Activation Of Different Caspases Research Articles

Eprinomectin: a derivative of ivermectin suppresses growth and metastatic phenotypes of prostate cancer cells by targeting the β-catenin signaling pathway.

Prostate cancer (PCa) is the second leading cause of cancerdeath among men in the USA. The emergence of resistance to androgen deprivation therapy gives rise to metastatic castration-resistant prostate cancer. Eprinomectin (EP) is a member of a family of drugs called avermectins with parasiticide and anticancer properties.The pupose of this study was to evaluate the anticancer effects of EP against metastatic PCa using cellular models. METHODS: In this study, we have investigated the effect of EP's anticancer properties and delineated the underlying mechanisms in the DU145 cellular model using several assays such as cell viability assay, colony formation assay, wound-healing assay, immunofluorescence, apoptosis assay, cell cycle analysis, and immunoblotting. Our results indicate that EP significantly inhibits the cell viability, colony formation, and migration capacities of DU145 cells. EP induces cell cycle arrest at the G0/G1 phase, apoptosis via the activation of different caspases, and autophagy through the increase in the generation of reactive oxygen species and endoplasmic reticulum stress. In addition, EP downregulates the expression of cancer stem cell markers and mediates the translocation of β-catenin from the nucleus to the cytoplasm, indicating its role in inhibiting downstream target genes such as c-Myc and cyclin D1. Our study shows that EP has tremendous potential to target metastatic PCa cells and provides new avenues for therapeutic approaches for advanced PCa.

Cannabinoids Reduce Melanoma Cell Viability and Do Not Interfere with Commonly Used Targeted Therapy in Metastatic Melanoma In Vivo and In Vitro.

Background: Cannabinoids are mainly used for recreational purposes, but also made their way into oncology, since these substances can be taken to increase appetite in tumour cachexia. Since there are some hints in the literature that cannabinoids might have some anti-cancerous effects, the aim of this study was to study if and how cannabinoids mediate pro-apoptotic effects in metastatic melanoma in vivo and in vitro and its value besides conventional targeted therapy in vivo. Methods: Several melanoma cell lines were treated with different concentrations of cannabinoids, and anti-cancerous efficacy was assessed by proliferation and apoptosis assays. Subsequent pathway analysis was performed using apoptosis, proliferation, flow cytometry and confocal microscopy data. The efficacy of cannabinoids in combination with trametinib was studied in NSG mice in vivo. Results: Cannabinoids reduced cell viability in multiple melanoma cell lines in a dose-dependent way. The effect was mediated by CB1, TRPV1 and PPARα receptors, whereby pharmacological blockade of all three receptors protected from cannabinoid-induced apoptosis. Cannabinoids initiated apoptosis by mitochondrial cytochrome c release with consecutive activation of different caspases. Essentially, cannabinoids significantly decreased tumour growth in vivo and were as potent as the MEK inhibitor trametinib. Conclusions: We could demonstrate that cannabinoids reduce cell viability in several melanoma cell lines, initiate apoptosis via the intrinsic apoptotic pathway by cytochrome c release and caspase activation and do not interfere with commonly used targeted therapy.

Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection.

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially described to occur by apoptosis, programmed cell death is now known to encompass three interconnected pathways: pyroptosis, apoptosis, and necroptosis, together coined as one process, PANoptosis. Influence A virus (IAV) infection induces PANoptosis in mammalian cells by inducing the activation of different caspases, which, in turn, cleave various host as well as viral proteins, leading to processes like the activation of the host innate antiviral response or the degradation of antagonistic host proteins. In this regard, caspase 3-mediated cleavage of host cortactin, histone deacetylase 4 (HDAC4), and histone deacetylase 6 (HDAC6) has been discovered in both animal and human epithelial cells in response to the IAV infection. To demonstrate this, inhibitors, RNA interference, and site-directed mutagenesis were employed, and, subsequently, the cleavage or resistance to cleavage and the recovery of cortactin, HDAC4, and HDAC6 polypeptides were measured by western blotting. These methods, in conjunction with RT-qPCR, form a simple yet effective strategy to identify the host as well as viral proteins undergoing caspase-mediated cleavage during an infection of IAV or other human and animal viruses. The present protocol elaborates the representative results of this strategy, and the ways to make it more effective are also discussed.

