Increased RhoA and RhoB protein accumulation in cultured human trabecular meshwork cells by lovastatin.

PURPOSE: This study aimed to determine the effect of lovastatin on Rho G-protein expression and activation in human trabecular meshwork (TM) cells. METHODS: Confluent cultures of low-passage (primary) or transformed (GTM3) human TM cells were incubated overnight with vehicle (0.01% ethanol) or activated lovastatin (10 microM). Changes in Rho mRNA, protein content, and activation were quantified by qRT-PCR, immunoblotting, and ELISA, respectively. F-actin organization was determined using Alexa Fluor 488-conjugated phalloidin. RESULTS: Low-passage or transformed TM cells treated with lovastatin exhibited marked increases in RhoA and RhoB mRNA and protein content. Actinomycin D prevented lovastatin-dependent increases in RhoB, but not RhoA, protein accumulation. In contrast, cycloheximide prevented lovastatin from increasing both RhoA and RhoB. Supplementation with mevalonate or geranylgeranyl pyrophosphate prevented, whereas inhibition of geranylgeranyl transferase mimicked, the effects of lovastatin on RhoA and RhoB accumulation. The effect of lovastatin was dose dependent, with newly synthesized protein accumulating in the cytosol. The amount of functionally active (GTP-bound) RhoA in cell lysates was significantly reduced by lovastatin. Lovastatin altered the morphology of TM cells by disrupting F-actin organization. CONCLUSIONS: Lovastatin enhances the accumulation of RhoA and RhoB in human TM cells, in part, by limiting geranylgeranyl isoprenylation of these G-proteins. We propose that post-translational geranylgeranylation serves as a regulator of both RhoA and RhoB protein expression and processing in human TM cells. Increased accumulation of unprenylated forms of RhoA and RhoB may disrupt Rho-dependent regulation of TM cell cytoskeletal organization.

Critical role for FoxO3a-dependent regulation of p21CIP1/WAF1 in response to statin signaling in cardiac myocytes.

Statins are widely used clinical drugs that exert beneficial growth-suppressive effects in patients with cardiac hypertrophy. We investigated the role of the cell cycle inhibitor p21(CIP1/WAF1) (p21) in statin-dependent inhibition of hypertrophic growth in postmitotic cardiomyocytes. We demonstrate that lovastatin fails to inhibit cardiac hypertrophy to angiotensin II in p21(-/-) mice and that reconstitution of p21 function by TAT.p21 protein transduction can rescue statin action in these otherwise normally developed animals. Lovastatin specifically recruits the forkhead box FoxO3a transcription factor to the p21 promoter, mediating transcriptional transactivation of the p21 gene as analyzed in isolated primary cardiomyocytes. Lovastatin also stimulates protein kinase B/Akt kinase activity, and Akt-dependent phosphorylation forces p21 in the cytoplasm, where it inhibits Rho-kinases contributing to the suppression of cardiomyocyte hypertrophy. Loss of p21 or FoxO3a by RNA interference causes a general inhibition of lovastatin signal transduction. These results suggest that p21 functions as FoxO3a downstream target to mediate an statin-derived anti-hypertrophic response. Taken together, our genetic and biochemical data delineate an essential function of p21 for statin-dependent inhibition of cardiac myocyte hypertrophy.

4,8-Dimethyldecanal, the aggregation pheromone of Tribolium castaneum, is biosynthesized through the fatty acid pathway.

