Doxorubicin (DOX), a widely used antitumour drug, causes dose-dependent cardiotoxicity. Cardiac mitochondria represent a critical target organelle of toxicity during DOX chemotherapy. Proposed mechanisms include generation of ROS (reactive oxygen species) and disturbances in mitochondrial calcium homoeostasis. In the present study, we probed the mechanistic link between mitochondrial ROS and calcium in the embryonic rat heart-derived H9c2 cell line and in adult rat cardiomyocytes. The results show that DOX stimulates calcium/calcineurin-dependent activation of the transcription factor NFAT (nuclear factor of activated T-lymphocytes). Pre-treatment of cells with an intracellular calcium chelator abrogated DOX-induced nuclear NFAT translocation, Fas L (Fas ligand) expression and caspase activation, as did pre-treatment of cells with a mitochondria-targeted antioxidant, Mito-Q (a mitochondria-targeted antioxidant consisting of a mixture of mitoquinol and mitoquinone), or with adenoviral-over-expressed antioxidant enzymes. Treatment with GPx-1 (glutathione peroxidase 1), MnSOD (manganese superoxide dismutase) or a peptide inhibitor of NFAT also inhibited DOX-induced nuclear NFAT translocation. Pre-treatment of cells with a Fas L neutralizing antibody abrogated DOX-induced caspase-8- and -3-like activities during the initial stages of apoptosis. We conclude that mitochondria-derived ROS and calcium play a key role in stimulating DOX-induced 'intrinsic and extrinsic forms' of apoptosis in cardiac cells with Fas L expression via the NFAT signalling mechanism. Implications of ROS- and calcium-dependent NFAT signalling in DOX-induced apoptosis are discussed.
CD44 is implicated in cell-cell and cell-matrix adhesion, cell migration, and signaling. CD44 cleavage correlates with the tumor burden and metastatic potential in various cancers. In this study, we demonstrate that matrix metalloproteinase-9 (MMP-9) acts as a processing enzyme for CD44 cleavage. Further, this processing event stimulates cell motility and inhibition of either CD44 or MMP-9 inhibited cell migration. MMP-9 and CD44 co-localization on the cell surface was observed in the histological sections of human glioblastoma (GBM) tissues. Confocal microscopy and co-immunoprecipitation studies in GBM xenograft cells further confirm this interaction. The interaction of MMP-9 with CD44 induced CD44 cleavage which was inhibited by both transcriptional knockdown of MMP-9 and with MMP-9 specific inhibitor. Further, supplementation of purified and activated human MMP-9 (hMMP-9) in MMP-9-knockdown cells resumed CD44 cleavage and migration. Additionally, activated hMMP-9 protein induced cleavage of recombinant human CD44 (rhCD44) in an in vitro assay. Selective overexpression of either extracellular domain (CD44ECD) or intracellular domain (CD44ICD) confirmed that CD44ECD played a role in cell migration and invasion. Taken together, our results suggest that MMP-9 is involved in the shedding of CD44 from cancer cells, which would promote the malignant potential of tumor cells.
SUMMARYWe previously showed that MMP-9 inhibition using an adenoviral-mediated delivery of MMP-9 siRNA (Ad-MMP-9), caused senescence in medulloblastoma cells. Regardless of whether or not, Ad-MMP-9 would induce apoptosis, the possible signaling mechanism is still obscure. In this report, we demonstrate that Ad-MMP-9 induced apoptosis in DAOY cells as determined by propidium iodide and TUNEL staining. Ad-MMP-9 infection induced the release of cytochrome-c, activation of caspases-9-3, and cleavage of PARP. Ad-MMP-9 infection stimulated ERK, and EMSA indicated an increase in NF-κB activation. ERK inhibition, using kinase dead mutant for ERK, ameliorated NF-κB activation and caspase-mediated apoptosis in Ad-MMP-9 infected cells. β1-integrin expression in Ad-MMP-9 infected cells also increased, and this increase was reversed by the reintroduction of MMP-9. We found that, addition of β1 blocking antibodies inhibited Ad-MMP-9-induced ERK activation. Taken together, our results indicate that MMP-9 inhibition induces apoptosis due to altered β1 integrin expression in medulloblastoma. In addition, ERK activation plays an active role in this process and functions upstream of NF-κB activation to initiate the apoptotic signal.