Macrophage migration inhibitory factor inhibition as a novel therapeutic approach against triple-negative breast cancer

Triple-negative breast cancer (TNBC), defined as loss of estrogen, progesterone, and Her2 receptors, is a subtype of highly aggressive breast cancer with worse prognosis and poor survival rate. Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine aberrantly expressed in many solid tumors and known to promote tumor progression and metastasis. However, its role in TNBC progression and metastasis is unexplored. Here we have shown that in TNBC patients, MIF expression was significantly enriched in the tumor compared to adjacent normal tissue. Using publically available patient datasets, we showed that MIF overexpression correlates with worse survival in TNBC compared to other hormonal status. Orthotopic implantation of TNBC cells into MIF knockout mice showed reduced tumor growth compared to wild-type mice. In addition, we have shown that MIF downregulation inhibits TNBC growth and progression in a syngeneic mouse model. We further showed that CPSI-1306, a small-molecule MIF inhibitor, inhibits the growth of TNBC cells in vitro. Mechanistic studies revealed that CPSI-1306 induces intrinsic apoptosis by alteration in mitochondrial membrane potential, cytochrome c (Cyt c) release, and activation of different caspases. In addition, CPSI-1306 inhibits the activation of cell survival and proliferation-related molecules. CPSI-1306 treatment also reduced the tumor growth and metastasis in orthotopic mouse models of mammary carcinoma. CPSI-1306 treatment of tumor-bearing mice significantly inhibited TNBC growth and pulmonary metastasis in a dose-dependent manner. Histological analysis of xenograft tumors revealed a higher number of apoptotic cells in CPSI-1306-treated tumors compared to vehicle controls. Our studies, for the first time, show that MIF overexpression in TNBC enhances growth and metastasis. Taken together, our results indicate that using small molecular weight MIF inhibitors could be a promising strategy to inhibit TNBC progression and metastasis.

Biological activity and apoptotic signaling pathway of C11-functionalized cephalostatin 1 analogues

Cephalostatin 1, a potent anti-cancer agent, is a natural bis-steroidal alkaloid that causes cell death in the subnanomolar to picomolar ranges via an atypical apoptosis pathway. Although cephalostatin 1 is a highly effective anticancer drug, its availability limits its utilization. We previously reported the synthesis of two 12′α-hydroxy derivatives of cephalostatin 1 that induce cell death by activating the ER stress apoptosis signaling pathway. For the current work, we synthesized six C11-functionalized cephalostatin 1 analogues (CAs) to evaluate their biological activity. For the cytotoxic compounds, the induced apoptotic pathway was investigated. The C11-functionalized cephalostatin 1 analogues 5 and 6 (CA5 and CA6) were found to exhibit cytotoxic activity against K-562 leukemia cells, MCF-7 breast cancer cells and DU-145 prostate cancer cells, while the remaining four analogues did not show anti-tumor activities against any of the cell lines. Our results indicated that CA5 and CA6 induced cell death via the atypical ER-dependent apoptosis pathway; they increased the expression of Smac/DIABLO, an inhibitor of inhibitors of apoptosis (IAPs), which in turn facilitated the activation of different caspases including the ER-caspase 4 without cytochrome c release from mitochondria. CA5 and CA6 are promising anticancer agents due to their low GI50, the remarkable apoptosis pathway they induce which can overcome chemoresistance, and their very low toxicity to normal cells making them cephalostatin 1 utilizable alternatives.

The role of apoptosis repressor with a CARD domain (ARC) in the therapeutic resistance of renal cell carcinoma (RCC): the crucial role of ARC in the inhibition of extrinsic and intrinsic apoptotic signalling

