Pifithrin-alpha protects against doxorubicin-induced apoptosis and acute cardiotoxicity in mice.

The present experiments were designed to evaluate the effects of pifithrin-alpha (PFT-alpha), which is a p53 inhibitor, on doxorubicin (DOX)-induced apoptosis and cardiac injury. Administration of DOX (22.5 mg/kg ip) in mice upregulated the mRNA levels of Bax and MDM2, whereas PFT-alpha attenuated those levels when administered at a total dose of 4.4 mg/kg at 30 min before and 3 h after DOX challenge. DOX treatment led to an upregulation of p53 protein levels, which was preceded by elevated levels of phosphorylated p53 at Ser15. PFT-alpha had no effect on the level of p53 or its phosphorylated form. The protein levels of Bax and MDM2 were elevated by DOX and attenuated by PFT-alpha. DOX gave rise to increased apoptosis-positive nuclei in cardiac cells, elevated serum creatine phosphokinase, ultrastructural alterations, and cardiac dysfunction. PFT-alpha offered protection against all of the aforementioned changes. Finally, PFT-alpha did not interfere with the antitumor potency of DOX. This study demonstrates that PFT-alpha effectively inhibits DOX-induced cardiomyocyte apoptosis, which suggests that PFT-alpha has the potential to protect cancer patients against DOX-induced cardiac injury.

Dexamethasone induces caspase activation in murine osteoblastic MC3T3-E1 cells.

Glucocorticoids are widely used as anti-inflammatory and chemotherapeutic agents. However, prolonged use of glucocorticoids leads to osteoporosis. This study was designed to examine the mechanism of dexamethasone (DEX)-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 10(-7) M DEX for 6 h. DEX exerted a variety of effects on apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that DEX upregulated mRNA levels of caspases-1, -3, -6, -8, -11, -12, and bcl-XL. Western blot analysis showed enhanced processing of these caspases, with the appearance of their activated enzymes 8 h after DEX treatment. In addition, DEX also induced the activation of caspase-9. DEX elevated the levels of cleaved poly(ADP-ribose) polymerase and lamin A, a caspase-3 and a caspase-6 substrate, respectively. Expression of bcl-XL protein level was upregulated by DEX. Cytochrome c release was detected in the cytosol of DEX-treated cells. Furthermore, caspase-3 enzyme activity was elevated by 2-fold after DEX treatment for 7 h. Finally, early apoptotic cells were detected in cells treated with DEX for 3 h. Our results demonstrate that DEX-induced apoptosis involves gene activation of a number of caspases.

TGF-beta1 inhibits multiple caspases induced by TNF-alpha in murine osteoblastic MC3T3-E1 cells.

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine that induces apoptosis in a number of cell systems, including osteoblasts. Transforming growth factor beta1 (TGF-beta1) is an abundant growth factor that is known to stimulate bone formation. This study was designed to examine the role of TGF-beta1 on TNF-alpha-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 20 ng/ml of TNF-alpha, 10 ng/ml of TGF-beta1, or combination, for 6 h. TNF-alpha exerted a variety of effects on the apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that TNF-alpha upregulated the mRNA levels of caspase-1, -7, -11, -12, and FAS. Western blot analysis showed enhanced processing of caspase-1, -7, -11, and -12, with the appearance of their activated enzymes 24 h after TNF-alpha treatment. In addition, caspase-3-like activity was significantly activated following TNF-alpha treatment. Levels of cleaved poly(ADP-ribose) polymerase and FAS protein were also elevated by TNF-alpha. Finally, Hoechst staining, terminal deoxynucleotidyl-transferase nick-end labeling (TUNEL) assay, and oligonucleosome ELISA all indicated that TNF-alpha induced apoptosis. In contrast, the addition of TGF-beta1 attenuated all of the aforementioned effects of TNF-alpha. Our results demonstrate that TGF-beta1 can decrease TNF-alpha-induced apoptosis in murine osteoblasts at least in part by attenuating TNF-alpha-induced caspase gene expression.