cancer cell lines that include increased expression of a panel of pro-apoptotic genes and decreased transcription of defensive anti-apoptotic XIAP and MCL1 genes. By a variety of mechanisms, thyroid hormone (T4) is an endogenous anti-apoptotic factor that may oppose chemotherapy-induced apoptosis in αvβ3-expressing cancer cells. It is possible to decrease this anti-apoptotic activity pharmacologically by reducing circulating levels of T4 or by blocking effects of T4 that are initiated at αvβ3.

Inhibition of Stat3 Activation Suppresses Caspase-3 and the Ubiquitin-Proteasome System, Leading to Preservation of Muscle Mass in Cancer Cachexia

Cachexia occurs in patients with advanced cancers. Despite the adverse clinical impact of cancer-induced muscle wasting, pathways causing cachexia are controversial, and clinically reliable therapies are not available. A trigger of muscle protein loss is the Jak/Stat pathway, and indeed, we found that conditioned medium from C26 colon carcinoma (C26) or Lewis lung carcinoma cells activates Stat3 (p-Stat3) in C2C12 myotubes. We identified two proteolytic pathways that are activated in muscle by p-Stat3; one is activation of caspase-3, and the other is p-Stat3 to myostatin, MAFbx/Atrogin-1, and MuRF-1 via CAAT/enhancer-binding protein δ (C/EBPδ). Using sequential deletions of the caspase-3 promoter and CHIP assays, we determined that Stat3 activation increases caspase-3 expression in C2C12 cells. Caspase-3 expression and proteolytic activity were stimulated by p-Stat3 in muscles of tumor-bearing mice. In mice with cachexia caused by Lewis lung carcinoma or C26 tumors, knock-out of p-Stat3 in muscle or with a small chemical inhibitor of p-Stat3 suppressed muscle mass losses, improved protein synthesis and degradation in muscle, and increased body weight and grip strength. Activation of p-Stat3 stimulates a pathway from C/EBPδ to myostatin and expression of MAFbx/Atrogin-1 and increases the ubiquitin-proteasome system. Indeed, C/EBPδ KO decreases the expression of MAFbx/Atrogin-1 and myostatin, while increasing muscle mass and grip strength. In conclusion, cancer stimulates p-Stat3 in muscle, activating protein loss by stimulating caspase-3, myostatin, and the ubiquitin-proteasome system. These results could lead to novel strategies for preventing cancer-induced muscle wasting.

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.

Electrochemical impedance spectroscopy for monitoring caspase-3 activity

Caspases play a key role in apoptosis and represent important therapeutic targets for treating cancer and inflammatory diseases. In this proof-of-concept study, an electrochemical impedance spectroscopy (EIS)-based biosensor was developed for the analysis of caspase-3 activity in non-diluted biological samples using screen-printed gold electrodes (SPGE) compatible with small sample volumes (i.e. 2μL). A caspase-3-specific peptide substrate was immobilized on the surface of the SPGE using N-hydroxysuccinimide (NHS)-activated lipoic acid esters. The proteolytic activity of caspase-3 was analyzed using EIS, in which the presence of the enzyme resulted in cleavage of substrate peptides. Changes in the surface-immobilized substrate peptide film were detected using the apparent charge transfer resistance (RAPP) in connection with [Fe(CN)6]3−/4− redox probe. The analytical performance of the biosensor was challenged with undiluted apoptotic human SH-SY5Y neuroblastoma cell lysates at 2-μL aliquots. Control experiments were performed with healthy SH-SY5Y cell lysates and did not display a significant decrease in RAPP values. EIS data were also confirmed using a commercially available optical kit. EIS-based biosensor demonstrated a promising potential to become a versatile tool for rapid screening of caspase-3 proteolytic activity in complex biological samples.

Human embryonic stem cells prevent T-cell activation by suppressing dendritic cells function via TGF-beta signaling pathway.

Human embryonic stem cells (hESCs) represent a potential source of transplantable cells for regenerative medicine, but development of teratoma even in syngenic recipients represents a critical obstacle to safe stem cell-based therapies. We hypothesized that hESCs escape the immune surveillance by regulating the environmental immune system. Using cocultures of hESCs with allogenic peripheral blood mononuclear cells, we demonstrated that hESCs prevent proliferation and activation of human CD4+ T lymphocytes, an effect dependent upon monocytes. Altered expression of key signaling molecules responsible for the crosstalk of monocytes with T cells was detected in the presence of hESCs. Analyzing the mechanism of action, we demonstrated that hESCs were able to downregulate intracellular glutathione levels in both monocytes and CD4+ cells by suppressing glutamate cysteine ligase expression and to alter MHCII and CD80 expression in monocytes. These effects were achieved at least partially via TGF-beta signaling, and both monocyte phenotype and GCLC expression were affected by Caspase-3 proteolytic activity. Altogether, our results demonstrate a novel immune-suppressive mechanism used by hESCs.

Ursolic acid inhibits the invasive phenotype of SNU-484 human gastric cancer cells.

Metastasis is a major cause of cancer-related mortality in patients with gastric cancer. Ursolic acid, a pentacyclic triterpenoid compound derived from medicinal herbs, has been demonstrated to exert anticancer effects in various cancer cell systems. However, to the best of our knowledge, the inhibitory effect of ursolic acid on the invasive phenotype of gastric cancer cells has yet to be reported. Therefore, the aim of the present study was to investigate the effect of ursolic acid on the invasiveness of SNU-484 human gastric cancer cells. Ursolic acid efficiently induced apoptosis, possibly via the downregulation of B-cell lymphoma 2 (Bcl-2), the upregulation of Bcl-2-associated X protein and the proteolytic activation of caspase-3. Furthermore, the activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase was increased by the administration of ursolic acid. In addition, ursolic acid significantly suppressed the invasive phenotype of the SNU-484 cells and significantly decreased the expression of matrix metalloproteinase (MMP)-2, indicating that MMP-2 may be responsible for the anti-invasive activity of ursolic acid. Taken together, the results of the present study demonstrate that ursolic acid induces apoptosis and inhibits the invasive phenotype of gastric cancer cells; therefore, ursolic acid may have a potential application as a chemopreventive agent to prevent the metastasis of gastric cancer or to alleviate the process of metastasis.

