Caspase protocols in mice.

Members of the caspase family of proteases are evolutionarily conserved cysteine proteases that play a crucial role as the central executioners of the apoptotic pathway. Since the discovery of caspases, many methods have been developed to detect their activation and are widely used in basic and clinical studies. In a mouse tissue, caspase activation can be monitored by cleavage of caspase-specific synthetic substrates and by detecting cleaved caspase by western blot analysis of the tissue extract. In tissue sections, active caspase can be detected by immunostaining using specific antibodies to the active caspase. In addition, among the myriads of caspase-specific substrates known so far, cleaved fragments produced by caspases from the substrates such as PARP, lamin A, and cytokeratin-18 can be monitored in tissue sections by immunostaining as well as western blots of tissue extracts. In general, more than one method should be used to ascertain detection of activation of caspases in a mouse tissue.

Meprin A and meprin alpha generate biologically functional IL-1beta from pro-IL-1beta.

The present study demonstrates that both oligomeric metalloendopeptidase meprin A purified from kidney cortex and recombinant meprin alpha are capable of generating biologically active IL-1beta from its precursor pro-IL-1beta. Amino-acid sequencing analysis reveals that meprin A and meprin alpha cleave pro-IL-1beta at the His(115)-Asp(116) bond, which is one amino acid N-terminal to the caspase-1 cleavage site and five amino acids C-terminal to the meprin beta site. The biological activity of the pro-IL-1beta cleaved product produced by meprin A, determined by proliferative response of helper T-cells, was 3-fold higher to that of the IL-1beta product produced by meprin beta or caspase-1. In a mouse model of sepsis induced by cecal ligation puncture that results in elevated levels of serum IL-1beta, meprin inhibitor actinonin significantly reduces levels of serum IL-1beta. Meprin A and meprin alpha may therefore play a critical role in the production of active IL-1beta during inflammation and tissue injury.

Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity.

One of the major side effects of cisplatin chemotherapy is toxic acute kidney injury due to preferential accumulation of cisplatin in renal proximal tubule epithelial cells and the subsequent injury to these cells. Apoptosis is known as a major mechanism of cisplatin-induced cell death in renal tubular cells. We have also recently demonstrated that autophagy induction is an immediate response of renal tubular epithelial cell exposure to cisplatin. Inhibition of cisplatin-induced autophagy blocks the formation of autophagosomes and enhances cisplatin-induced caspase-3, -6, and -7 activation, nuclear fragmentation and apoptosis. The switch from autophagy to apoptosis by autophagic inhibitors suggests that autophagy induction was responsible for a pre-apoptotic lag phase observed on exposure of renal tubular cells to cisplatin. Our studies provide evidence that autophagy induction in response to cisplatin mounts an adaptive response that suppresses and delays apoptosis. The beneficial effect of autophagy has a potential clinical significance in minimizing or preventing cisplatin nephrotoxicity.

Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells.

Autophagy has emerged as another major "programmed" mechanism to control life and death much like "programmed cell death" is for apoptosis in eukaryotes. We examined the expression of autophagic proteins and formation of autophagosomes during progression of cisplatin injury to renal tubular epithelial cells (RTEC). Autophagy was detected as early as 2-4 h after cisplatin exposure as indicated by induction of LC3-I, conversion of LC3-I to LC3-II protein, and upregulation of Beclin 1 and Atg5, essential markers of autophagy. The appearance of cisplatin-induced punctated staining of autophagosome-associated LC3-II upon GFP-LC3 transfection in RTEC provided further evidence for autophagy. The autophagy inhibitor 3-methyladenine blocked punctated staining of autophagosomes. The staining of normal cells with acridine orange displayed green fluorescence with cytoplasmic and nuclear components in normal cells but displayed considerable red fluorescence in cisplatin-treated cells, suggesting formation of numerous acidic autophagolysosomal vacuoles. Autophagy inhibitors LY294002 or 3-methyladenine or wortmannin inhibited the formation of autophagosomes but induced apoptosis after 2-4 h of cisplatin treatment as indicated by caspase-3/7 and -6 activation, nuclear fragmentation, and cell death. This switch from autophagy to apoptosis by autophagic inhibitors further suggests that the preapoptotic lag phase after treatment with cisplatin is mediated by autophagy. At later stages of cisplatin injury, apoptosis was also found to be associated with autophagy, as autophagic inhibitors and inactivation of autophagy proteins Beclin 1 and Atg5 enhanced activation of caspases and apoptosis. Our results demonstrate that induction of autophagy mounts an adaptive response, suppresses cisplatin-induced apoptosis, and prolongs survival of RTEC.

