[In vitro study over statins effects on cellular growth curves and its reversibility with mevalonate]. / Estudio in vitro sobre los efectos de las estatinas en las curvas de crecimiento celular y su reversión con mevalonato.

HMG-CoA-Reductase inhibitors, also known as statins, are currently the most powerful cholesterol-lowering drugs available on the market. Clinical trials and experimental evidence suggest that statins have heavy anti-atherosclerotic effects. These are in part consequence of lipid lowering but also result from pleiotropic actions of the drugs. These so-called pleiotropic properties affect various aspects of cell function, inflammation, coagulation, and vasomotor activity. These effects are mediated either indirectly through LDL-c reduction or via a direct effect on cellular functions. Although many of the pleiotropic properties of statins may be a class effect, some may be unique to certain agents and account for differences in their pharmacological activity. So, although statins typically have similar effects on LDL-c levels, differences in chemical structure and pharmacokinetic profile can lead to variations in pleiotropic effects. In this paper we analize the in vitro effects of different statins over different cell lines from cells implicated in atherosclerotic process: endothelial cells, fibroblasts, and vascular muscular cells. In relation with our results we can proof that the effects of different dosis of different statins provides singular effects over growth curves of different cellular lines, a despite of a class-dependent effects. So, pleiotropic effects and its reversibility with mevalonate are different according with the molecule and the dosis.

Simvastatin-induced compartmentalisation of doxorubicin sharpens up nuclear topoisomerase II inhibition in human rhabdomyosarcoma cells.

Tumours, which are initially sensitive to cytotoxic agents, often develop resistance to a broad spectrum of structurally unrelated drugs. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been shown to inhibit ATP-binding cassette (ABC) transporters but have also impact on glycosylation of such proteins. Doxorubicin is a substrate for ABC transporters like P-glycoprotein (ABCB1) which is present in human RD rhabdomyosarcoma cells. It was therefore the aim of this study to identify the compartmentalisation and action of doxorubicin in simvastatin-treated RD cells. Due to autofluorescence of doxorubicin, intracellular distribution was monitored by confocal microscopy. The biological effects were traced on the level of colony formation, caspase activation and DNA injury. Here we show that simvastatin treatment leads to ABCB1 inhibition and down-regulation of the transporter. Consequently, these cells accumulate significant amounts of doxorubicin, predominantly in the nucleus and lysosomes. While clearance of the anthracycline into lysosomes is not altered by simvastatin treatment, it significantly enhanced nuclear accumulation in a HMG-CoA reductase-independent manner. Thus, in such treated cells, topoisomerase II activity is significantly inhibited, which is further corroborated by augmented double-strand DNA breaks. Moreover, colony formation was synergistically inhibited by the combination of simvastatin and doxorubicin. Given the fact that ABCB1 expression correlates with an adverse prognosis in many tumours, adjuvant chemotherapy including statins might represent a novel therapeutic concept to overcome ABCB1-mediated multidrug resistance by direct inhibition and down-regulation.

Mitochondria-targeted nitroxides exacerbate fluvastatin-mediated cytostatic and cytotoxic effects in breast cancer cells.

Mito-CP11, a mitochondria-targeted nitroxide formed by conjugating a triphenylphosphonium cation to a five-membered nitroxide, carboxy-proxyl (CP), has been used as a superoxide dismutase (SOD) mimetic. In this study, we investigated the antiproliferative and cytotoxic properties of submicromolar levels of Mito-CP11 alone and in combination with fluvastatin, a well known cholesterol lowering drug, in breast cancer cells. Mito-CP11, but not CP or CP plus the cationic ligand, methyl triphenylphosphonium (Me-TPP+), inhibited MCF-7 breast cancer cell proliferation. Mito-CP11 had only minimal effect on MCF-10A, non-tumorigenic mammary epithelial cells. Mito-CP11, however, significantly enhanced fluvastatin-mediated cytotoxicity in MCF-7 cells. Mito-CP11 alone and in combination with fluvastatin inhibited nuclear factor kappa-B activity mainly in MCF-7 cells. We conclude that mitochondria-targeted nitroxide antioxidant molecules (such as Mito-CP11) that are non-toxic to non-tumorigenic cells could enhance the cytostatic and cytotoxic effects of statins in breast cancer cells. This strategy of combining mitochondria-targeted non-toxic molecules with cytotoxic chemotherapeutic drugs may be successfully used to enhance the efficacy of antitumor therapies in breast cancer treatment.

