We investigated mitochondrial toxicity of four lipophilic stains (cerivastatin, fluvastatin, atorvastatin, simvastatin) and one hydrophilic statin (pravastatin). In L6 cells (rat skeletal muscle cell line), the four lipophilic statins (100 micromol/l) induced death in 27-49% of the cells. Pravastatin was not toxic up to 1 mmol/l. Cerivastatin, fluvastatin and atorvastatin (100 micromol/l) decreased the mitochondrial membrane potential by 49-65%, whereas simvastatin and pravastatin were less toxic. In isolated rat skeletal muscle mitochondria, all statins, except pravastatin, decreased glutamate-driven state 3 respiration and respiratory control ratio. Beta-oxidation was decreased by 88-96% in the presence of 100 micromol/l of the lipophilic statins, but only at higher concentrations by pravastatin. Mitochondrial swelling, cytochrome c release and DNA fragmentation was induced in L6 cells by the four lipophilic statins, but not by pravastatin. Lipophilic statins impair the function of skeletal muscle mitochondria, whereas the hydrophilic pravastatin is significantly less toxic.
Cyclic nucleotide phosphodiesterase (PDE) isoforms can influence disease pathogenesis and be novel therapeutic targets. Because lower cAMP levels may contribute to the decreased apoptosis that occurs in chronic lymphocytic leukemia (CLL), we assessed the expression levels of PDE isoforms in peripheral blood mononuclear cells (PBMC) of healthy adults and patients with CLL. We found a unique PDE mRNA signature in CLL: higher levels than in normal PBMC of PDE7B (increased Ϸ23-fold) and lower levels of PDE3B, 4D, 5A, and 9A mRNA (each decreased Ϸ30-fold). Increased PDE7B mRNA in CLL correlates with a 10-fold-higher expression of PDE7B protein and results in an increased contribution of PDE7 to total PDE activity. Consistent with the higher level of PDE7B expression, inhibitors of PDE7 (BRL-50481, IR-202) and a dual PDE4/PDE7 inhibitor (IR-284) selectively increase apoptosis in CLL cells compared with normal PBMC or B cells. Apoptosis of CLL cells promoted by inhibitors of PDE7 and PDE4/7 is attenuated by PKA inhibition, occurs via a mitochondrial-dependent process, and is associated with increased cAMP accumulation and down-regulation of the antiapoptotic protein survivin and of PDE7B. The increase in PDE7B expression and PDE7 inhibitor-promoted apoptosis implicates PDE7B as a drug target in CLL. Our findings identify a unique PDE signature in CLL and illustrate the utility of broad analyses of PDE isoform expression in human disease.apoptosis ͉ B cell ͉ cAMP ͉ survivin
Statins are widely used to prevent cardiovascular diseases. They are well-tolerated, with side-effects mainly seen in skeletal muscle. How these side-effects are caused is unknown. We compared isolated primary mouse skeletal muscle myocytes, C2C12 myotubes and liver HepG2 cells to detect differences that could uncover why statins are toxic in skeletal muscle but less so in the liver. 10μM simvastatin caused a decrease in mitochondrial respiration in the primary mouse myocytes and C2C12 myotubes, but had no effect in the HepG2 cells. Mitochondrial integrity is maintained by multiple signaling pathways. One of these pathways, Igf-1/Akt signaling, is also heavily implicated in causing statin-induced toxicity by upregulating atrogin-1. We found that phosphorylated Akt was reduced in C2C12 myotubes but not in HepG2 cells. HepG2 mitochondrial respiration became susceptible to simvastatin-treatment after Akt inhibition, and mitochondrial respiration was rescued in Igf-1-treated C2C12 myotubes. These results suggest that disruption of Igf-1/Akt signaling is a causative factor in simvastatin-induced mitochondrial dysfunction in C2C12 myotubes, whereas HepG2 cells are protected by maintaining Igf-1/Akt signaling. We conclude that phosphorylation of Akt is a key indicator of susceptibility to statin-induced toxicity. How statins can disrupt Igf-1/Akt signaling is unknown. Statins reduce geranylgeranylation of small GTPases, such as Rap1. Previous studies implicate Rap1 as a link between cAMP/Epac and Igf-1/Akt signaling. Transient transfection of constitutively active Rap1 into C2C12 myotubes led to a partial rescue of simvastatin-induced inhibition of mitochondrial respiration, providing a novel link between signaling and respiration.