BackgroundRenal cell carcinomas (RCCs) display broad resistance against conventional radio- and chemotherapies, which is due at least in part to impairments in both extrinsic and intrinsic apoptotic pathways. One important anti-apoptotic factor that is strongly overexpressed in RCCs and known to inhibit both apoptotic pathways is ARC (apoptosis repressor with a CARD domain).MethodsExpression and subcellular distribution of ARC in RCC tissue samples and RCC cell lines were determined by immunohistochemistry and fluorescent immunohistochemistry, respectively. Extrinsic and intrinsic apoptosis signalling were induced by TRAIL (TNF-related apoptosis-inducing ligand), ABT-263 or topotecan. ARC knock-down was performed in clearCa-12 cells using lentiviral transduction of pGIPZ. shRNAmir constructs. Extrinsic respectively intrinsic apoptosis were induced by TRAIL (TNF-related apoptosis-inducing ligand), ABT263 or topotecan. Potential synergistic effects were tested by pre-treatment with topotecan and subsequent treatment with ABT263. Activation of different caspases and mitochondrial depolarisation (JC-1 staining) were analysed by flow cytometry. Protein expression of Bcl-2 family members and ARC in RCC cell lines was measured by Western blotting. Statistical analysis was performed by Student’s t-test.ResultsRegarding the extrinsic pathway, ARC knockdown strongly enhanced TRAIL-induced apoptosis by increasing the activation level of caspase-8. Regarding the intrinsic pathway, ARC, which was only weakly expressed in the nuclei of RCCs in vivo, exerted its anti-apoptotic effect by impairing mitochondrial activation rather than inhibiting p53. Topotecan- and ABT-263-induced apoptosis was strongly enhanced following ARC knockdown in RCC cell lines. In addition, topotecan pre-treatment enhanced ABT-263-induced apoptosis and this effect was amplified in ARC-knockdown cells.ConclusionTaken together, our results are the first to demonstrate the importance of ARC protein in the inhibition of both the extrinsic and intrinsic pathways of apoptosis in RCCs. In this context, ARC cooperates with anti-apoptotic Bcl-2 family members to exert its strong anti-apoptotic effects and is therefore an important factor not only in the therapeutic resistance but also in future therapy strategies (i.e., Bcl-2 inhibitors) in RCC. In sum, targeting of ARC may enhance the therapeutic response in combination therapy protocols.

Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems

The frontline drug doxorubicin has been used for treating cancer for over 30 years. While providing a cure in select cases, doxorubicin causes toxicity to most major organs, especially life-threatening cardiotoxicity, which forces the treatment to become dose-limiting. Doxorubicin is known to bind to DNA-associated enzymes, intercalate with DNA base pairs, and target multiple molecular targets to produce a range of cytotoxic effects. For instance, it causes the activation of various molecular signals from AMPK (AMP-activated protein kinase inducing apoptosis) to influence the Bcl-2/Bax apoptosis pathway. By altering the Bcl-2/Bax ratio, downstream activation of different caspases can occur resulting in apoptosis. Doxorubicin also induces apoptosis and necrosis in healthy tissue causing toxicity in the brain, liver, kidney and heart. Over the years, many studies have been conducted to devise a drug delivery system that would eliminate these adverse affects including liposomes, hydrogel and nanoparticulate systems, and we highlight the pros and cons of these drug delivery systems. Overall the future for the continued use of doxorubicin clinically against cancer looks set to be prolonged, provided certain enhancements as listed above are made to its chemistry, delivery and toxicity. Increased efficacy depends on these three aims being met satisfactorily as discussed in turn in this review.

Photodynamic therapy and the role of heat shock protein 70

Photodynamic therapy (PDT) is an effective treatment for superficial epithelial skin cancers and is also being utilised for skin rejuvenation. PDT with porphyrin-derived photosensitisers may be capable of inducing rapid cytochrome c release initiating apoptotic cascade via an activation of different caspases. Hsp70 has been demonstrated to directly bind to the caspase recruitment domain (CARD) of apoptotic-protease activating factor 1 (Apaf-1), thereby preventing the recruitment of oligomerisation of Apaf-1 and association of Apaf-1 with procaspase 9. Further, cytoplasmic Hsp70 rapidly translocates to the cell surface where it stabilises damaged membranes and preserves their integrity depending on the PDT dose. The induced cell surface expression and release of Hsp70 and its relevance for tumor response or skin rejuvenation is not fully understood, but seems to be of interest for monitoring, predicting or optimising treatment regimens. This review aims to summarise the current knowledge on PDT mediated cell signalling.

A human ribonuclease induces apoptosis associated with p21WAF1/CIP1induction and JNK inactivation

BackgroundRibonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase.MethodsCytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot.ResultsWe show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAPConclusionsWe conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK. This mechanism is significantly different from that found for onconase.