Simvastatin-induced endothelial cell detachment and microparticle release are prenylation dependent.

Statins reduce cardiovascular disease risk and affect endothelial function by cholesterol-dependent and independent mechanisms. Recently, circulating (detached) endothelial cells and endothelial microparticles (EMP) have been associated with endothelial functioning in vitro and in vivo. We investigated whether simvastatin affects endothelial detachment and release of EMP. Human umbilical vein endothelial cells (HUVECs) were incubated with clinically relevant concentrations of simvastatin (1.0 and 5.0 microM), with or without mevalonic acid (100 microM) or geranylgeranylpyrophosphate (GGPP; 20 microM) for 24 hours, and analyzed by flowcytometry and Western blot. Simvastatin at 1.0 and 5.0 microM increased cell detachment from 12.5+/-4.1% to 26.0+/-7.6% (p=0.013) and 28.9 +/- 2.2% (p=0.002) as well as EMP release (p=0.098 and p=0.041, respectively). The majority of detached cells was apoptotic, although the fraction of detached cells that showed signs of apoptosis (>70%) was unaffected by simvastatin. Detached cells and EMP contained caspase 3 and caspase 8, but not caspase 9. Restoring either cholesterol biosynthesis and prenylation (mevalonate) or prenylation alone (GGPP) reversed all simvastatin-induced effects on cell detachment and EMP release. Adherent cells showed no signs of simvastatin-induced apoptosis. Simvastatin promotes detachment and EMP release by inhibiting prenylation, presumably via a caspase 8-dependent mechanism. We hypothesize that by facilitating detachment and EMP release, statins improve the overall condition of the remaining vascular endothelium.

Pravastatin improves renal ischemia-reperfusion injury by inhibiting the mevalonate pathway.

Statins are known to lessen the severity of renal ischemia-reperfusion injury. The present study was undertaken to define the mechanism of renoprotective actions of statins using a mouse kidney injury model. Treatment of mice with pravastatin, a widely used statin, improved renal function after renal ischemia-reperfusion without lowering the plasma cholesterol level. Administration of pravastatin with mevalonate, a product of HMG-CoA reductase, eliminated renal protection suggesting an effect of pravastatin on mevalonate or its metabolism. In hypercholestrolemic apolipoprotein E knockout mice with reduced HMG-CoA reductase activity; the degree of injury was less severe than in control mice, however, there was no protective action of pravastatin on renal injury in the knockout mice. Treatment with a farnesyltransferase inhibitor (L-744832) mimicked pravastatin's protective effect but co-administration with the statin provided no additional protection. Both pravastatin and L-744832 inhibited the injury-induced increase in plasma IL-6 concentration to a similar extent. Our results suggest the protective effect of pravastatin on renal ischemia-reperfusion injury is mediated by inhibition of the mevalonate-isoprenoid pathway independent of its lipid lowering action.

Mevalonate sensitizes the nociceptive transmission in the mouse spinal cord.

