Lovastatin induces mitotic abnormalities in various cell lines.

We examined the effects of lovastatin, a common anti-atherosclerotic drug and a blocker of the cell cycle, on the process of mitosis. It is known that lovastatin induces an arrest or a retardation of the cell cycle in many cell types not only at the G(1)phase, but also at the G(2)/M transition. After 24-48 h incubation of epithelial PtK(2), T24, HeLa cells and fibroblastic L929 cells in the presence of 1. 0-60.0 microm lovastatin, diverse mitotic perturbations have been observed. The most noteworthy phenomena recorded were prometaphase retardation and chromosome lagging during metaphase and anaphase. After the recovery in lovastatin-free media, the cells continued mitosis without any disturbances. Mevalonic acid prevented the effects of lovastatin. We conclude that the effects were specific for lovastatin-induced inhibition of mevalonic acid synthesis. Immunofluorescence studies with anticentromeric antibodies suggested that one of the possible causes of the lovastatin-induced mitotic disorder could be an interference with the development and function of the centromeres.     

Statin-inhibited endothelial permeability could be associated with its effect on PECAM-1 in endothelial cells

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are known to inhibit leukocyte recruitment to endothelium but the mechanism is less understood. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an endothelial junction protein involved in leukocyte diapedesis. We hypothesize that in endothelial cells, statins may well recruit PECAM-1 to exert their inhibitory effect on leukocyte trans-endothelial migration (TEM). In lovastatin-treated resting human umbilical vein endothelial cells (HUVECs), increased levels of mRNA and protein of PECAM-1 as well as its bio-synthesis (all approximately 2-fold) were observed by real-time PCR, Western blotting and 35S-labeled methionine incorporation assay, respectively. Moreover, in lovastatin treated resting cells as well as TNF-alpha activated endothelial cells, unanimously decreased Triton X-100 insoluble and soluble PECAM-1 ratio was observed. Such changes were accompanied by decreased TEM of U-937 cells (a promonocyte cell line). All lovastatin's effects were abrogated by mevalonic acid. In resting HUVECs, geranylgeranyl pyrophosphate (GGPP), but not farnesyl pyrophosphate (FPP) (both are isoprenoid intermediates in the cholesterol biosynthesis pathway) compromised the effect of lovastatin on PECAM-1 expression, whereas C3 toxin, an inhibitor of small G proteins, exerted statin-like effect. CONCLUSION: Statin-reduced endothelial permeability could be attributed to altered intracellular distribution of PECAM-1 in endothelial cells. We speculate that lovastatin regulates PECAM-1 expression in HUVECs through the mevalonate-GGPP pathway by inhibiting of Rho small GTPase.     

Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb

Background

Among the epigenetic alterations occurring in cancer, DNA hypermethylation and histone hypoacetylation are the focus of intense research because their pharmacological inhibition has shown to produce antineoplastic activity in a variety of experimental models. The objective of this study was to evaluate the combined antineoplastic effect of the DNA methylation inhibitor hydralazine and the histone deacetylase inhibitor valproic acid in a panel of cancer cell lines.

Results

Hydralazine showed no growth inhibitory effect on cervical, colon, breast, sarcoma, glioma, and head & neck cancer cell lines when used alone. On the contrary, valproic acid showed a strong growth inhibitory effect that is potentiated by hydralazine in some cell lines. Individually, hydralazine and valproic acid displayed distinctive effects upon global gene over-expression but the number of genes over-expressed increased when cells were treated with the combination. Treatment of HeLa cells with hydralazine and valproic acid lead to an increase in the cytotoxicity of gemcitabine, cisplatin and adriamycin. A higher antitumor effect of adriamycin was observed in mice xenografted with human fibrosarcoma cells when the animals were co-treated with hydralazine and valproic acid.

Conclusion

Hydralazine and valproic acid, two widely used drugs for cardiovascular and neurological conditions respectively have promising antineoplastic effects when used concurrently and may increase the antitumor efficacy of current cytotoxic agents.     

Lovastatin inhibits G1/S transition of normal human B-lymphocytes independent of apoptosis.