4,8-Dimethyldecanal (4,8-DMD) is the aggregation pheromone produced by male red flour beetles (RFB), Tribolium castaneum. To elucidate the biosynthetic origin of 4,8-DMD, the following studies were performed: (1) effects of juvenile hormone (JH) III, and pathway inhibitors mevastatin, an inhibitor of the mevalonate pathway, and 2-octynoic acid, an inhibitor of the fatty acid pathway, were tested to determine whether 4,8-DMD is derived from the fatty acid pathway or the mevalonate pathway; (2) incorporation of 13C-labeled acetate, propionate, and mevalonolactone into 4,8-DMD was measured to directly determine the biosynthetic origin of 4,8-DMD; and (3) incorporation of deuterium-labeled precursors, including 2-methylbutanoate (C5D), 4-methylhexanoate (C7D), 2,6-dimethyloctanoate (C10D), and 4,8-dimethyldecanoate (C12D) was tested to determine whether 4,8-DMD is biosynthesized in the sequence Ac-Pr-Ac-Pr-Ac (Ac; acetate, Pr; propionate). JH III was topically applied to males at various doses. Inhibitors and isotopically labeled substrates were administered orally by feeding the beetles flour treated with the substrates of interest, after which volatiles were collected from both sexes of RFBs. The amount of 4,8-DMD produced was significantly reduced with increasing doses of JH III. Also, 2-octynoic acid inhibited the production of 4,8-DMD, but mevastatin did not. Exposure of RFBs to [1-(13)C]acetate and [1-(13)C]propionate, but not [2-(13)C]mevalonolactone, resulted in incorporation of the labeled compounds into 4,8-DMD. RFBs fed flour treated with deuterium-labeled C5D, C10D, and C12D, but not C7D, incorporated these compounds into 4,8-DMD. The findings that the production of 4,8-DMD was inhibited by 2-octynoic acid but unaffected by mevastatin, combined with the high incorporation of [1-(13)C]acetate and [1-(13)C]propionate into 4,8-DMD and the incorporation of deuterated precursors, unambiguously demonstrated that 4,8-DMD is of fatty acid rather than terpene biosynthetic origin, and that the biosynthesis of 4,8-DMD proceeds in the sequence Ac-Pr-Ac-Pr-Ac.

Mevastatin induces apoptosis in HL60 cells dependently on decrease in phosphorylated ERK.

Mevastatin which is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol synthesis, suppress cell proliferation and induce apoptosis. However, the molecular mechanism of apoptosis induction is not well understood. So, in the present study, we attempted to clarify the mechanism by which mevastatin induces apoptosis in HL60 cells. It was found that mevastatin induced apoptosis. At that time, we observed an increase in caspase-3 activity and morphological fragmentation of the nuclei. The apoptosis induced by mevastatin was not inhibited by the addition of farnesyl pyrophosphate (FPP), squalene, ubiquinone, and isopentenyladenine, but was inhibited by the addition of geranylgeranyl pyrophosphate (GGPP). When we examined the survival signals at the time of apoptotic induction, we also observed that the administration of mevastatin had caused a remarkable decrease in the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). However, other survival signals, such as nuclear factor kappa B (NF-kappaB), protein kinase B (Akt), and p38 mitogen-activated protein kinase (p38), exhibited no change. In addition, no quantitative change was observed in Bcl-2, which was an anti-apoptosis protein. It was also observed that apoptosis was induced when U0126, an MEK inhibitor, was added to the cells to inhibit ERK. These results suggested that mevastatin induced apoptosis when it inhibited GGPP biosynthesis and consequently decreased the level of phosphorylated ERK, which was a survival signal; moreover, at that time, there was no influence on NF-kappaB, Akt, p38, and Bcl-2. The results of this study also suggested that mevastatin could be used as an anticancer agent.

Statins induce suppressor of cytokine signaling-3 in macrophages.

Our previous study has shown that lipophilic 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors of statins can inhibit interferon-gamma-induced inducible nitric oxide synthase gene expression in RAW264.7 macrophages. In this study, we showed that lovastatin and fluvastatin are able to upregulate the mRNA expression of the suppressor of cytokine signaling-3 (SOCS-3) gene. This effect is specific for SOCS-3 and could be blocked by mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, while it was not affected by inhibitors of protein kinase C and A, mitogen-activated protein/extracellular signal-regulated kinase kinase, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, Src, Raf and Rho kinase. SOCS-3 expression results in the inhibition of interferon-gamma-, interleukin-6- and macrophage colony-stimulating factor-elicited signal transducer and activator of transcription phosphorylation, suggesting a novel anti-inflammatory mechanism of statins to down-modulate the functions of interferon-gamma-activated macrophages.

Microarray and biochemical analysis of lovastatin-induced apoptosis of squamous cell carcinomas.