Isoprenylation is crucial for the biological activation of small molecular G proteins. Activation of Rho/Rho kinase (ROCK) signaling has been reported to be involved in the initiation and maintenance of hyperalgesia caused by nerve injury and inflammation. The present study was then undertaken to examine whether the protein isoprenylation could affect thermal nociceptive threshold in the mouse spinal cord. Intrathecal administration of mevalonate (0.05-5.0 micromol) dose-dependently decreased the paw-withdrawal latencies for the thermal stimulation, indicating that mevalonate induces thermal hyperalgesia. Intrathecal pretreatment with a geranylgeranyl transferase I inhibitor GGTI-2133 (0.001-1.0 nmol) or a ROCK inhibitor Y27632 (0.001-1.0 nmol) completely blocked the mevalonate-induced thermal hyperalgesia. On the other hand, mevalonate-induced thermal hyperalgesia was only slightly attenuated by a farnesyl transferase inhibitor FTI-277 (0.01-1.0 nmol). Intrathecal injection of mevalonate increased the amount of geranylgeranylated RhoA in the spinal cord, which was completely blocked by intrathecal pretreatment with GGTI-2133. Intrathecal injection of mevalonate also produced RhoA translocation from cytosol to plasma membrane. This mevalonate-induced RhoA translocation was also blocked by intrathecal pretreatment with GGTI-2133, indicating that the RhoA translocation is triggered by RhoA geranylgeranylation. Moreover, inhibition of mevalonate synthesis by HMG-CoA reductase inhibition with simvastatin attenuated the second phase, but not the first phase, of nociceptive response to formalin. Our present results suggest that mevalonate sensitizes the spinal nociceptive transmission, which is mediated by the activation of ROCK following the RhoA geranylgeranylation.

Simvastatin inactivates beta1-integrin and extracellular signal-related kinase signaling and inhibits cell proliferation in head and neck squamous cell carcinoma cells.

The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, also called statins, are commonly used as lipid-lowering drugs that inhibit cholesterol biosynthesis. An anticancer effect, as a pleiotropic function of certain statins, has been hypothesized. In the present study, we investigated the effect of simvastatin, one of the natural statins, on cell proliferation, cell cycle, invasive activity, and molecular expressions associated with cell-extracellular matrix adhesion, signal transduction, and DNA synthesis in Tu167 and JMAR cells from head and neck squamous cell carcinoma. The addition of simvastatin resulted in a dose-dependent inhibition of cell growth and migration into the extracellular matrix. Considerable morphological changes occurred after treatment with simvastatin, demonstrating loss of cell adhesion and disruption of actin filaments in cytoplasm. The inhibitory effect of simvastatin on cell proliferation seemed to be associated with cell cycle arrest and increased expression of p21, p27, and activated caspase-3. The expression of beta1-integrin, a counter adhesion for the extracellular matrix, phosphorylated FAK, and phosphorylated ERK was decreased by treatment with simvastatin. The proapoptotic effect of simvastatin was inhibited by treatment with mevalonate. cDNA microarray assay demonstrated that molecular changes resulting from treatment with simvastatin included the up-regulation of cell cycle regulators and apoptosis-inducing factors and the down-regulation of integrin-associated molecules and cell proliferation markers. Of down-regulated genes induced by simvastatin treatment, a significant depletion of thymidylate synthase was confirmed using western blot analysis. These results imply that simvastatin has the potential to be effective for the prevention of the growth and metastasis of cancer cells.

Rho/ROCK pathway as a target of tumor therapy.

This study emphasizes the importance of Rho/ROCK pathway in lovastatin-induced apoptosis as replenishment with exogenous isoprenoid, geranylgeranylpyrophosphate (GGPP), resulted in inhibition of apoptosis in cultured tumor cells. Treatment of C6 glioma cells with Toxin B and exoenzyme C3 resulted in cell death suggesting the role of geranylgeranylated protein(s) in the survival of glioma cells. Relative apoptotic death observed in cells transfected with dominant negative constructs of RhoA, Rac, and cdc42 imply Rho A as playing the major role in cell survival. Furthermore, the inhibition of Rho A kinase (ROCK), a direct downstream effector of Rho A, by Y-27632 or dominant negative of ROCK, induced apoptosis in glioma cells. These findings indicate that RhoA/ROCK pathway is involved negatively in the regulation of glioma cell death pathway. Moreover, in vivo studies of lovastatin treatment in animals implanted with C6 glioma cell tumors also resulted in smaller tumor size and induced apoptosis in the tumor tissue. The implantation of stably transfected C6 glioma cells with expression vector of C3 exoenzyme, dominant negative of RhoA and ROCK, resulted in significant smaller tumor mass, further establishing the importance of geranylgeranylated proteins, specifically RhoA and its downstream effecter ROCK, in cell survival and tumor genesis.