Lovastatin is a potent inhibitor of protein prenylation, and it has been reported to have pleiotropic cellular effects. In the present study we have elucidated the effects of lovastatin on cell cycle progression and apoptosis of normal human B-lymphocytes. When added to B-lymphocytes stimulated with anti-immunoglobulin (anti-mu) and SAC, lovastatin (20 microM) inhibited the cells in the late G1 phase of the cell cycle. Thus, no early activation parameters such as Ca(2+) flux or MYC induction were affected by lovastatin, whereas progression of cells into the second cell cycle as well as DNA synthesis was markedly reduced. We therefore examined the effects of lovastatin on components of the cell cycle machinery responsible for regulating the G1/S transition. We demonstrated that pRB phosphorylation, cdk2 activity needed for this phosphorylation, and the levels of cyclin A, D, and E were inhibited after 24 h of lovastatin treatment, while the levels of p27(Kip1) were elevated. There was no effect on p21(Cip1), cyclin D2, cdk4, and cdk6. These data are consistent with the cells being inhibited by lovastatin between 24 and 32 h into G1. Lovastatin added to stimulated B-cells in late G1 still inhibited the DNA synthesis by 60%, but at this point only minor effects were noted on the cell cycle machinery. We therefore looked for induced apoptosis as an explanation for reduced S-phase entry of the cells. However, despite the ability to enhance the apoptosis of unstimulated B-cells from 48 to 61% as judged by the TUNEL method, lovastatin only marginally affected apoptosis when administered to stimulated B-cells. Thus, it appears that accelerated apoptosis cannot account for the effect of lovastatin on cell cycle progression.     

Effects of anticonvulsants on cholinergic and GABAergic properties in the neuronal cell clone NG108-15

The effects of anticonvulsant drugs on growth, cholinergic, and GABAergic properties were examined in the neuronal cell clone NG108-15. Cells were exposed for 4 days to valproic acid, phenobarbital, phenytoin, or carbamazepine in concentrations equivalent to therapeutic free levels in human serum. Experiments were also performed with varying concentrations of a recently proposed antiepileptic, gamma-vinyl GABA. Of these five anticonvulsants, cell growth (total protein and cell counts) was decreased with valproic acid and phenytoin but only valproic acid and gamma-vinyl GABA altered neurotransmitter markers. Therapeutic concentrations of valproic acid increased choline acetyltransferase activity to 142% of control but had no effect on either the activity of glutamate decarboxylase or the level of GABA. The effects of a higher (toxic) concentration of valproic acid (200 g/ml) were similar to those induced by the differentiating agent dibutyryl cyclic AMP: both decreased cell growth, enhanced the activity of choline acetyltransferase and reduced the activity of glutamate decarboxylase. Gamma-vinyl GABA had no effect on cholinergic markers but, at 1300 g/ml, increased GABA levels to 135% of control despite the reduction of glutamate decarboxylase to 68% of control. In the NG108-15 cell clone, anticonvulsants have varying effects on cell growth, differentiation, and neurotransmitter systems. Our findings do not support the proposal that the mechanism of action for valproic acid, phenobarbital, phenytoin, and carbamazepine is via alteration of GABA levels.     

Lack of correlation between 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and lovastatin resistance in nerve growth factor treated PC-12 cells