The conversion of clopidogrel to its active metabolite, R-130964, is a two-step cytochrome P450 (CYP)-dependent process. The current investigations were performed to characterize in vitro the effects of different CYP inhibitors on the biotransformation and on the antiplatelet effect of clopidogrel. EXPERIMENTAL APPROACHClopidogrel biotransformation was studied using human liver microsomes (HLM) or specific CYPs and platelet aggregation using human platelets activated with ADP. KEY RESULTSExperiments using HLM or specific CYPs (3A4, 2C19) revealed that at clopidogrel concentrations >10 mM, CYP3A4 was primarily responsible for clopidogrel biotransformation. At a clopidogrel concentration of 40 mM, ketoconazole showed the strongest inhibitory effect on clopidogrel biotransformation and clopidogrel-associated inhibition of platelet aggregation with IC50 values of 0.03 Ϯ 0.07 mM and 0.55 Ϯ 0.06 mM respectively. Clarithromycin, another CYP3A4 inhibitor, impaired clopidogrel biotransformation and antiplatelet activity almost as effectively as ketoconazole. The CYP3A4 substrates atorvastatin and simvastatin both inhibited clopidogrel biotransformation and antiplatelet activity, less potently than ketoconazole. In contrast, pravastatin showed no inhibitory effect. As clopidogrel itself inhibited CYP2C19 at concentrations >10 mM, the CYP2C19 inhibitor lansozprazole affected clopidogrel biotransformation only at clopidogrel concentrations Յ10 mM. The carboxylate metabolite of clopidogrel was not a CYP substrate and did not affect platelet aggregation. CONCLUSIONS AND IMPLICATIONSAt clopidogrel concentrations >10 mM, CYP3A4 is mainly responsible for clopidogrel biotransformation, whereas CYP2C19 contributes only at clopidogrel concentrations Յ10 mM. CYP2C19 inhibition by clopidogrel at concentrations >10 mM may explain the conflicting results between in vitro and in vivo investigations regarding drug interactions with clopidogrel.
Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B‐cells. We have shown that CLL is associated with increased mRNA expression of phosphodiesterase 7B (PDE7B), which catalyzes the hydrolysis of cAMP, and that PDE7 inhibitors promote apoptosis of CLL cells in a cAMP/protein kinase A‐dependent manner. In addition, we find that the expression of Exchange protein directly activated by cAMP (Epac), a downstream mediator of cAMP, is higher in CLL cells than in normal‐PBMC and B‐cells; its activation protects CLL cells from apoptosis. We investigated if the increase in PDE7B mRNA in CLL correlates with that of Epac‐1 and if targeted simultaneously would increase cAMP‐promoted apoptosis of CLL cells. We find that patients with CLL show a significant correlation between their fold‐increases in PDE7B and Epac‐1 mRNA (P<0.001, r=0.5395, n = 45). Inhibiting Epac‐1 or Epac‐1 signaling via siRNA (48 hr) or Rap‐1 inhibition (GGT1‐298, 5 μM, 48 hr), respectively, induced apoptosis in CLL cells and enhanced the pro‐apoptotic effect of PDE4/7 inhibition (IR‐284, 48 hr, 10nM). These data suggest that studies to define the regulation of PDE7B and Epac‐1 may provide insights into the pathophysiology of CLL and that decreasing the expression and function of Epac‐1 may be beneficial for treatment of CLL by increasing the pro‐apoptotic effects of PDE7 inhibitors. Supported by the Lymphoma and Leukemia Society and NIH.
Chronic lymphocytic leukemia (CLL), the major form of adult leukemia, is characterized by accumulation of B‐cells. We have found drugs that raise cAMP can kill malignant lymphocytes and (compared to normal peripheral blood mononuclear cells (PBMC) and normal B‐cells) CLL cells overexpress PDE7B, which selectively catalyzes the hydrolysis of cAMP. Furthermore, we found that CLL cells are more sensitive to PDE7 and PDE4/7 inhibitor‐induced apoptosis compared to normal B‐cells. Using CLL cells we sought to investigate the mechanism of PDE7 and PDE4/7‐induced apoptosis. Inhibition of PDE7 (30 μ]M BRL‐50481, 10 μM IR‐202) and dual inhibition of PDE4 and PDE7 (100 nM IR‐284) increased basal and forskolin‐stimulated cAMP accumulation in CLL cells, suggesting that their pro‐apoptotic effect occurs via a cAMP‐dependent mechanism. BRL‐50481, IR‐202 and IR‐284 all induced mitochondrial depolarization, assessed using a MitoProbe JC‐1 assay kit, (13 ± 4 %; 17 ± 6 %; 39 ± 5 % depolarization, respectively vs. vehicle) and cytochrome c release from the mitochondria into the cytosol, visualized by immunofluoresence. We conclude that PDE inhibitor‐induced apoptosis of CLL cells occurs via a cAMP‐mitochondrial‐dependent mechanism and that PDE7 inhibition may be a novel therapeutic approach in CLL. Supported by the Lymphoma and Leukemia Society, NIH and Swiss Cancer League.
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