Expression of tissue factor in prostate cancer correlates with malignant phenotype.

Tissue factor (TF), apart from its established role in hemostasis, has been implicated in promoting angiogenesis and metastasis in a wide array of tumors including prostate cancer. Expression of TF was evaluated in freshly-resected prostate specimens obtained from patients with localized (n=9) and androgen ablated (n=6) disease using real-time reverse transcription-polymerase chain reaction and Western blot analysis. TF was detected in all specimens in both stages of the disease. We further analyzed for correlations between TF expression and those of several angiogenic growth factors and their receptors. TF RNA expression correlated significantly with expression of vascular endothelial growth factor-A in these specimens (s=0.621, P=0.013). Eighty-one prostate specimens from patients with benign prostatic hyperplasia (n=27), localized prostate cancer (ES, n=32), and advanced disease (n=22) were also evaluated using immunohistochemistry and findings were correlated with clinical parameters. TF expression was detected on epithelial cells of the malignant glands. Furthermore, its expression levels correlated significantly with Gleason score (s=0.58, P=0.0001) and with the stage of the disease (s=0.441, P=0.0001) in these specimens. These data support the role of TF in angiogenesis and disease progression.

Isolated thrombocytopenia induced by thalidomide in a patient with multiple myeloma: case report and review of literature.

Thalidomide is being increasingly used in hematology and oncology. Its use is associated with neuropathy, sedation, edema, fatigue, constipation, and deep venous thrombosis. Cytopenias are unusual, but there are case reports. However, there are no reports of isolated thrombocytopenia. We describe here a case of prolonged isolated thrombocytopenia most likely caused by thalidomide. The patient's in platelet count decreased promptly after an increase in the dose and improved after discontinuation of the drug. The relevant literature is reviewed.

Mcl-1 is downregulated in cisplatin-induced apoptosis, and proteasome inhibitors restore Mcl-1 and promote survival in renal tubular epithelial cells.

Mcl-1 is an antiapoptotic member of the Bcl-2 family that plays an important role in cell survival. We demonstrate that proteasome-dependent regulation of Mcl-1 plays a critical role in renal tubular epithelial cell injury from cisplatin. Protein levels of Mcl-1 rapidly declined in a time-dependent manner following cisplatin treatment of LLC-PK(1) cells. However, mRNA levels of Mcl-1 were not altered following cisplatin treatment. Expression of other antiapoptotic members of the Bcl-2 family such as Bcl-2 and BclxL was not affected by cisplatin treatment. Cisplatin-induced loss of Mcl-1 occurs at the same time as the mitochondrial release of cytochrome c, activation of caspase-3, and initiation of apoptosis. Treatment of cells with cycloheximide, a protein synthesis inhibitor, revealed rapid turnover of Mcl-1. In addition, treatment with cycloheximide in the presence or absence of cisplatin demonstrated that cisplatin-induced loss of Mcl-1 results from posttranslational degradation rather than transcriptional inhibition. Overexpression of Mcl-1 protected cells from cisplatin-induced caspase-3 activation and apoptosis. Preincubating cells with the proteasome inhibitor MG-132 or lactacystin not only restored cisplatin-induced loss of Mcl-1 but also resulted in an accumulation of Mcl-1 that exceeded basal levels; however, Bcl-2 and BclxL levels did not change in response to MG-132 or lactacystin. The proteasome inhibitors effectively blocked cisplatin-induced mitochondrial release of cytochrome c, caspase-3 activation, and apoptosis. These studies suggest that proteasome regulation of Mcl-1 is crucial in the cisplatin-induced apoptosis via the mitochondrial apoptotic pathway and that Mcl-1 is an important therapeutic target in cisplatin injury to renal tubular epithelial cells.