Summary 1. The relationships among the mevalonic acid (MVA) forming enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (CoA) reductase, cell growth and differentiation, and the cytotoxic effects of the reductase inhibitor lovastatin were studied in PC-12 cells, exposed to growth factors.2. When added individually, nerve growth factor (NGF), basic fibroblast growth factor, and epidermal growth factor induce an increase in HMG-CoA reductase activity in cells grown in serum-containing medium. In the presence of serum, the effect of NGF on HMG-CoA reductase is persistent.3. Short-term serum starvation and long-term NGF treatment, in combination, have an additive effect, resulting in a high reductase activity.4. Unlike serum and MVA, which downregulate levels of HMG-CoA reductase by accelerating its degradation, NGF upregulates reductase by slowing the rate of its degradation. This mechanism, however, appears to operate only in the presence of serum, as after prolonged growth with NGF in serum-free medium, cells have a low reductase activity.5. PC-12 cells grown in the absence of NGF are highly sensitive to lovastatin (25 µM) and more than 70% of the cells die after 48 hr. NGF confers lovastatin resistance on cells grown in the presence or in the absence of serum (only 30–40% cell death after 48 hr with lovastatin).6. NGF-induced resistance on lovastatin develops with time and is apparent only in the well-differentiated PC-12 cells whether or not the cells express a high reductase activity.7. Thus, levels of HMG-CoA reductase activity and lovastatin resistance in PC-12 cells are not directly correlated, though clearly inversed lovastatin cytotoxicity and elevated reductase activities are expressed during the period of cell proliferation.8. These data suggest that fully differentiated neuronal cells may not be affected by prolonged high doses of lovastatin.     

Lovastatin does not accentuate but is rather additive to palmitate-induced apoptosis in cardiomyocytes

Fatty acids such as palmitate have been observed to induce apoptosis in cardiomyocytes but the mechanism of this cytotoxicity is unresolved. The present study sought to determine whether an aspect of fatty acid metabolism is responsible for palmitate-induced apoptosis in cardiomyocytes. As palmitate metabolism increases acetyl CoA production via increased beta oxidation within the mitochondria, we hypothesized that increased acetyl CoA entering the cholesterol biosynthesis pathway might produce intermediates or end products that would be toxic to the cell. To test this hypothesis, cardiomyocytes from embryonic chick cardiomyocytes were treated with the 3-hydroxy-3-methylgutaryl CoA (HMG-CoA) reductase inhibitor lovastatin that inhibits the cholesterol biosynthesis pathway downstream of the acetyl CoA trimerization into HMG-CoA. Lovastatin did not inhibit palmitate-induced apoptosis. Rather, lovastatin induced significant apoptosis itself and when combined with palmitate, the level of apoptosis was equal to the sum of palmitate alone and lovastatin alone. This observation suggests that palmitate and lovastatin are inducing apoptosis by two independent mechanisms. A role for mitochondrial metabolism via carnitine palmitoyl transferase (CPT) in palmitate-induced apoptosis was suggested since capric acid, a fatty acid that is metabolized within the mitochondria but does not utilize CPT-1, did not induce apoptosis. Palmitate-induced apoptosis was further related to the metabolism of saturated fatty acids as the unsaturated fatty acid oleic acid did not induce apoptosis. These data suggest that a unique feature about palmitate metabolism independent of its role in cholesterol biosynthesis is responsible for palmitate-induced apoptosis and the effects of palmitate are additive to those of lovastatin to induce cardiac apoptosis.     

Role of cholesterol synthesis in regulation of bile acid synthesis and biliary cholesterol secretion in humans

We used lovastatin, a specific inhibitor of HMG-CoA reductase, to study the role of cholesterol synthesis in regulation of both bile acid synthesis, measured by release of 14CO2 from [26-14C]cholesterol, and biliary cholesterol secretion, measured by standard marked perfusion techniques, in humans. Six volunteers were studied in each of four periods: a) control; b) 6-10 hours after a single 40 mg oral dose of lovastatin to study acute effects; c) after 5-6 weeks of lovastatin 40 mg orally twice a day to study steady-state effects; and d) 24 h after cessation of chronic lovastatin. Mean bile acid synthesis fell to 69% of control (P less than 0.01) after single-dose lovastatin and remained at 83% of control after 5-6 weeks on lovastatin (P less than 0.05). After withdrawal of lovastatin, mean bile acid synthesis was 88% of control (NS). Mean biliary cholesterol secretion did not change after single-dose lovastatin (103% of control), but fell to 81% of control during chronic lovastatin treatment (P less than 0.05). After withdrawal of lovastatin, mean cholesterol secretion remained at 80% of control (P less than 0.05). These data suggest that in humans cholesterol synthesis is an immediate regulator of bile acid synthesis. Cholesterol synthesis also regulates biliary cholesterol secretion, but the effect is not immediate and therefore may be indirect.     