Simvastatin inhibits human saphenous vein neointima formation via inhibition of smooth muscle cell proliferation and migration.

OBJECTIVE: Migration and proliferation of vascular smooth muscle cells (SMCs) contributes to intimal hyperplasia in saphenous vein (SV) bypass grafts, which leads to patency-threatening stenosis. Evidence for the involvement of basement membrane-degrading matrix metalloproteinases (MMPs) and growth factors in mediating SMC migration and proliferation has been presented in a number of in vitro and in vivo models. 3-Hydroxy-3 methylglutaryl CoA reductase inhibitors (statins) are widely used in patients with atherosclerosis and are claimed to have additional effects beyond cholesterol reduction. We therefore examined the effects of simvastatin, a commonly prescribed statin, on the proliferation and migration of cultured human SV SMC and on neointima formation and MMP activity in human SV organ cultures. To clarify its mode of action, we studied in parallel the effects of a specific MMP inhibitor, marimastat. STUDY DESIGN: Human SV specimens were obtained from patients who underwent coronary artery bypass grafting, and were cultured for 14 days in the presence of three concentrations of simvastatin and subsequently processed for measurement of MMP activity and neointimal thickness measurements. Cultured SV SMCs were used to construct growth curves in the presence of 10% fetal calf serum or 10% fetal calf serum supplemented with simvastatin or marimastat. Migration through a Matrigel basement-membrane matrix (invasion) was quantified with modified Boyden chambers. RESULTS: Simvastatin dose dependently reduced neointima formation (P =.004) in association with reduced MMP-9 activity (P =.03). SMC proliferation and invasion also were inhibited with simvastatin (P <.007 and P <.009, respectively). Marimastat dose dependently inhibited SMC invasion (P <.001) but importantly had no effect on SMC proliferation (P >.36). CONCLUSION: For effective control of neointimal development in vivo, a pharmacologic strategy should inhibit both SMC migration and proliferation. The ancillary properties of 3-Hydroxy-3 methylglutaryl CoA reductase inhibitors typified by simvastatin may be important in this regard.

Key regulatory oxysterols in liver: analysis as delta4-3-ketone derivatives by HPLC and response to physiological perturbations.

A number of oxysterols have been implicated in metabolic regulation. Key among these are (24S),25-epoxycholesterol and (24S)-hydroxycholesterol, high affinity ligands for the nuclear transcription factor liver X receptor alpha; 27-hydroxycholesterol, a bile acid synthetic intermediate; and 25-hydroxycholesterol, which has been used to study regulation of lipid metabolism by the sterol regulatory element-binding protein family of transcription factors. Investigation of the physiological importance of these compounds in vivo has been hampered by lack of analytical methods to reproducibly and accurately determine their concentrations in tissues. This article describes a method designed to determine quantitatively the amounts of these important side-chain oxysterols by derivatization to the Delta4-3-ketones followed by high performance liquid chromatography. The method was validated with known standards and then was used to determine the concentrations of these oxysterols in rodent liver under various physiological conditions. All four oxysterols were present in the picogram per milligram protein range and have distinct subcellular distributions and responses to physiological perturbations in vivo.

Intracellular trafficking of the free cholesterol derived from LDL cholesteryl ester is defective in vivo in Niemann-Pick C disease: insights on normal metabolism of HDL and LDL gained from the NP-C mutation.