Thrombin receptor expression is upregulated in prostate cancer.

BACKGROUND: Aberrant expression of protease-activated receptors (PARs) has been associated with increased angiogenesis, tumor growth, and metastasis of various cancers. We assessed the status of PAR1 expression in prostate cancer. METHODS: The study compared the abundance levels of PAR1 RNA and protein using real-time reverse-transcriptase polymerase chain reaction and immunoblotting in freshly resected prostate tissues from early localized-stage disease (n=9) to those from patients with advanced metastatic disease (n=7). PAR1 expression and localization was evaluated using immunohistochemical staining of prostate specimens with benign prostatic hyperplasia (n=27), early- (n=32) and advanced-stage (n=22) prostate cancer. Association analyses of PAR1 expression with expression of VEGF-family of growth factors, their receptors, and clinicopathological characteristics of the patients were also performed. RESULTS: PAR1 RNA expression in advanced-stage prostate was 2.39-fold higher (P=0.024) and its protein expression was 2.75-fold higher (P=5.89x10(-5)) when compared with early-stage prostate cancer. PAR1 expression was localized to endothelial cells in vascular network of prostate tumor areas. The expression of PAR1 correlated statistically significantly with advanced disease stage (P=0.0006) and pre-operative PSA levels (P=0.005) in these samples. CONCLUSIONS: These findings demonstrate that PAR1 expression is increased in prostate cancer. Its predominant expression in vascular network suggests that it may play a direct and crucial role in angiogenesis and could be a relevant target for therapeutic interventions to control or to prevent disease progression.

p53-dependent caspase-2 activation in mitochondrial release of apoptosis-inducing factor and its role in renal tubular epithelial cell injury.

We demonstrate the role of p53-mediated caspase-2 activation in the mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-treated renal tubular epithelial cells. Gene silencing of AIF with its small interfering RNA (siRNA) suppressed cisplatin-induced AIF expression and provided a marked protection against cell death. Subcellular fractionation and immunofluorescence studies revealed cisplatin-induced translocation of AIF from the mitochondria to the nuclei. Pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone or p53 inhibitor pifithrin-alpha markedly prevented mitochondrial release of AIF, suggesting that caspases and p53 are involved in this release. Caspase-2 and -3 that were predominantly activated in response to cisplatin provided a unique model to study the role of these caspases in AIF release. Cisplatin-treated caspase-3 (+/+) and caspase-3 (-/-) cells exhibited similar AIF translocation to the nuclei, suggesting that caspase-3 does not affect AIF translocation, and thus, caspase-2 may be involved in the translocation. Caspase-2 inhibitor benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone or down-regulation of caspase-2 by its siRNA significantly prevented translocation of AIF. Caspase-2 activation was a critical response from p53, which was markedly induced and phosphorylated in cisplatin-treated cells. Overexpression of p53 not only resulted in caspase-2 activation but also mitochondrial release of AIF. The p53 inhibitor pifithrin-alpha or p53 siRNA prevented both cisplatin-induced caspase-2 activation and mitochondrial release of AIF. Caspase-2 activation was dependent on the p53-responsive gene, PIDD, a death domain-containing protein that was induced by cisplatin in a p53-dependent manner. These results suggest that caspase-2 activation mediated by p53 is an important pathway involved in the mitochondrial release of AIF in response to cisplatin injury.

Stage-specific characterization of the vascular endothelial growth factor axis in prostate cancer: expression of lymphangiogenic markers is associated with advanced-stage disease.