Protective effects of valproic acid against airway hyperresponsiveness and airway remodeling in a mouse model of allergic airways disease

Airway remodeling and airway hyperresponsiveness are major aspects of asthma pathology that are not targeted optimally by existing anti-inflammatory drugs. Histone deacetylase inhibitors have a wide range of effects that may potentially abrogate aspects of remodeling. One such histone deacetylase inhibitor is valproic acid (2-propylvaleric acid). Valproic acid is used clinically as an anti-epileptic drug and is a potent inhibitor of class I histone deacetylases but also inhibits class II histone deacetylases. We used valproic acid as a molecular model of histone deacetylase inhibition in vivo in chronic allergic airways disease mice with airway remodeling and airway hyperresponsiveness. Wild-type Balb/c mice with allergic airways disease were treated with valproic acid or vehicle control. Airway inflammation was assessed by bronchoalveolar lavage fluid cell counts and examination of lung tissue sections. Remodeling was assessed by morphometric analysis of histochemically stained slides and lung function was assessed by invasive plethysmography measurement of airway resistance. Valproic acid treatment did not affect inflammation parameters; however, valproic acid treatment resulted in reduced epithelial thickness as compared to vehicle treated mice

(p < 0.01), reduced subepithelial collagen deposition (p < 0.05) and attenuated airway hyperresponsiveness (p < 0.05 and p < 0.01 for the two highest doses of methacholine, respectively). These findings show that treatment with valproic acid can reduce structural airway remodeling changes and hyperresponsiveness, providing further evidence for the potential use of histone deacetylase inhibitors for the treatment of asthma.     

Effect of valproic acid on radiation-induced DNA damage in euchromatic and heterochromatic compartments

The distinction between heterochromatin and euchromatin in the double-strand break (DSB) damage pathway is of interest, recent reports indicate that chromatin is not created equally nor is it acquiescent to DSBs. Using the classical histone deacetylase inhibitor, Trichostatin A, we have previously demonstrated that chromatin represents a heterogeneous substrate with respect to histone tail modification by histone deacetylase inhibitors and consequent responses to DNA damage and repair. Here, we extended the initial findings by investigating the radiation sensitizing properties of the widely used antiepileptic, valproic acid. Clonogenic survival assays confirm that valproic acid is an efficient sensitizer of radiation-induced cell death. The radiosensitizing effect is correlated with valproic acid-mediated histone hyperacetylation, chromatin decondensation and enhanced formation of radiation-induced γH2AX preferentially on euchromatic alleles. Heterochromatin was much more resistant to histone tail modification, changes in chromatin architecture and DNA damage. These findings are consolidated by studies with the structurally related analogue, valpromide, which does not inhibit histone deacetylase enzymes. At a relatively low concentration (1 mM) valpromide did not cause chromatin modifications and radiation sensitivity, providing further evidence that the radiation sensitizing properties of valproic acid are at least in part, due to histone modification-dependent effects on euchromatin. When higher concentrations (5 mM) were used, both compounds resulted in significant radiation sensitivity, albeit, with differing efficacy (dose modifying factors of 1.5 and 1.2 for valproic acid and valpromide, respectively). The findings imply that histone-modification independent mechanisms also contribute to the radiation sensitizing properties of valproic acid. Overall, our findings are consistent with the emerging interest in the use histone deacetylase inhibitors in combination with radiotherapy for the treatment of cancer.     

Protective effects of valproic acid against airway hyperresponsiveness and airway remodeling in a mouse model of allergic airways disease