Fas-induced apoptosis increases hepatocyte tissue factor procoagulant activity in vitro and in vivo.

Hepatocyte (HPC) apoptosis occurs in association with hepatotoxic responses and chronic liver disease, and is coupled to activation of the blood coagulation cascade. HPCs have been shown to express tissue factor (TF), the primary activator of blood coagulation, in a form that lacks procoagulant activity. In this study, we determined the effect of inducing HPC apoptosis on the procoagulant activity of TF. Treatment of primary mouse HPCs with the Fas death receptor agonist (anti-CD95 antibody, Jo2) triggered apoptosis as shown by cleavage of caspase-3, increased caspase-3 proteolytic activity, and cell surface exposure of phosphatidylserine (PS). Jo2-induced apoptosis significantly increased TF-dependent factor Xa generation by HPCs. Moreover, Jo2 treatment was associated with increased levels of microparticle-associated TF procoagulant activity in the culture medium. Pretreatment with a caspase-3 inhibitor significantly reduced Jo2-induced HPC TF activity and prevented the increase in microparticle-associated TF procoagulant activity. Application of the high-affinity PS-binding protein lactadherin inhibited TF-dependent factor Xa generation by Jo2-treated HPCs and dramatically reduced microparticle-associated TF procoagulant activity. Treatment of wild-type mice with a sublethal dose of Jo2 was associated with a robust increase in the activation of coagulation as measured by plasma thrombin-antithrombin (TAT) levels; whereas mice with liver-specific TF deficiency had significantly lower TAT levels. Overall, the results indicate that Fas-initiated, caspase-3-dependent HPC apoptosis increases TF procoagulant activity through a mechanism involving PS externalization. This suggests that activation of liver TF likely contributes to the procoagulant state associated with HPC apoptosis in liver toxicity and disease.

Gemcitabine sensitizes lung cancer cells to Fas/FasL system‐mediated killing

Gemcitabine is a chemotherapy agent commonly used in the treatment of non-small cell lung cancer (NSCLC) that has been demonstrated to induce apoptosis in NSCLC cells by increasing functionally active Fas expression. The aim of this study was to evaluate the Fas/Fas ligand (FasL) system involvement in gemcitabine-induced lung cancer cell killing. NSCLC H292 cells were cultured in the presence or absence of gemcitabine. FasL mRNA and protein were evaluated by real-time PCR, and by Western blot and flow cytometry, respectively. Apoptosis of FasL-expressing cells was evaluated by flow cytometry, and caspase-8 and caspase-3 activation by Western blot and a colorimetric assay. Cytotoxicity of lymphokine-activated killer (LAK) cells and malignant pleural fluid lymphocytes against H292 cells was analysed in the presence or absence of the neutralizing anti-Fas ZB4 antibody, by flow cytometry. Gemcitabine increased FasL mRNA and total protein expression, the percentage of H292 cells bearing membrane-bound FasL (mFasL) and of mFasL-positive apoptotic H292 cells, as well as caspase-8 and caspase-3 cleavage. Moreover, gemcitabine increased CH11-induced caspase-8 and caspase-3 cleavage and proteolytic activity. Cytotoxicity of LAK cells and pleural fluid lymphocytes was increased against gemcitabine-treated H292 cells and was partially inhibited by ZB4 antibody. These results demonstrate that gemcitabine: (i) induces up-regulation of FasL in lung cancer cells triggering cell apoptosis via an autocrine/paracrine loop; (ii) induces a Fas-dependent apoptosis mediated by caspase-8 and caspase-3 activation; (iii) enhances the sensitivity of lung cancer cells to cytotoxic activity of LAK cells and malignant pleural fluid lymphocytes, partially via Fas/FasL pathway. Our data strongly suggest an active involvement of the Fas/FasL system in gemcitabine-induced lung cancer cell killing.

Myricetin Induces Apoptosis in HepG2 Cells Through Akt/p70S6K/Bad Signaling and Mitochondrial Apoptotic Pathway

The present investigation was undertaken to gain insight into the molecular mechanism by which myricetin induces apoptosis in human hepatocarcinoma HepG2 cells. Myricetin caused the disruption of mitochondrial membrane potential in a dose-dependent manner. Moreover, myricetin triggered translocation of the pro-apoptotic protein Bax to the mitochondria, downregulation of anti-apoptotic Bcl-2 expression and upregulated the expression of pro-apoptotic protein Bad in the mitochondria. The present study also showed that myricetin promoted the release of cytochrome C from mitochondria into the cytosol followed by an increase in the proteolytic activation of caspase-3 and the concomitant degradation of PARP protein. Additionally, western blot analysis showed that the Akt/p70s6k1 pathway was inhibited in myricetin-treated HepG2 cells, accordingly the phosphorylation of Bad at Ser136 was downregulated. Collectively, these findings indicate that myricetin induced apoptosis in HepG2 cell through mitochondria apoptotic pathway and Akt/p70s6k1/Bad signaling. Present results provide new information on the possible mechanisms for the anti-cancer activity of myricetin.