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PURPOSE - Apoptosis, or programmed cell death, can be leveraged as a surrogate measure of response to therapeutic interventions in medicine. Cysteine aspartic acid-specific proteases, or caspases, are essential determinants of apoptosis signaling cascades and represent promising targets for molecular imaging. Here, we report development and in vivo validation of [(18)F]4-fluorobenzylcarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone ([(18)F]FB-VAD-FMK), a novel peptide-based molecular probe suitable for quantification of caspase activity in vivo using positron emission tomography (PET).
EXPERIMENTAL DESIGN - Supported by molecular modeling studies and subsequent in vitro assays suggesting probe feasibility, the labeled pan-caspase inhibitory peptide, [(18)F]FB-VAD-FMK, was produced in high radiochemical yield and purity using a simple two-step, radiofluorination. The biodistribution of [(18)F]FB-VAD-FMK in normal tissue and its efficacy to predict response to molecularly targeted therapy in tumors was evaluated using microPET imaging of mouse models of human colorectal cancer.
RESULTS - Accumulation of [(18)F]FB-VAD-FMK was found to agree with elevated caspase-3 activity in response to Aurora B kinase inhibition as well as a multidrug regimen that combined an inhibitor of mutant BRAF and a dual PI3K/mTOR inhibitor in (V600E)BRAF colon cancer. In the latter setting, [(18)F]FB-VAD-FMK PET was also elevated in the tumors of cohorts that exhibited reduction in size.
CONCLUSIONS - These studies illuminate [(18)F]FB-VAD-FMK as a promising PET imaging probe to detect apoptosis in tumors and as a novel, potentially translatable biomarker for predicting response to personalized medicine.
BACKGROUND - Tumor necrosis factor alpha (TNFalpha) is associated with trauma-induced hearing loss. Local treatment of cochleae of trauma-exposed animals with a glucocorticoid is effective in reducing the level of hearing loss that occurs post-trauma (e.g., electrode insertion trauma-induced hearing loss/dexamethasone treatment).
HYPOTHESIS - Dexamethasone (Dex) protects auditory hair cells (AHCs) from trauma-induced loss by activating cellular signal pathways that promote cell survival.
MATERIALS AND METHODS - Organ of Corti explants challenged with an ototoxic level of TNFalpha was the trauma model with Dex the otoprotective drug. A series of inhibitors were used in combination with the Dex treatment of TNFalpha-exposed explants to investigate the signal molecules that participate in Dex-mediated otoprotection. The otoprotective capacity of Dex against TNFalpha ototoxicity was determined by hair cell counts obtained from fixed explants stained with FITC-phalloidin labeling with investigators blinded to specimen identity.
RESULTS - The general caspase inhibitor Boc-d-fmk prevented TNFalpha-induced AHC death. There was a significant reduction (p<0.05) in the efficacy of Dex otoprotection against TNFalpha ototoxicity when the following cellular events were blocked: (1) glucocorticoid receptors (Mif); (2) PI3K (LY294002); (3) Akt/PKB (SH-6); and (4) NFkappaB (NFkappaB-I).
CONCLUSION - Dex treatment protects hair cells against TNFalpha apoptosis in vitro by activation of PI3K/Akt and NFkappaB signaling.
Diallyl disulfide (DADS), an important component of garlic (Allium sativum) derivative, has been demonstrated to exert a potential molecular target against human cancers. We investigated DADS-induced expressions of Apaf1, cystatin B, caspase-3 and FADD (fas-associated protein with death domain) in breast, prostate and lung cancer cells. These showed coincident data when further examined by quantitative reverse transcription-polymerase chain reaction and Western blot analysis. Furthermore, DADS induced a marked amount of Bax translocation, cytochrome c release and activation of caspase-3 and caspase-9. DADS-treated tumor cells triggered mitochondria-mediated signaling pathways that led to a significant increase in apoptosis induction. Further studies with caspase-3 and caspase-9 inhibitors (zDEVD-fmk and zLEHD-fmk, respectively) proved that DADS induces apoptosis through a caspase-3-dependent pathway. DADS is only an agent used in the study. The molecular mechanism presented therefore provides strong additional support to the hypothesis that DADS is a strong inducer of apoptosis through a Bax-triggered mitochondria-mediated and caspase-3-dependent pathway. This study shows clearly that DADS causes caspase-dependent apoptosis in human cancer cells through a Bax-triggered mitochondrial pathway. Therefore, the mitochondrial pathway might be the target for cancer chemoprevention and/or chemotherapy by DADS.
Published by Elsevier Inc.