We recently identified 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of the mevalonate pathway, as a potential therapeutic target of the head and neck squamous cell carcinomas (HNSCC) and cervical carcinomas (CC). The products of this complex biochemical pathway, including de novo cholesterol, are vital for a variety of key cellular functions affecting membrane integrity, cell signaling, protein synthesis, and cell cycle progression. Lovastatin, a specific inhibitor of HMG-CoA reductase, induces a pronounced apoptotic response in a specific subset of tumor types, including HNSCC and CC. The mediators of this response are not well established. Identification of differentially expressed genes represents a feasible approach to delineate these mediators as lovastatin has the potential to modulate transcription indirectly by perturbing levels of sterols and other mevalonate metabolites. Expression analysis following treatment of the HNSCC cell lines SCC9 or SCC25 with 10 microM lovastatin for 1 day showed that less than 2% (9 cDNAs) of the 588 cDNAs on this microarray were affected in both cell lines. These included diazepam-binding inhibitor/acyl-CoA-binding protein, the activated transcription factor 4 and rhoA. Because the biosynthesis of mevalonate leads to its incorporation into more than a dozen classes of end products, their role in lovastatin-induced apoptosis was also evaluated. Addition of the metabolites of all the major branches of the mevalonate pathway indicated that only the nonsterol moiety, geranylgeranyl pyrophosphate (GGPP), significantly inhibited the apoptotic effects of lovastatin in HNSCC and CC cells. Because rhoA requires GGPP for its function, this links the microarray and biochemical data and identifies rhoA as a potential mediator of the anticancer properties of lovastatin. Our data suggest that the depletion of nonsterol mevalonate metabolites, particularly GGPP, can be potential mediators of lovastatin-induced apoptosis of HNSCC and CC cells.

The role of cytochrome P450-mediated drug-drug interactions in determining the safety of statins.

The objectives of this review are to discuss the role of cytochrome P450 (CYP450) isoforms in drug metabolism, to explain differences in metabolism among the HMG-CoA reductase inhibitors (HMGs, statins), to review drug-drug and drug-food interaction studies dealing with the HMGs, to present case reports dealing with HMG-related myopathy, to discuss major clinical implications of these case reports and to express an opinion of use of HMGs in clinical practice.

Lovastatin-induced proliferation inhibition and apoptosis in C6 glial cells.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the rate-limiting enzyme in cholesterol biosynthesis. HMG-CoA reductase converts HMG-CoA to mevalonate, which is then converted into cholesterol or various isoprenoids through multiple enzymatic steps. In this study, we examined the cytotoxic effects of lovastatin, an HMG-CoA reductase inhibitor, in C6 glial cells. Lovastatin at concentrations higher than 10 microM suppressed cell proliferation and induced cell death, which were prevented completely by mevalonate (300 microM). The data from lactate dehydrogenase assay and fluorescence microscopic assay using Hoechst 33342 and propidium iodide showed that mevalonate at a concentration of 100 microM could prevent lovastatin-induced cell death, whereas it could not prevent lovastatin-induced inhibition of cell proliferation. These data suggest that the lovastatin-induced interruption of cell cycle transition was not sufficient to induce cell death in C6 glial cells. In the presence of lovastatin at concentrations higher than 10 microM, DNA laddering, the typical finding of apoptosis, was identified. Lovastatin-induced apoptosis was prevented by mevalonate (100 microM). Both cycloheximide (0.5 microgram/ml) and actinomycin D (0.1 microgram/ml) prevented lovastatin-induced DNA laddering. In this study, we demonstrated that the cytotoxic effects of lovastatin fall into two categories: suppression of cell growth and induction of apoptosis in C6 glial cells.

Lovastatin induces fibroblast apoptosis in vitro and in vivo. A possible therapy for fibroproliferative disorders.