Ion channel inhibitors block caspase activation by mechanisms other than restoring intracellular potassium concentration

Ion fluxes at the plasma membrane have an important role in early stages of apoptosis. Accordingly, plasma membrane depolarization and gain of Na+ and loss of K+ are initial events in apoptosis. We have studied the effect of staurosporine (STS), a well-established apoptosis inducer, on the membrane potential of HeLa cells to determine the nature of STS-activated ion conductances and their role in the activation of different caspases. We observed that STS can activate tetraethylammonium (TEA+) and 4-aminopyridine-sensitive K+ channels and flufenamic-sensitive cation channels as an early response. The combination of these ion channel inhibitors significantly reduced cytochrome c (cyt c) release and activation of caspase-9, -3 and -8. STS also induced a large reduction in the intracellular [K+] that was not blocked by the ion channel inhibitors. Our data suggest that reduction in the [K+]i is necessary but not sufficient and that ion channel inhibitors block activation of caspase-3 by two different mechanisms: the inhibitors of K+ channels by reducing cyt c release while flufenamic acid by a different, unrelated mechanism that does not involve cation channels at the plasma membrane. Our data also imply that these ion channels activated by STS are not responsible for the reduction in the [K+]i associated with apoptosis.

Resveratrol Inhibits Uveal Melanoma Tumor Growth via Early Mitochondrial Dysfunction

To test the efficacy of resveratrol, a nontoxic plant product, in the treatment of uveal melanoma. The effect of oral administration and peritumor injection of resveratrol was tested on tumor growth in two animal models of uveal melanoma. The mechanism of resveratrol action on uveal melanoma cells was studied in vitro in a cell-viability assay: with JC-1 dye, to measure mitochondrial membrane potential; by Western blot analysis, to analyze the cellular redistribution of cytochrome c and Smac/diablo; and in a fluorescence assay with specific substrates, to measure activation of different caspases. Resveratrol treatment inhibited tumor growth in animal models of uveal melanoma. Since oral administration resulted in relatively low bioavailability of resveratrol, the effect of increased local levels was tested by peritumor injection of the drug. This method resulted in tumor cell death and tumor regression. In vitro experiments with multiple uveal melanoma cell lines demonstrate that resveratrol causes a decrease in cell viability, resulting at least in part from an increase in apoptosis through a mitochondrial pathway. An early event in drug action is the direct targeting of mitochondria by resveratrol, which leads to a decrease in mitochondrial membrane potential and the eventual activation of caspase-3. These data suggest that resveratrol can inhibit tumor growth and can induce apoptosis via the intrinsic mitochondrial pathway and that by further increasing bioavailability of resveratrol the potency of the drug can be increased, leading to tumor regression. The nontoxic nature of the drug at levels needed for therapy make resveratrol an attractive candidate for the treatment of uveal melanoma.

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole induces apoptosis and necrosis with activation of different caspases in rat splenocytes.

A dietary carcinogen, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) at 20 microM activates caspase-3-like proteases as an apoptotic marker in rat splenocytes. The present study demonstrated 100 microM Trp-P-1 induced necrosis with activation of caspase-3-like proteases. The activation in necrosis and apoptosis resulted from the activation of caspase-9 and caspase-8, respectively. Thus, Trp-P-1 induces apoptosis and necrosis with the activation of different caspases.

Caspase activation pathways induced by staurosporine and low potassium: role of caspase-2.

Apoptotic death is a physiological process with regulatory mechanisms that are under the control of different molecules such as caspases. These are classified as initiators, such as caspases-8 and -9, and effectors, such as caspases-3 and -7. The participation of caspase-2 in the effector phase of apoptosis has been commonly observed in many cell types; however, it is able to act as an initiator caspase, depending on the apoptotic stimulus. Cerebellar granule cells (CGCs) undergo apoptosis when they are transferred from high potassium (K25) to low potassium (K5); this process seems to be mediated by caspase-3 activation. Staurosporine (STS), a full strength inhibitor of kinase proteins, also induces apoptosis in these cells. To characterize the caspase cascade induced by two stimuli in the same cell type we studied the activation of different caspases in CGCs treated with STS or K5. We found that both K5 and STS induce the activation of caspase-3. This result was confirmed by the proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), an endogenous caspase-3 substrate. Caspase-2 was activated preferentially by STS, which showed a temporal course suggesting that this caspase was induced before caspase-3. The initiator caspase-9 was also activated by both K5 and STS, as well as cytochrome-c release. The results obtained in this study suggest that STS and K5 induced different activation caspase pathways for apoptotic cell death of CGCs.

Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells.

To investigate the mechanisms underlying apoptosis in breast cancer cells, staurosporine was used as an apoptotic stimulus in the human breast cancer cell lines MCF-7 and T47D. Staurosporine induced dose and time dependent increases in DNA fragmentation which was abrogated by z-VAD-fmk. MCF-7 cells did not express caspase-3, suggesting that DNA fragmentation occurred in the absence of caspase-3 and that other caspases may be involved. Staurosporine induced DEVDase activity in T47D cells suggesting the involvement of caspase-3 and/or caspase-7, yet there was no DEVDase activity in MCF-7 cells, probably ruling out the involvement caspase-7. However, staurosporine induced the cleavage of pro-caspase-6 in MCF-7 cells, but not in T47D cells. Caspase dependent PARP cleavage was detected in MCF-7 cells at 3 h, whereas only partial PARP cleavage was detected in T47D cells and then only after 24 h. Moreover, staurosporine led to cytochrome c release at 2 h in MCF-7 cells and 6 h in T47D cells. In addition, a time dependent and caspase-independent reduction of the mitochondrial transmembrane potential was observed; which appeared to occur after the release of cytochrome c. Translocation of Bax from the cytosol to mitochondria was observed in both cell types, and this preceded cytochrome c release in both T47D and MCF-7 cells. Apoptotic events in both cell types differ temporally, involving activation of different caspases and mitochondrial changes.British Journal of Cancer (2002) 87, 909–917. doi:10.1038/sj.bjc.6600541 www.bjcancer.com© 2002 Cancer Research UK

Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis.

The proto-oncogene Bcl-2 is expressed in membranes of mitochondria and endoplasmic reticulum and mediates resistance against a broad range of apoptotic stimuli. Although several mechanisms of Bcl-2 action have been proposed, its role in different cellular organelles remains elusive. Here, we analyzed the function of Bcl-2 targeted specifically to certain subcellular compartments in Jurkat cells. Bcl-2 expression was restricted to the outer mitochondrial membrane by replacing its membrane anchor with the mitochondrial insertion sequence of ActA (Bcl-2/MT) or the ER-specific sequence of cytochrome b5 (Bcl-2/ER). Additionally, cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/DeltaTM) were employed. Apoptosis induced by ionizing radiation or by the death receptors for CD95L or TRAIL was analyzed by determination of the mitochondrial membrane potential (DeltaPsi(m)) and activation of different caspases. Bcl-2/WT and Bcl-2/MT strongly inhibited radiation-induced apoptosis and caspase activation, whereas Bcl-2/DeltaTM had completely lost its anti-apoptotic effect. Interestingly, Bcl-2/ER conferred protection against radiation-induced mitochondrial damage and apoptosis similarly to Bcl-2/MT. The finding that ER-targeted Bcl-2 interfered with mitochondrial DeltaPsi(m) breakdown and caspase-9 activation indicates the presence of a crosstalk between both organelles in radiation-induced apoptosis. By contrast, Bcl-2 in either subcellular position did not influence CD95- or TRAIL-mediated apoptosis.

Protease Activation in Apoptosis Induced by MAL

The proteolytic caspase cascade plays a central role in the signaling and execution steps of apoptosis. This study investigated the activation of different caspases in apoptosis induced by MAL (a folding variant of human α-lactalbumin) isolated from human milk. Our results show that the caspase-3-like enzymes, and to a lesser extent the caspase-6-like enzymes, were activated in Jurkat and A549 cells exposed to MAL. Activated caspases subsequently cleaved several protein substrates, including PARP, lamin B, and α-fodrin. A broad-range caspase inhibitor, zVAD-fmk, blocked the caspase activation, the cleavage of proteins, and DNA fragmentation, indicating an important role for caspase activation in MAL-induced apoptosis. Since an antagonistic anti-CD95 receptor antibody, ZB4, did not influence the MAL-induced killing, we conclude that this process does not involve the CD95-mediated pathway. While MAL did not directly activate caspases in the cytosol, it colocalized with mitochondria and induced the release of cytochrome c. Thus, these results demonstrate that caspases are activated and involved in apoptosis induced by MAL and that direct interaction of MAL with mitochondria leads to the release of cytochrome c, suggesting that this release is an important step in the initiation and/or amplification of the caspase cascade in these cells.