The mechanism of mitochondrial damage, a key contributor to renal tubular cell death during acute kidney injury, remains largely unknown. Here, we have demonstrated a striking morphological change of mitochondria in experimental models of renal ischemia/reperfusion and cisplatin-induced nephrotoxicity. This change contributed to mitochondrial outer membrane permeabilization, release of apoptogenic factors, and consequent apoptosis. Following either ATP depletion or cisplatin treatment of rat renal tubular cells, mitochondrial fragmentation was observed prior to cytochrome c release and apoptosis. This mitochondrial fragmentation was inhibited by Bcl2 but not by caspase inhibitors. Dynamin-related protein 1 (Drp1), a critical mitochondrial fission protein, translocated to mitochondria early during tubular cell injury, and both siRNA knockdown of Drp1 and expression of a dominant-negative Drp1 attenuated mitochondrial fragmentation, cytochrome c release, caspase activation, and apoptosis. Further in vivo analysis revealed that mitochondrial fragmentation also occurred in proximal tubular cells in mice during renal ischemia/reperfusion and cisplatin-induced nephrotoxicity. Notably, both tubular cell apoptosis and acute kidney injury were attenuated by mdivi-1, a newly identified pharmacological inhibitor of Drp1. This study demonstrates a rapid regulation of mitochondrial dynamics during acute kidney injury and identifies mitochondrial fragmentation as what we believe to be a novel mechanism contributing to mitochondrial damage and apoptosis in vivo in mouse models of disease.
IFN regulatory factor 8 (IRF8) has been shown to suppress tumor development at least partly through regulating apoptosis of tumor cells; however, the molecular mechanisms underlying IRF8 regulation of apoptosis are still not fully understood. Here, we showed that disrupting IRF8 function resulted in inhibition of cytochrome c release, caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase cleavage in soft tissue sarcoma (STS) cells. Inhibition of the mitochondrion-dependent apoptosis signaling cascade is apparently due to blockage of caspase-8 and Bid activation. Analysis of signaling events upstream of caspase-8 revealed that disrupting IRF8 function dramatically increases FLIP mRNA stability, resulting in increased IRF8 protein level. Furthermore, primary myeloid cells isolated from IRF8-null mice also exhibited increased FLIP protein level, suggesting that IRF8 might be a general repressor of FLIP. Nuclear IRF8 protein was absent in 92% (55 of 60) of human STS specimens, and 99% (59 of 60) of human STS specimens exhibited FLIP expression, suggesting that the nuclear IRF8 protein level is inversely correlated with FLIP level in vivo. Silencing FLIP expression significantly increased human sarcoma cells to both FasL-induced and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, and ectopic expression of IRF8 also significantly increased the sensitivity of these human sarcoma cells to FasL- and TRAIL-induced apoptosis. Taken together, our data suggest that IRF8 mediates FLIP expression level to regulate apoptosis and targeting IRF8 expression is a potentially effective therapeutic strategy to sensitize apoptosis-resistant human STS to apoptosis, thereby possibly overcoming chemoresistance of STS, currently a major obstacle in human STS therapy.
Podocyte injury and loss contribute to progressive glomerulosclerosis. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear hormone receptor, which we have found to be increased in podocytes in a variety of kidney diseases. It is not known if PPAR-gamma contributes to renal injury or if it serves as a countermeasure to limit renal injury during disease progression. We tested these possibilities utilizing the puromycin aminonucleoside (PAN) model of renal injury in immortalized mouse podocytes. The cultured podocytes expressed PPAR-gamma mRNA at baseline but this was decreased by PAN. Pioglitazone, a pharmacologic agonist of PPAR-gamma, increased both PPAR-gamma mRNA and activity in injured podocytes, as assessed by a reporter plasmid assay. Further, pioglitazone significantly decreased PAN-induced podocyte apoptosis and necrosis while restoring podocyte differentiation. The PPAR-gamma agonist significantly restored expression of the cyclin-dependent kinase inhibitor p27 and the antiapoptotic molecule Bcl-xL while significantly decreasing proapoptotic caspase-3 activity. Pioglitazone tended to decrease PAN-induced transforming growth factor-beta (TGF-beta) mRNA expression. Our study shows that PPAR-gamma is normally expressed by podocytes and its activation is protective against PAN-induced apoptosis and necrosis. We postulate that this protective effect may be mediated in part by effects on p27 and TGF-beta expression.
Ku70 plays an important role in DNA damage repair and prevention of cell death. Previously, we reported that apoptosis caused a decrease in cellular Ku70 levels. In this study, we analyzed the mechanism of how Ku70 levels decrease during drug-induced apoptosis. In HeLa cells, staurosporin (STS) caused a decrease in Ku70 levels without significantly affecting Ku70 mRNA levels. We found that Ku70 protein was highly ubiquitinated in various cell types, such as HeLa, HEK293T, Dami (a megakaryocytic cell line), endothelial, and rat kidney cells. An increase in ubiquitinated Ku70 protein was observed in apoptotic cells, and proteasome inhibitors attenuated the decrease in Ku70 levels in apoptotic cells. These results suggest that the ubiquitin-proteasome proteolytic pathway plays a role in decreasing Ku70 levels in apoptotic cells. Ku70 forms a heterodimer with Ku80, which is required for the DNA repair activity of Ku proteins. We also found that Ku80 levels decreased in apoptotic cells and that Ku80 is a target of ubiquitin. Ubiquitinated Ku70 was not found in the Ku70-Ku80 heterodimer, suggesting that modification by ubiquitin inhibits Ku heterodimer formation. We propose that the ubiquitin-dependent modification of Ku70 plays an important role in the control of cellular levels of Ku70.
