Sumio Matzno

Statin-induced apoptosis linked with membrane farnesylated Ras small G protein depletion, rather than geranylated Rho protein.

Rhabdomyolysis is a severe adverse effect of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase inhibitors (statins). This myopathy is strongly enhanced by the combination with statins and fibrates, another hypolipidaemic agent. We have evaluated the initial step of statin‐induced apoptosis by the detection of membrane flip‐flop using flow cytometric analysis. L6 rat myoblasts were treated with various statins (atorvastatin (3 μm), cerivastatin (3 μm), fluvastatin (3 μm), pravastatin (3 mm), or simvastatin (3 μm)) for 2, 4 or 6 h followed by reacting with FITC‐conjugated annexin V for the detection of initial apoptosis signal (flip‐flop). Various statin‐treated myoblasts were significantly stained with FITC‐annexin V at 6 h, whereas they were not detected at 2 h. Moreover, immunoblot analysis indicated that when the cells were treated with cerivastatin (3 μm), membrane‐associated Ras protein was activated and detached until 6 h, resulting in cell death through the consequent activation of caspase‐8. On the other hand, since cytosolic Ras activation did not activate, there is still an unknown mechanism in statin‐related Ras depletion. In conclusion, statin‐induced apoptosis in muscular tissue was directly initiated by the farnesyl‐anchored Ras protein depletion from cell membrane with subsequent apoptosis.

Evaluation of the synergistic adverse effects of concomitant therapy with statins and fibrates on rhabdomyolysis

Rhabdomyolysis is a severe adverse effect of hypolipidaemic agents such as statins and fibrates. We evaluated this muscular cytotoxicity with an in‐vitro culture system. Cellular apoptosis was determined using phase‐contrast and fluorescein microscopic observation with Hoechst 33342 staining. L6 rat myoblasts were treated with various statins and bezafibrate under various conditions. With statins only, skeletal cytotoxicity was ranked as cerivastatin > fluvastatin > simvastatin > atorvastatin > pravastatin in order of decreasing potency. Combined application of fibrates enhanced ator‐vastatin‐induced myopathy, which causes little apoptosis alone. These results suggest that statins and fibrates synergistically aggravate rhabdomyolysis.

Synergistic action of statins and nitrogen-containing bisphosphonates in the development of rhabdomyolysis in L6 rat skeletal myoblasts.

Objectives Nitrogen‐containing bisphosphonates, which are widely used to treat osteoporosis, act as inhibitors of farnesyl pyrophosphate synthase, one of the key enzymes of the mevalonate pathway, and thus may have the potential to enhance the effect of statins (inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase). In this study, we evaluated the synergistic effect of two nitrogen‐containing bisphosphonates, alendronate and risedronate, in statin‐induced apoptosis in rat skeletal L6 myoblasts.

Effects of quinine on the intracellular calcium level and membrane potential of PC 12 cultures

The mechanism for the perception of bitterness appears to be quite complicated, even for quinine, which is a model bitter substance, and thus has yet to be completely elucidated. To investigate the possibility of being able to predict the bitterness of quinine solutions, we examined the effects of quinine on intracellular calcium ion concentration ([Ca2+]i) and membrane potentials in PC 12 cultures. [Ca2+]i and membrane potentials were analysed by fluorescence confocal microscopic imaging using the Ca2+‐sensitive probe Calcium Green 1/AM and the membrane potential‐sensitive probe bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol (DiBAC4(3)). Quinine elicited an increase in the membrane potential along with a concentration‐dependent increase in [Ca2+]i. These increases were inhibited by extracellular Ca2+‐free conditions, thapsigargin, which is a Ca2+‐pump inhibitor, and U73122, which is a phospholipase C inhibitor. The quinine‐induced increase in [Ca2+]i levels was inhibited by nifedipine, an L‐type Ca2+‐channel blocker, ω‐conotoxin, a T‐type Ca2+‐channel blocker, and BMI‐40, which is a bitterness‐masking substance. These results suggest that responses in PC 12 cultures may be used as a simple model of bitterness perception.

FLOW CYTOMETRIC EVALUATION OF SYNERGISTIC PRO‐APOPTOTIC EFFECTS OF STATINS AND CLOFIBRATES IN IM‐9 HUMAN LYMPHOBLASTS

1 In the present study, we evaluated fibrate‐mediated potentiation of statin‐induced apoptosis in IM‐9 human lymphoblasts. 2 The pro‐apoptotic effects of statin and fibrate were measured by flow cytometry with biotin–annexin V, followed by addition of avidin–fluorescein isothiocyanate and propidium iodide. Apoptosis was confirmed using karyopyknotic staining, as well as detection of DNA fragmentation and caspase 3 activation. 3 Incubation of IM‐9 cells with both 0.1 µmol/L cerivastatin and 200 µmol/L clofibrate had a synergistic effect compared with 0.1 µmol/L cerivastatin alone or 200 µmol/L clofibrate alone. The magnitude of apoptosis induced by various combinations of statins and clofibrate were as follows: cerivastatin (0.1 µmol/L) + clofibrate (200 µmol/L) > atorvastatin (0.1 µmol/L) + clofibrate (200 µmol/L) > pravastatin (100 µmol/L) + clofibrate (200 µmol/L). Other fibrates (bezafibrate and clinofibrate) did not show any synergistic effect. Furthermore, karyopyknotic staining, caspase 3 activation and DNA fragmentation demonstrated synergistic pro‐apoptotic effects of statin and fibrate. 4 The results of the present study suggest that simultaneous treatment with statins and clofibrate could provide improved therapeutic efficacy in leukaemia patients.