PURPOSE: The vascular endothelial growth factor (VEGF) family plays a critical role in tumor angiogenesis and lymphangiogenesis. We characterized, at the mRNA and protein levels, the expression of VEGF-A and VEGF-D and their cognate receptors, VEGFR-1, VEGFR-2, and VEGFR-3 in early- and advanced-stage prostate cancer specimens. EXPERIMENTAL DESIGN: The levels of VEGF-A and VEGF-D mRNA in early- and advanced-stage specimens were compared using an angiogenic gene array and were confirmed by quantitative real-time PCR. Receptor protein levels and activation status were determined by immunoblotting. Spatial expression of the proteins was evaluated using immunohistochemistry with fresh and archival tissues from benign prostatic hypertrophy specimens, early-stage prostate specimens, and advanced-stage metastatic specimens. Circulating plasma levels of these growth factors were measured using ELISAs. RESULTS: We observed that expression patterns of VEGF isotypes corresponded to the prostate cancer stage: high expression of angiogenic growth factor VEGF-A was observed in early-stage prostate specimens, whereas high expression of lymphangiogenic growth factor VEGF-D was associated with advanced-stage metastatic disease. All VEGF receptors were present at variable levels in all specimens, but their activation states varied in a stage-specific manner. VEGFR-1 and, to a limited extent, VEGFR-2 were activated in early-stage specimens, whereas VEGFR-2 and VEGFR-3 were activated in advanced-stage specimens. CONCLUSIONS: Our results suggest that lymphangiogenic markers, such as VEGF-D and VEGFR-2 and VEGFR-3, may be better than angiogenic markers as targets of therapeutic intervention in advanced-stage prostate disease.

Regulation of caspase-3 and -9 activation in oxidant stress to RTE by forkhead transcription factors, Bcl-2 proteins, and MAP kinases.

Cytotoxicity to renal tubular epithelial cells (RTE) is dependent on the relative response of cell survival and cell death signals triggered by the injury. Forkhead transcription factors, Bcl-2 family member Bad, and mitogen-activated protein kinases are regulated by phosphorylation that plays crucial roles in determining cell fate. We examined the role of phosphorylation of these proteins in regulation of H(2)O(2)-induced caspase activation in RTE. The phosphorylation of FKHR, FKHRL, and Bcl-2 family member Bad was markedly increased in response to oxidant injury, and this increase was associated with elevated levels of basal phosphorylation of Akt/protein kinase B. Phosphoinositol (PI) 3-kinase inhibitors abolished this phosphorylation and also decreased expression of antiapoptotic proteins Bcl-2 and BclxL. Inhibition of phosphorylation of forkhead proteins resulted in a marked increase in the proapoptotic protein Bim. These downstream effects of PI 3-kinase inhibition promoted the oxidant-induced activation of caspase-3 and -9, but not caspase-8 and -1. The impact of enhanced activation of caspases by PI 3-kinase inhibition was reflected on accelerated oxidant-induced cell death. Oxidant stress also induced marked phosphorylation of ERK1/2, P38, and JNK kinases. Inhibition of ERK1/2 phosphorylation but not P38 and JNK kinase increased caspase-3 and -9 activation; however, this activation was far less than induced by inhibition of Akt phosphorylation. Thus the Akt-mediated phosphorylation pathway, ERK signaling, and the antiapoptotic Bcl-2 proteins distinctly regulate caspase activation during oxidant injury to RTE. These studies suggest that enhancing renal-specific survival signals may lead to preservation of renal function during oxidant injury.

Differential toxicity of anthracyclines on cultured endothelial cells.

Anthracyclines are known for their endothelial toxicity. Newer derivatives may have fewer toxic effects on endothelium. The authors therefore evaluated the effects of doxorubicin, doxorubicin analogs (daunorubicin, idarubicin), and pegylated liposomal doxorubicin (doxil) in human coronary artery endothelial cells (HCAECs). Endothelial viability did not change significantly with doxil, but was decreased with doxorubicin, daunorubicin, or idamycin. Similarly caspase-3 activity was significantly elevated in HCAECs treated with doxorubicin, daunorubicin, and idamycin. In contrast, doxil did not cause significant increase in caspase activity. The authors also characterized the levels of antiapoptotic and prosurvival proteins using Western blot analysis. There was no significant difference in the expression levels of Bcl-2, Bax, and phospho-Akt in endothelial cells treated with anthracycline derivatives. However, the expression levels of Mcl-l protein were unaltered in endothelial cells treated with doxil but were significantly decreased when treated with other anthracycline analogs. Doxil minimally affected the expression levels of p53, whereas other anthracyclines induced p53 protein levels to a significant level, resulting in endothelial cell apoptosis. The authors conclude that the liposomal anthracycline protects endothelial cells from injury by preventing caspase-3 activation and maintaining the expression of antiapoptotic molecule Mcl-1.

Role of coagulation and fibrinolytic system in prostate cancer.