A proposed mechanism for cardiotoxicity of doxorubicin (DOX) involves apoptosis in cardiomyocytes. Here we investigated the molecular basis for the differences in DOX-induced toxicity in adult rat cardiomyocytes (ARCM), neonatal rat cardiomyocytes (NRCM), and rat embryonic H9c2 cardiomyoblasts. Activation of caspase-9 and -3 was considerably lower in DOX-treated ARCM as compared to NRCM and H9c2 cardiomyoblasts. Addition of cytochrome c caused the activation of caspase-9 and -3 in permeabilized NRCM and H9c2 cardiomyoblasts but not in permeabilized ARCM. Expression of proapoptotic proteins, apoptotic protease activating factor-1 (Apaf1) and procaspase-9 was significantly lower, and abundance of antiapoptotic X-linked inhibitor of apoptosis protein (XIAP) was higher in ARCM, as compared to immature cardiac cells. Despite the abundance of XIAP in ARCM, its role in the inhibition of apoptosome function was dismissed, as second mitochondria-derived activator of caspases. (Smac)-N7 peptide, had no effect on caspase activation in response to cytochrome c in these cells. Adenoviral expression of Apaf1 exacerbated the activation of caspase-9 and -3 in DOX-treated NRCM, but did not increase their activities in DOX-treated ARCM. This finding points to a major difference in the apoptotic signaling between immature and adult cardiomyocytes. Mitochondrial apoptotic pathway is limited in ARCM treated with DOX.
Nuclear Factor of Activated T cells (NFAT), a family of transcription factors, has been implicated in many cellular processes, including some cancers. For the first time, the present study characterizes the role of NFAT3 in doxorubicin (DOX) mediated apoptosis, migration, and invasion in SNB19 and U87 glioma cells. This study demonstrates specific knockdown of NFAT3 results in a dramatic inhibition of the apoptotic effect, induced by DOX, and favors cell survival. Inhibition of NFAT3 activation by shNFAT3 (shNF3) significantly downregulated TNF-α induction, its receptor TNFR1, caspase 10, caspase 3 and PARP, abrogating DOX-mediated apoptosis in glioma cells. DOX treatment resulted in NFAT3 translocation to the nucleus. Similarly, shNF3 treatment in SNB19 and U87 cells reversed DOX-induced inhibition of cell migration and invasion as determined by wound healing and matrigel invasion assays. Taken together, these results indicate that NFAT3 is a prerequisite for the induction of DOX-mediated apoptosis in glioma cells.
We have assessed the capacity of human umbilical cord blood (hUCB)-derived stem cells to differentiate into cardiomyocytes and repair Angiotensin II induced insult in culture and in mouse hearts when injected. hUCB were able to differentiate into cardiomyocyte-like cells, when induced with 5-Azacytidine or co-cultured with rat neonatal cardiomyocytes (NRCM). When co-cultured, hUCB reversed the pathological effects induced by angiotensin II (Ang-II) in NRCM and in mice injected after Ang-II infusion. As assessed by increased heart weight to body mass ratio and Ang-IIinduced fibrosis, cardiac hypertrophy was also reduced after hUCB were injected. hUCB also reversed the pathological heart failure markers induced by Ang-II in mice. Further, we observed a shift from pathological hypertrophy towards physiological hypertrophy by hUCB in Ang-IIchallenged mice. Our findings support hUCB as a feasible model for experimentation in stem cell therapy and emphasize the relevance of the hUCB in reversing heart failure conditions.
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