Diseases associated with pathological fibroproliferation represent a major cause of morbidity and mortality. Despite the importance of this class of disorders, current therapy is of limited value, and no therapy is available to reduce the fibroblast population size within existing fibrotic lesions. In this regard, constitutive expression of growth-promoting genes can sensitize cells to undergo apoptosis. Studies in our laboratory have demonstrated that lovastatin potently induces apoptosis in fibroblasts constitutively expressing Myc, and that lung fibroblasts isolated from fibrotic lesions constitutively express growth-promoting genes. In this study, we sought to determine if nontransformed lung fibroblasts would manifest susceptibility to lovastatin-induced apoptosis similar to that observed in fibroblasts ectopically expressing Myc. Here we show that clinically achievable concentrations of lovastatin induce apoptosis in normal and fibrotic lung fibroblasts in vitro, as evidenced by acridine orange staining, terminal transferase nick end translation (TUNEL), and DNA laddering. Apoptosis of human lung fibroblasts was dose- and time-dependent, and blocked by exogenous mevalonic acid. Furthermore, apoptosis was associated with decreased levels of mature Ras, a molecule directly implicated in fibroblast rescue from apoptosis. The ability of lovastatin to induce fibroblast apoptosis in vivo was examined using a guinea pig wound chamber model. Lovastatin (5 microM, 8 d) reduced granulation tissue formation in the wound chambers by 64.7%, with associated ultrastructural evidence of fibroblast apoptosis. These findings support further study of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors as potential therapy for patients with fibroproliferative disorders.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase fibrinolytic activity in rat aortic endothelial cells. Role of geranylgeranylation and Rho proteins.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (HRIs) have been recently shown to prevent atherosclerosis progression. Clinical benefit results from combined actions on various components of the atherosclerotic lesion. This study was designed to identify the effects of HRI on one of these components, the endothelial fibrinolytic system. Aortas isolated from rats treated for 2 days with lovastatin (4 mg/kg body wt per day) showed a 3-fold increase in tissue plasminogen activator (tPA) activity. In a rat aortic endothelial cell line (SVARECs) and in human nontransformed endothelial cells (HUVECs), HRI induced an increase in tPA activity and antigen in a time- and concentration-dependent manner. In SVARECs, the maximal response was observed when cells were incubated for 48 hours with 50 micromol/L HRI. An increase of tPA mRNA was also in evidence. In contrast, HRI inhibited plasminogen activator inhibitor-1 activity and mRNA. The effects of HRI were reversed by mevalonate and geranylgeranyl pyrophosphate, but not by LDL cholesterol and farnesyl pyrophosphate, and were not induced by alpha-hydroxyfarnesyl phosphonic acid, an inhibitor of protein farnesyl transferase. C3 exoenzyme, an inhibitor of the geranylgeranylated-activated Rho protein, reproduced the effect of lovastatin on tPA and plasminogen activator inhibitor-1 activity and blocked its reversal by geranylgeranyl pyrophosphate. The effect of HRI was associated with a disruption of cellular actin filaments without modification of microtubules. A disrupter of actin filaments, cytochalasin D, induced the same effect as lovastatin on tPA, whereas a disrupter of microtubules, nocodazole, did not. In conclusion, HRI can modify the fibrinolytic potential of endothelial cells, likely via inhibition of geranylgeranylated Rho protein and disruption of the actin filaments. The resulting increase of fibrinolytic activity of endothelial cells may contribute to the beneficial effects of HRI in the progression of atherosclerosis.

Alpha1-adrenoreceptor stimulation causes vascular smooth muscle cell hypertrophy: a possible role for isoprenoid intermediates.

We investigated whether contraction-induced agonists such as alpha1-adrenoceptor agonists are important regulators of smooth muscle cell hypertrophy by examining the effects of one potent agonists, phenylephrine, on the hypertrophy. Under the experimental conditions used, we found that phenylephrine was potent in inducing alpha1-adrenoreceptor-dependent hypertrophy of vascular smooth muscle cells as defined by increased incorporation of [14C]leucine in a dose-dependent fashion. Further, we assessed the effect of lovastatin, an 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on hypertrophy of cultured vascular smooth muscle cells as defined by the increased incorporation of [14C]leucine caused by phenylephrine. Lovastatin (5-15 microM) caused a significant dose-dependent reduction in [14C]leucine incorporation which was completely prevented in the presence of exogenous mevalonate (100 microM). Exogenous low density lipoprotein (100 microg/ml) and cholesterol (15 microg/ml) did not prevent lovastatin inhibition of [14C]leucine incorporation. In contrast, the isoprenoid farnesol largely prevented inhibition of [14C]leucine incorporation by the lovastatin. We conclude that mevalonate metabolites are essential for phenylephrine-induced smooth muscle cell hypertrophy, possibly through the production of the isoprenoid farnesol.

Inhibition of proliferation of human smooth muscle cells by various HMG-CoA reductase inhibitors; comparison with other human cell types.