Patients with prostate carcinoma paradoxically have both a hypercoagulable state and a bleeding diathesis. Hypercoagulability manifested by venous and arterial thrombosis has been documented in several large clinical trials. However, many investigators have reported a high risk of postoperative bleeding in prostate cancer patients. Disseminated intravascular coagulopathy has also been commonly noted at different clinical stages of this indolent cancer. In this article we review clinical, laboratory, and experimental evidence for abnormalities of various components of the coagulation and plasminogen pathways and analyze their contribution in prostate cancer growth, progression, and angiogenesis. Finally, we propose potential therapeutic antiangiogenic strategies in patients with prostate cancer.

Potential anticancer effects of statins: fact or fiction?

Deregulation of any of the steps in cell growth, proliferation and apoptosis may result in its malignant transformation. Statins, along with their lipid-lowering potential, modify several processes in the cell cycle. These agents inhibit cell proliferation and arrest cell cycle progression by interrupting growth-promoting signals. Statins selectively induce proapoptotic protential in tumor cells and synergistically enhance proapoptotic potential of several cytotoxic agents. Statins alter angiogenic potential of cells by modulating apoptosis inhibitory effects of VEGF and decrease secretion of metalloproteases. Statins also alter adhesion and migration of tumor cells, thereby inhibiting tumor invasion and metastasis. Statins suppress rate of activation of multiple coagulation factors and thus prevent coagulation-mediated angiogenesis. Statins have been shown to have anti-tumor activity in experimental models. Various anti-neoplastic properties of statins are probably a result of inhibition of posttranslational modifications of growth regulatory proteins. Molecular mechanisms of antiproliferative, proapoptotic and antiangiogenic effects of statins are reviewed in this chapter.

Cloning and expression of rat caspase-6 and its localization in renal ischemia/reperfusion injury.

BACKGROUND: Caspase-6 is an important member of the executioner caspases in the caspase family of cell death proteases. The executioner caspases are the major active caspases detected in apoptotic cells and are generally considered to mediate the execution of apoptosis by cleaving and inactivating intracellular proteins. However, the complete characterization of mRNA and protein of caspase-6 in rat and its expression in normal kidney and in disease state has not been previously elucidated. METHODS: A rat kidney cortex lambdagt10 cDNA library was screened to isolate the full-length caspase-6 cDNA. The recombinant caspase-6 protein was characterized by expression in bacteria and by transient transfection in mammalian cells. The expression in various tissues was analyzed by Northern blot, and localization in normal and ischemic kidney was performed by immunohistochemistry. RESULTS: The predicted amino acid sequence of rat caspase-6 contains 277 amino acids, with two potential glycosylation sites, an integrin binding site (KGD), the caspase active site pentapeptide QACRG and the caspase family signature, HX2-4(S,C) X4(L,I,V,M,F)2(S,T)HG (HVDADCFVCVFLSHG). Rat caspase-6 is unique among known caspases by possessing a relatively long 5' untranslational region. Among various tissues tested, cas-pase-6 was expressed in varying levels in kidney, liver, spleen, heart, muscle, testis, and lung. Bacterial expression of recombinant rat caspase-6 resulted in production of both of the pro-form and active form of the enzyme suggesting autoactivation. Transient overexpression of rat caspase-6 in COS-1 cells induced DNA fragmentation, a hallmark of apoptosis. We also examined the localization and expression of caspase-6 by immunohistochemistry in kidneys subjected to 40 minutes of ischemia followed by 24 hours of reperfusion injury. Normal kidney showed mostly cytoplasmic and some nuclear staining of the tubules. Kidneys 24 hours after 40 minutes of ischemia showed more intense and diffused cytoplasmic staining with prominent nuclear staining, indicating increased expression and translocation from the cytoplasm to the nuclei. The staining in glomeruli was negative in both normal and ischemic kidney. CONCLUSIONS: These studies demonstrate cloning, expression and characterization of the full-length rat caspase-6 and its localization in normal kidneys and kidneys subjected to ischemia/reperfusion injury. Since caspase-6 is involved in the degradation of nuclear matrix proteins and in activation of caspase-3, it may play an important role during renal ischemic injury.