Airway remodeling and airway hyperresponsiveness are major aspects of asthma pathology that are not targeted optimally by existing anti-inflammatory drugs. Histone deacetylase inhibitors have a wide range of effects that may potentially abrogate aspects of remodeling. One such histone deacetylase inhibitor is valproic acid (2-propylvaleric acid). Valproic acid is used clinically as an anti-epileptic drug and is a potent inhibitor of class I histone deacetylases but also inhibits class II histone deacetylases. We used valproic acid as a molecular model of histone deacetylase inhibition in vivo in chronic allergic airways disease mice with airway remodeling and airway hyperresponsiveness. Wild-type Balb/c mice with allergic airways disease were treated with valproic acid or vehicle control. Airway inflammation was assessed by bronchoalveolar lavage fluid cell counts and examination of lung tissue sections. Remodeling was assessed by morphometric analysis of histochemically stained slides and lung function was assessed by invasive plethysmography measurement of airway resistance. Valproic acid treatment did not affect inflammation parameters; however, valproic acid treatment resulted in reduced epithelial thickness as compared to vehicle treated mice (p < 0.01), reduced subepithelial collagen deposition (p < 0.05) and attenuated airway hyperresponsiveness (p < 0.05 and p < 0.01 for the two highest doses of methacholine, respectively). These findings show that treatment with valproic acid can reduce structural airway remodeling changes and hyperresponsiveness, providing further evidence for the potential use of histone deacetylase inhibitors for the treatment of asthma.     

Pattern- and age-dependency of the antiepileptic effects induced by valproic acid in the rat hippocampus.

The effects induced by the antiepileptic drug valproic acid were studied in the CA3 subfield of in vitro hippocampal slices obtained from young (16- to 27-day-old) and adult (over 60-day-old) rats. Spontaneous epileptiform discharges were induced by the addition of the convulsant 4-aminopyridine to the medium. Valproic acid (0.5 mM) selectively blocked the ictal epileptiform discharges in slices obtained from young rats. Interictal epileptiform discharges disappeared during perfusion with higher doses of valproic acid (2 mM). This blockade of interictal epileptiform activity was not observed when valproic acid (0.5-5 mM) was tested in hippocampal slices from adult rats. Thus, in the hippocampus of young rats, 4-aminopyridine-induced ictal activity is more sensitive to valproic acid than are interictal discharges. Moreover, valproic acid is effective in controlling interictal discharges in the young, but not in the adult rat hippocampus.     

Inhibition of growth of cultured tobacco cells at low concentrations of lovastatin is reversed by cytokinin

De novo synthesis of mevalonic acid, which is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase, is the first committed step in the formation of isoprenoid compounds. Various studies have shown that mevalonic acid-derived compounds are required for growth of plant and animal cells, a conclusion supported by the observation that cells treated with lovastatin (a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase) cease growth. We show that Nicotiana tabacum BY-2 cells, which require exogenous auxin for growth in culture but do not require exogenous cytokinin, are growth inhibited by 1 μm lovastatin. However, these cells are capable of growing in the presence of 1 μm lovastatin if 8 μm zeatin is supplied in the medium. Furthermore, benzyladenine, kinetin, and thidiazuron effectively reverse the inhibition of growth of these cells at 1 μm lovastatin, whereas adenine and 6-methyladenine have no effect. These results demonstrate that restoration of growth to lovastatin-treated cells is cytokinin specific and is not caused by metabolism of cytokinin into other isoprenoid compounds. Cytokinin does not effectively reverse the effects of higher concentrations of lovastatin, but mevalonic acid does, consistent with the hypothesis that cytokinin biosynthesis is more sensitive to lovastatin than the biosynthesis of other essential isoprenoid compounds in tobacco cells. This observation suggests that lovastatin can be used to induce cytokinin dependence in cytokinin-autonomous tobacco cell cultures.     

Effect of cyclin E overexpression on lovastatin-induced G1 arrest and RhoA inactivation in NIH3T3 cells.