The effects of 6 HMG-CoA reductase inhibitors: pravastatin, lovastatin, simvastatin, atorvastatin, fluvastatin and cerivastatin were analyzed in cultured human smooth muscle cells, fibroblasts, endothelial cells and myoblasts. In vascular smooth muscle cells, pravastatin was a much weaker inhibitor of cholesterol synthesis than the 5 other drugs which displayed equally strong inhibitory potency. The anti-proliferative effects of these 6 drugs were analyzed by measuring cell number and mitochondrial dehydrogenase activity (MTT assay) after 3 days of incubation. IC25 values for inhibition of proliferation were very similar among the 4 cell types and were in the following order of magnitude: pravastatin << lovastatin = simvastatin = atorvastatin = fluvastatin << cerivastatin. Only in the case of pravastatin was proliferation inhibited at lower concentration in smooth muscle cells than in the other cell types. Proliferation was also assessed by measuring DNA synthesis in these cells. A 3 day-incubation with 1 microM of pravastatin had no effect on this parameter in all 4 cell types. However, 1 microM of simvastatin or lovastatin caused either an inhibition (in smooth muscle cells and endothelial cells) or stimulation (in fibroblasts) of this process. The effects of simvastatin on cell number, mitochondrial dehydrogenase activity and DNA synthesis were counteracted by simultaneous mevalonate addition. Simvastatin treatment was also associated with a change in the post-translational modification of the ras protein in smooth muscle cells, probably by inhibition of its farnesylation. Moreover, simvastatin treatment blocked the PDGF and bFGF-induced DNA synthesis in synchronized smooth muscle cells, whereas it does not affect the fetal calf serum-induced DNA synthesis in synchronized fibroblasts, suggesting that simvastatin blocks various steps of the cell cycle and that this effect depends on the cell type and the growth signalling pathway activated.

Biochemical changes and morphological alterations of liver and kidney in hamsters after administration of the HMG-coenzyme A reductase inhibitor, simvastatin: prevention and reversibility by mevalonate.

The present study analyses the effects of simvastatin, a specific inhibitor of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA reductase) in male Syrian hamsters fed a standard diet or a diet supplemented with 0.12% cholesterol and 20% coconut oil. In hamsters fed the standard diet, gastric administration of simvastatin (10 mg/kg/day) during 12 days was found to be lethal and to have hepatotoxic and nephrotoxic effects. This toxicity was exacerbated in hamsters fed a hyperlipidaemic diet and was preceded by a progressive anorexia and loss of body weight. Marked elevations in serum aspartate and alanine aminotransferase activities were associated with the organ lesions. All elevated biochemical changes and morphological alterations were prevented or reversed by coadministration of mevalonate, the product of the HMG-CoA reductase. It is suggested that the dramatic effect of simvastatin could result from depletion of a non-sterol metabolite of mevalonate in spite of a lack of protective effects of farnesol and geranylgeraniol in the following study. The toxicity of simvastatin could indeed result from the low basal activity of HMG-CoA reductase in hamster liver coupled with a prolonged inhibition of mevalonate synthesis.

Lovastatin enhances the photocytotoxicity of UVA radiation towards cultured N.C.T.C. 2544 human keratinocytes: prevention by cholesterol supplementation and by a cathepsin inhibitor.

The effect of the hydroxymethylglutaryl-CoA (HMG-CoA) inhibitor lovastatin on the UVA-induced photocytotoxicity has been investigated in cultured human N.C.T.C. 2544 keratinocytes. In the absence of irradiation, 5 x 10(-7) M lovastatin did not exhibit any significant cytotoxic effect towards this cell line. Although the drug cannot act as a photosensitizer, because it does not absorb in the UVA range, it markedly increased the UVA-induced cellular damage (about 70% reduction in cell viability at 5 x 10(-7) M). This effect was not accompanied by an increase in the lipid peroxidation product content of cells as compared with treatment with UVA alone. Medium supplementation with 0.01 mg/ml free cholesterol totally prevented the enhancement of UVA photocytotoxicity induced by lovastatin. A protective effect was also observed when cells were supplemented with an amount of low-density lipoprotein giving the same cholesterol concentration in the culture medium. Finally, E64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane], a lysosomal cathepsin inhibitor, also prevents the cell death induced by UVA in cells treated with lovastatin. These results suggest that HMG-CoA reductase inhibitors could increase the sensitivity of skin cells to UVA radiation, and that this phenomenon is related to lysosomal enzyme release.

Intracellular alkalinization suppresses lovastatin-induced apoptosis in HL-60 cells through the inactivation of a pH-dependent endonuclease.