Tamoxifen (TAM), a synthetic nonsteroidal antiestrogen effectively and widely used for breast cancer treatment, is known to have antioxidant and cardioprotective effects, but whether the beneficial cardiovascular effect of TAM is linked to its antioxidant effect is unknown. In this study, we investigated the effect of TAM on the levels of manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, in cardiac tissues and cardiomyocytes. TAM treatment induced MnSOD expression in vitro and in vivo. Cardiomyocytes isolated from TAM-pretreated mice also had higher MnSOD levels and fewer apoptotic cells compared to cardiomyocytes from control mice after adriamycin (ADR) treatment. To further confirm the role of MnSOD in the protection against ADR in cardiomyocytes, we used cardiomyocytes isolated from MnSOD knock-out (MnSOD(+/-)), wild-type (NTg) and human MnSOD transgenic (TgH) mice. TUNEL assay indicated that the percentage of cells undergoing apoptosis after ADR treatment was significantly greater in MnSOD(+/-) than in NTg or TgH cardiomyocytes. 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay showed that basal level of mitochondrial function was lower in MnSOD(+/-) cardiomyocytes than in NTg or TgH, and that MnSOD(+/-) was more sensitive to ADR. ADR treatment increased caspase activity, which was significantly higher in MnSOD(+/-) than in NTg or TgH cardiomyocytes. These results suggested that TAM-induced MnSOD expression is at least, in part, contribute to the cardioprotective effects of TAM.
Different molecular pathways are believed to be involved in the pathogenesis of classic Hodgkin lymphoma as opposed to non-Hodgkin lymphoma. Antiapoptotic mechanisms have been proposed for classic Hodgkin lymphoma, including expression of the cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (c-FLIP), which plays a critical role in resistance to CD95/Fas-mediated apoptosis. In this study, we compare the expression of c-FLIP in the neoplastic cells of classic Hodgkin lymphoma and nodular lymphocyte-predominant Hodgkin lymphoma cases. Sixteen cases of classic Hodgkin lymphoma and 19 cases of nodular lymphocyte-predominant Hodgkin lymphoma were reviewed. Of 16 classic Hodgkin lymphoma cases, 13 cases (81%) were c-FLIP-positive, compared with 6 of 19 (32%) nodular lymphocyte-predominant Hodgkin lymphoma cases. Strong cytoplasmic staining was seen in 7 of 13 c-FLIP-positive classic Hodgkin lymphoma cases, in contrast with only 2 of 6 c-FLIP-positive nodular lymphocyte-predominant Hodgkin lymphoma cases. The 2 cases of nodular lymphocyte-predominant Hodgkin lymphoma with strong c-FLIP expression were associated with transformation to large B-cell lymphoma. An additional 15 cases of diffuse large B-cell lymphoma were studied for c-FLIP expression. All but 1 were c-FLIP-positive. In conclusion, we detected c-FLIP in a significantly lower proportion of nodular lymphocyte-predominant Hodgkin lymphoma cases compared with classic Hodgkin lymphoma cases. Therefore, c-FLIP expression may not be the major mechanism of pathogenesis in nodular lymphocyte-predominant Hodgkin lymphoma. However, strong c-FLIP expression in nodular lymphocyte-predominant Hodgkin lymphoma was associated with transformation to large B-cell lymphoma in 2 cases. c-FLIP expression is not limited to Hodgkin lymphoma, because the majority of diffuse large B-cell lymphoma cases tested were strongly c-FLIP-positive.
Caspases are a family of cysteine proteases that are expressed as inactive zymogens and undergo proteolytic maturation in a sequential manner in which initiator caspases cleave and activate the effector caspases 3, 6 and 7. Effector caspases cleave structural proteins, signaling molecules, DNA repair enzymes and proteins which inhibit apoptosis. Activation of effector, or executioner, caspases has historically been viewed as a terminal event in the process of programmed cell death. Emerging evidence now suggests a broader role for activated caspases in cellular maturation, differentiation and other non-lethal events. The importance of activated caspases in normal cell development and signaling has recently been extended to the CNS where these proteases have been shown to contribute to axon guidance, synaptic plasticity and neuroprotection. This review will focus on the adaptive roles activated caspases in maintaining viability, the mechanisms by which caspases are held in check so as not produce apoptotic cell death and the ramifications of these observations in the treatment of neurological disorders.
Copyright 2004 Kluwer Academic Publishers