The HMG-CoA reductase inhibitor, lovastatin, blocks targeting of the Rho and Ras families of small GTPases to their active sites by inhibiting protein prenylation. Control NIH3T3 cells, and those overexpressing human cyclin E protein were treated with lovastatin for 24 h to determine the effects of cyclin E overexpression on lovastatin-induced growth arrest and cell rounding. Lovastatin treatment (10 microM) of control 3T3 cells resulted in growth arrest at G1 accompanied by actin stress fiber disassembly, cell rounding, and decreased active RhoA from the membranous protein fraction. By contrast, in NIH3T3 cells overexpressing cyclin E, lovastatin did not cause loss of RhoA from the membrane (active) protein fraction, actin stress fiber disassembly, cell rounding or growth arrest within 24 h. Analysis of cell cycle proteins showed that 24 h of lovastatin treatment in the control cells caused an elevation in the levels of the cyclin-dependent kinase inhibitor p27(kip1), inhibition of both cyclin E- and cyclin A-dependent kinase activity, and decreased levels of hyperphosphorylated retinoblastoma protein (pRb). By contrast, lovastatin treatment of the cyclin E overexpressors did not suppress either cyclin E- or cyclin A-dependent kinase activity, nor did it alter the level of maximally phosphorylated pRb, despite increased levels of p27(kip1). However, by 72 h, the cyclin E overexpressors rounded up but remained attached to the substratum, indicating a delayed response to lovastatin. In contrast with lovastatin, inactivation of membrane-bound Rho proteins (i.e., GTP-bound RhoA, RhoB, RhoC) with botulinum C3 transferase caused cell rounding and G1 growth arrest in both cell types but did not inhibit cyclin E-dependent histone kinase activity in the cyclin E overexpressors. In addition, 24 h of cycloheximide treatment caused depletion of RhoA from the membrane (active) fraction in neo cells, but in the cells overexpressing cyclin E, RhoA remained in the active (membrane-associated) fraction. Our observations suggest that (1) RhoA activation occurs downstream of cyclin E-dependent kinase activation, and (2) overexpression of cyclin E decreased the turnover rate of active RhoA.     

Effect of inhibition of cholesterol synthetic pathway on the activation of Ras and MAP kinase in mesangial cells

Intermediary metabolites of cholesterol synthetic pathway are involved in cell proliferation. Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, blocks mevalonate synthesis, and has been shown to inhibit mesangial cell proliferation associated with diverse glomerular diseases. Since inhibition of farnesylation and plasma membrane anchorage of the Ras proteins is one suggested mechanism by which lovastatin prevents cellular proliferation, we investigated the effect of lovastatin and key mevalonate metabolites on the activation of mitogen-activated protein kinase (MAP kinase) and Ras in murine glomerular mesangial cells. The preincubation of mesangial cells with lovastatin inhibited the activation of MAP kinase stimulated by either FBS, PDGF, or EGF. Mevalonic acid and farnesyl-pyrophosphate, but not cholesterol or LDL, significantly prevented lovastatin-induced inhibition of agonist-stimulated MAP kinase. Lovastatin inhibited agonist-induced activation of Ras, and mevalonic acid and farnesylpyrophosphate antagonized this effect. Parallel to the MAP kinase and Ras data, lovastatin suppressed cell growth stimulated by serum, and mevalonic acid and farnesylpyrophosphate prevented lovastatin-mediated inhibition of cellular growth. These results suggest that lovastatin, by inhibiting the synthesis of farnesol, a key isoprenoid metabolite of mevalonate, modulates Ras-mediated cell signaling events associated with mesangial cell proliferation.     

Evaluation of the mood-stabilizing agent valproic acid as a preventative for toxoplasmosis in mice and activity against tissue cysts in mice