Protein isoprenylation is a post-translational modification essential for the biological activity of G-proteins. Inhibition of protein isoprenylation by lovastatin (LOV) induces apoptosis in HL-60 cells, a process of active cell death characterized by the internucleosomal degradation of genomic DNA. In this article we show that LOV-induced apoptosis is associated with intracellular acidification and that activation of the Na+/H+ antiporter induces a raise in pHi which is sufficient to prevent or arrest DNA digestion. First, LOV induced a decrease in pHi which was dose-dependent and correlated with the extent of DNA degradation. Flow cytometry analysis revealed that this acidification was due to the appearance of a subpopulation of cells whose pHi was 0.9 pH units below control values. Cell sorting experiments demonstrated that DNA degradation had occurred only in those cells which had suffered intracellular acidification. LOV-induced apoptosis could be suppressed by mevalonate supplementation, inhibition of protein synthesis, and protein kinase C activation by phorbol myristate acetate. In all three cases, intracellular acidification was abolished. Inhibition of the Na+/H+ antiporter by 5-N-ethyl-N-isopropyl amiloride induced DNA degradation in HL-60 cells per se and suppressed the protective effect of phorbol myristate acetate. LOV-induced intracellular acidification was not due to a complete inhibition of the Na+/H+ antiporter. In fact, LOV-treated cells were able to respond to phorbol myristate acetate stimulation of the Na+/H+ antiporter with a marked increase in pHi. This effect was accompanied by a rapid arrest of DNA digestion. These observations illustrate the strong pH dependence of LOV-induced DNA degradation, thus providing a connection between the activation of the Na+/H+ antiporter and the suppression of apoptosis.

Inhibition of human vascular smooth muscle cell proliferation by lovastatin: the role of isoprenoid intermediates of cholesterol synthesis.

Restenosis remains the largest single obstacle to the long-term success of invasive vascular interventions. Lovastatin, an HMG-CoA reductase inhibitor, has been shown to reduce myointimal hyperplasia in animal models of restenosis and in one clinical coronary restenosis trial. We have assessed the effect of lovastatin on the growth of cultured human vascular smooth muscle cells derived from saphenous vein and vascular graft stenoses. Lovastatin (2 microM) inhibited proliferation over 14 days in saphenous vein (and graft stenoses) derived vascular smooth muscle cells by 42% and 32% respectively: this was not significantly different. Lovastatin (10 microM) reduced [methyl 3H]-thymidine uptake by 51% in saphenous vein-derived cells. These concentrations were significantly higher than those achieved in plasma during therapeutic dosage. Lovastatin-induced inhibition of vascular smooth muscle cell proliferation and [methyl 3H]-thymidine uptake was completely reversed by adding mevalonate (100 microM) but cholesterol (10-40 micrograms ml-1) had no effect. Isopentenyl adenine (25-50 microM) did not affect the inhibition of [methyl 3H]-thymidine uptake by lovastatin (10 microM), but farnesol (20 microM), another isoprenoid precursor of cholesterol synthesis, reversed the antiproliferative effect.

PKC activity in rat C6 glioma cells: changes associated with cell cycle and simvastatin treatment.

The parallel effects of simvastatin on cell cycle and PKC activity in rat C6 glioma cells were investigated. Simvastatin, 2.5 microM, for 24 h resulted in cell growth arrest in early G1 phase of the cell cycle and in a significant increase of total PKC activity (283 +/- 42 vs 470 +/- 61 pmoles/min/mg protein p = 0.002 for control cells and simvastatin-treated cells, respectively). The effect of simvastatin was fully prevented by mevalonate. A time dependent increase of PKC activity was observed in control exponentially free-growing C6 cells approaching confluency: a highly significant negative correlation (r = -0.91 p < 0.0001) between PKC activity and growth rate was calculated. PKC activity was high in cells arrested in G0 by serum starvation (0.4%). Following addition of complete medium (17.5% serum) the PKC activity progressively decreased and reached a minimum when cells traversed the G2/M phase, as determined by DNA analysis distribution. PKC activity dropped 30% in simvastatin-arrested early G1 cells; 44% in hydroxyurea-arrested cells at the G1/S boundary; and 73% in Colcemid mitosis-blocked cells. The results show that C6 glioma cell PKC activity is maximal in a G0 quiescent state and varies at different points of the cell cycle.

Simvastatin and intracellular pH regulation by the Na+/H+ antiport of SV40-virus-transformed human MRC5 fibroblasts.

1. Inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase by simvastatin leads to inhibition of both cell growth and Na+/H+ antiport activity. The effect of simvastatin on intracellular pH and Na+/H+ antiport activity was therefore studied on an adherent cell line, the SV40-virus-transformed MRC5 human fibroblast. 2. Simvastatin led to a dose-dependent decrease in intracellular pH, attributed to a reduction in Na+/H+ exchange, together with a rounding of cell shape. Mevalonate (1 mmol/l) prevented these effects of simvastatin, and when added after inhibition of the antiport by simvastatin, reversed these changes within 1-2h. 3. The phenomenon of mevalonate reversal of antiport inhibition by simvastatin was not sensitive to cycloheximide, indicating its post-translational nature. This was also consistent with the short period of incubation with mevalonate leading to reversal of antiport inhibition (1-2 h). These changes in intracellular pH regulation were not due to alterations in cell cholesterol content. 4. A variety of inhibitors of post-translational processes, such as N-linked glycosylation (tunicamycin), phosphorylation (staurosporine), isoprenylation (farnesol, limonene), and of pertussis-toxin-sensitive G-proteins or calmodulin (W7), had no effect on the reversal by mevalonate of simvastatin-induced changes in Na+/H+ antiport activity. 5. N-Ethylmaleimide (50 mumol/l for 5 min) prevented mevalonate reversing the effects of simvastatin, suggesting the importance of thiol groups in the phenomenon of reversal of the inhibition of Na+/H+ antiport activity by simvastatin. Furthermore, concurrent incubation of simvastatin-treated cells with dithiothreitol (1 mmol/l) and N-ethylmaleimide restored the ability of mevalonate to reverse the inhibitory effects of simvastatin on Na+H+ antiport activity.

Mevalonate controls cytoskeleton organization and cell morphology in thyroid epithelial cells.

Blockade of mevalonate synthesis by the 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitor mevinolin (lovastatin) causes FRTL-5 thyroid cells to undergo significant morphological changes; these include a transition from a flat, polygonal to a round shape, the development of cytoplasmic arborizations, and the loss of contact between neighboring cells. Immunofluorescence studies of cytoskeletal structures show that, at early times after administering the drug, and before the round phenotype develops, stress fibers disassemble while the peripheral actin filaments, which are adjacent to the cytoplasmic face of the plasma membrane, appear largely unaffected. Subsequently, when this cortical actin network becomes fragmented, cells start to round up and become separated from neighbors. Microtubules become disconnected from the plasma membrane and retract toward the cell center, although they do not appear depolymerized; indeed, at this stage, cytoplasmic elongations contain mostly intact microtubules. After exposure to mevinolin FRTL-5 cells also lose vinculin-related substrate contacts. Treatment of cells with either cycloheximide or colchicine abolishes morphological changes induced by mevinolin, suggesting that ongoing protein synthesis and microtubule integrity are prerequisites for the drug to be effective. Both cytoskeletal and morphological perturbations can be reversed by mevalonate, but not by cholesterol or the non-sterol derivatives of mevalonate such as dolichol, ubiquinone, and isopentenyladenine, individually or in combination. It is suggested that mevalonate deficiency may impair formation of isoprenylated proteins important for cytoskeletal organization and stability.

Reversal of ras-induced inhibition of gap-junctional intercellular communication, transformation, and tumorigenesis by lovastatin.

The plasma-membrane association and transforming activity of the ras oncoprotein p21 are dependent upon posttranslational farnesylation. Farnesyl synthesis and p21 ras farnesylation are inhibited by hydroxymethylglutaryl-CoA reductase inhibitors such as lovastatin. In this study, we examined whether lovastatin could reverse the transformed phenotype of a v-Ha-ras-transformed rat liver epithelial cell line (WB-ras cells) and if changes were associated with the enhancement of gap-junctional intercellular communication (GJIC). WB-ras cells grow in soft agar, have reduced GJIC, and are highly tumorigenic. Membrane association of p21 ras in these cells was inhibited after in vitro treatment with lovastatin (0.1-0.5 microM) for 48 h. Concomitantly, the cells displayed a more normal morphology, decreased growth in soft agar, and enhanced GJIC. These changes were prevented by cotreatment with mevalonic acid. The morphology and GJIC of rat liver epithelial cells transformed with other oncogenes (src, neu, and raf/myc) were not affected by lovastatin. Intrahepatic WB-ras tumors were induced in male rats by intraportal-vein injection of WB-ras cells. The size and DNA labeling index of these tumors were decreased approximately 75% by administration of lovastatin (5 mg/kg orally twice daily for 2 wk). These results suggest that lovastatin reversed the transformed phenotype of WB-ras cells by inhibiting p21 ras plasma membrane association. Furthermore, the concomitant enhancement of GJIC in lovastatin-treated cells suggests a role for reduced GJIC in the expression of the transformed phenotype.