Niemann-Pick C disease (NP-C) is a rare inborn error of metabolism with hepatic involvement and neurological sequelae that usually manifest in childhood. Although in vitro studies have shown that the lysosomal distribution of LDL-derived cholesterol is defective in cultured cells of NP-C subjects, no unusual characteristics mark the plasma lipoprotein profiles. We set out to determine whether anomalies exist in vivo in the cellular distribution of newly synthesized, HDL-derived or LDL-derived cholesterol under physiologic conditions in NP-C subjects. Three affected and three normal male subjects were administered [14C]mevalonate as a tracer of newly synthesized cholesterol and [3H]cholesteryl linoleate in either HDL or LDL to trace the distribution of lipoprotein-derived free cholesterol. The rate of appearance of free [14C]- and free [3H]cholesterol in the plasma membrane was detected indirectly by monitoring their appearance in plasma and bile. The plasma disappearance of [3H]cholesteryl linoleate was slightly faster in NP-C subjects regardless of its lipoprotein origin. Appearance of free [14C] cholesterol ill the plasma (and in bile) was essentially identical in normal and affected individuals as was the initial appearance of free [3H]cholesterol derived from HDL, observed before extensive exchange occurred of the [3H]cholesteryl linoleate among lipoproteins. In contrast, the rate of appearance of LDL-derived free [3H]cholesterol in the plasma membrane of NP-C subjects, as detected in plasma and bile, was retarded to a similar extent that LDL cholesterol metabolism was defective in cultured fibroblasts of these affected subjects. These findings show that intracellular distribution of both newly synthesized and HDL-derived cholesterol are essentially unperturbed by the NP-C mutation, and therefore occur by lysosomal-independent paths. In contrast, in NP-C there is defective trafficking of LDL-derived cholesterol to the plasma membrane in vivo as well as in vitro. The in vivo assay of intracellular cholesterol distribution developed herein should prove useful to quickly evaluate therapeutic interventions for NP-C.

Inhibition of isoprenoid biosynthesis and arterial smooth-muscle cell proliferation.

Recently, we provided in vitro and in vivo evidence that several vastatins with different potencies decrease arterial smooth-muscle cell (SMC) proliferation independently of their hypocholesterolemic properties. In this study, the in vivo dose-dependent antiproliferative activity of fluvastatin on neointimal formation induced by the insertion of a collar around one carotid artery was investigated in normocholesterolemic rabbits (five animals per treatment group). Intraperitoneal fluvastatin treatment progressively inhibited intimal to medial tissue ratios (I/M) by 5, 48, and 64% versus controls at doses of 3, 5, or 10 mg/kg/day, respectively. Local arterial delivery by an Alzet pump of mevalonate (8 mg/kg/day) at the site of collar placement fully prevented a fluvastatin (5 mg/kg/day) inhibitory effect on both I/M and SMC proliferation, as assessed by direct incorporation of bromodeoxyuridine (BrdU) into replicating DNA. The results suggest that vastatins exert a direct antiproliferative effect on intimal myocytes beyond their effects on plasma lipids, probably through local inhibition of isoprenoid biosynthesis.

Isoprenoid regulation of cell growth: identification of mevalonate-labelled compounds inducing DNA synthesis in human breast cancer cells depleted of serum and mevalonate.

Growth arrest induced by serum depletion and/or treatment with mevinolin (an inhibitor of mevalonate synthesis) in the human breast cancer cell line Hs578T was overcome by exogenous mevalonate, indicating that some product or metabolite of mevalonate may be involved in the mediation of serum-regulated growth of these cells. In the search for such compounds we first tested a variety of known end products of mevalonate with respect to their ability to counteract the inhibition of DNA synthesis caused by serum-free medium and mevinolin. Thereby high doses (10 micrograms/ml) of dolichol-20 were found to cause a partial counteraction. After straight-phase HPLC purification of endogenous lipids, isolated from 3H- or 14C-mevalonate-labelled Hs578T cultures, we found that non-sterol lipids co-eluting with dolichols efficiently induced DNA synthesis. After further purification with reverse-phase HPLC it was confirmed that virtually all of this effect was achieved by compounds(s) (seen as a single UV and radioactive peak) co-eluting with dolichol-20. Nanogram doses, at most, of this (these) compound(s) elicited a substantial stimulation of DNA synthesis. The lipid(s) also counteracted the inhibition by mevinolin of N-linked glycosylation, indicating that it (they) also interfere(s) with this processing. Since treatment with tunicamycin (an inhibitor of N-linked glycosylation) abolished this growth-stimulative effect, N-linked glycosylation seems to be a necessary event in the processes leading to lipid-induced initiation of DNA synthesis.

Central role of high density lipoprotein in plasma free cholesterol metabolism.

This study was designed to provide direct information on the in vivo metabolism in man of free (unesterified) cholesterol in the major lipoprotein classes. Five human subjects were administered one or two (simultaneous) of the following; [2-(14)C] mevalonic acid, high density lipoprotein (HDL)-free [(14)C] cholesterol, low density lipoprotein (LDL)-free [(14)C] cholesterol, and very low density lipoprotein (VLDL)-free [(3)H]cholesterol. Blood was then obtained at frequent intervals for at least 9 h, and the alpha(HDL) and beta(LDL + VLDL) lipoproteins were quickly separated by heparin-manganese precipitation to prevent ex vivo exchange of free cholesterol. After the administration of [(14)C]mevalonic acid the specific activity (disintegrations per minute/micromole) of free cholesterol in the alpha- and beta-lipoproteins increased for 3 h. During this period the alpha-free cholesterol specific activity was higher than the beta specific activity. After administration of VLDL and LDL labeled with free cholesterol, the alpha-free cholesterol specific activity reached a peak value within 20 min, at which time it was considerably lower than the beta-free cholesterol specific activity. When HDL labeled with free cholesterol was administered, a precursor product relationship was observed between the alpha-free cholesterol (precursor) and beta-free cholesterol (product) specific activities.A multicompartmental model was developed that contained the simplest structure necessary to fit all of the data obtained. The kinetic analysis revealed the presence of extensive exchange (20-85 mumol/min) of free cholesterol between HDL and a tissue pool(s) enriched with newly synthesized free cholesterol. It was found that virtually all (>95%) of the free cholesterol in the beta-lipoproteins (LDL+VLDL) cycles directly through HDL. The free cholesterol in LDL appears to behave in the same fashion as the free cholesterol in VLDL. The results show that there are marked differences in the kinetic behavior of the free cholesterol fractions of alpha- and beta-lipoproteins. There is extensive recycling of free cholesterol between HDL and tissue pools, and between HDL and the beta-lipoproteins; this recycling has been quantitated. The findings support the view that in vivo, the free cholesterol in HDL plays a central role in exchange reactions and in the vascular-tissue cholesterol transport system.

On the mechanism for the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, of cholesterol 7alpha-hydroxylase and of acyl-coenzyme A:cholesterol acyltransferase by free cholesterol.

The administration of mevalonic acid to rats by intravenous injection resulted in a dose- and time-dependent increase in the activity of cholesterol 7alpha-hydroxylase in the liver microsomal fraction, a decrease in the microsomal activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and no significant change in the activity of acyl-coenzyme A:cholesterol acyltransferase or in the concentration of free and of esterified cholesterol in the liver microsomal fraction. However, the increased hepatic cholesterogenesis that follows the injection of mevalonic acid resulted in an increase of the size of the intracellular pool of cholesterol that is in the environment of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and acts as substrate for cholesterol 7alpha-hydroxylase. The administration of mevalonic acid to rats by stomach tube resulted in an increase in the activity of cholesterol 7alpha-hydroxylase and of acyl-coenzyme A:cholesterol acyltransferase and in the concentration of cholesterol esters in the liver microsomal fraction, while there was a considerable decrease in the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Metabolism of mevalonate in rats and man not leading to sterols.

C-5 of mevalonate appears as C0-2 in the breath of rats and men almost immediately after administration either by injection or by mouth. Adult rats exhaled up to 6.5% of a dose of RS-[5-14C]mevalonate (13% of the utilizable R-enantiomer) in the breath in 100 min. The 14-C02 was not derived either from the matabolism of cholesterol biosynthesized from [5-14C]mevalonate or from the metabolism of the unnatural S-enantiomer of mevalonate. The amount of 14-C02 expired in the breath was the same whether the [5-14C]mevalonate was given intravenously or in a drink of water to man. One normocholesterolemic man dissipated 12%, a mildly non-familial hypercholesterolemic man dissipated 10%, and a familial hypercholesterolemic man dissipated 7% of a dose of [5-14C]mevalonate in 24 hours (calculated as a per cent of the R-enantiomer). The observations support the hypothesis of the existence of a metabolic shunt of intermediates of sterol biosynthesis, derived from mevalonate, not leading to sterols.