Toxoplasma gondii is a common intracellular protozoan infection of humans worldwide. Severe disease can occur in immunocompromised individuals and the in the fetuses of nonimmune pregnant women. Chronic infection is associated with vision and hearing problems, and functional mental alterations, including schizophrenia. The mood-stabilizing agent valproic acid has been shown to inhibit the development of T. gondii in vitro at dosages that are normally achieved in the serum and cerebral spinal fluid of human patients and to have positive effects on the behavior of rats chronically infected with T. gondii. The present study was done to examine the in vivo activity of valproic acid against acute toxoplasmosis in mice. Two studies were done with valproic acid given in the drinking water at concentrations of 1.5 mg/ml (Experiment 1) or 3.0 mg/ml (Experiment 2). In a third experiment (Experiment 3), valproic acid was injected intraperitoneally (i.p.) at doses of 200 or 300 mg/kg every 12 hr. Valproic acid was not effective in preventing acute toxoplasmosis. All mice treated with valproic acid died or were killed and did not (P > 0.05) live significantly longer than the controls. Tachyzoites were demonstrated in the tissues of infected valproic-acid-treated mice. A fourth study was done to determine if valproic acid has activity against T. gondii tissue cysts in chronically infected mice. Mice were chronically infected with the ME-49 strain of T. gondii for 8 wk and then treated orally with valproic acid at approximately 6.6 mg/ml (800 mg/kg/day) in the drinking water for 10 wk (amount was varied due to increasing mouse weights). No significant differences (P > 0.05) were present in tissue cyst numbers in valproic-acid-treated T. gondii chronically infected mice and in mice chronically infected with T. gondii but not given valproic acid. Our results indicate that valproic acid, although effective in vitro against T. gondii tachyzoites, is not effective as a preventative in mice inoculated with T. gondii tachyzoites. Additionally, no activity against tissue cysts was observed in chronically T. gondii-infected valproic-acid-treated mice.     

Influence of valproic acid on hepatic carbohydrate and lipid metabolism

Valproic acid (dipropylacetic acid), an antiepileptic agent known to be hepatotoxic in some patients, caused inhibition of lactate gluconeogenesis, fatty acid oxidation, and fatty acid synthesis by isolated hepatocytes. The latter process was the most sensitive to valproic acid, 50% inhibition occurring at ca. 125 microM with cells from meal-fed female rats. The medium-chain acyl-CoA ester fraction was increased whereas coenzyme A (CoA), acetyl-CoA, and the long chain acyl-CoA fractions were decreased by valproic acid. The increase in the medium chain acyl-CoA fraction was found by high-pressure liquid chromatography to be due to the accumulation of valproyl-CoA plus an apparent CoAester metabolite of valproyl-CoA. Salicylate inhibited valproyl-CoA formation and partially protected against valproic acid inhibition of hepatic metabolic processes. Octanoate had a similar protective effect, suggesting that activation of valproic acid in the mitosol is required for its inhibitory effects. It is proposed that either valproyl-CoA itself or the sequestration of CoA causes inhibition of metabolic processes. Valproyl-CoA formation also appears to explain valproic acid inhibition of gluconeogenesis by isolated kidney tubules. No evidence was found for the accumulation of valproyl-CoA in brain tissue, suggesting that the effects of valproic acid in the central nervous system are independent of the formation of this metabolite.     

Therapeutic levels of the hydroxmethylglutaryl-coenzyme A reductase inhibitor lovastatin activate ras signaling via phospholipase D2

Hydroxmethylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) lower serum cholesterol but exhibit pleiotropic biological effects that are difficult to ascribe solely to cholesterol depletion. Here, we investigated the effect of lovastatin on protein prenylation and cell signaling. We show that high concentrations (50 μM) of lovastatin inhibit Ras, Rho, and Rap prenylation but that therapeutic levels of lovastatin (50 nM to 500 nM) do not. In contrast, depletion of cellular cholesterol by therapeutic levels of lovastatin increased Ras GTP loading and mitogen-activated protein kinase (MAPK) activation in human umbilical vein endothelial cells and rodent fibroblasts. Elevated Ras signaling was not seen in statin-treated cells if cholesterol levels were maintained by supplementation. Activation of Ras-MAPK signaling was a consequence of, and dependent on, activation of phospholipase D2 (PLD2). Expression of dominant interfering PLD2 or biochemical inhibition of PLD2 abrogated Ras and MAPK activation induced by lovastatin. In contrast, ectopic expression of wild-type PLD2 enhanced Ras and MAPK activation in response to therapeutic levels of lovastatin. Statin-induced cholesterol depletion also modestly activated the epidermal growth factor receptor (EGFR), resulting in downregulation of EGFR expression. These results suggest that statins modulate key cell signaling pathways as a direct consequence of cholesterol depletion and identify the EGFR-PLD2-Ras-MAPK axis as an important statin target.     

Characterization of lovastatin–docosahexaenoate anticancer properties against breast cancer cells

Lovastatin (LOV) and docosahexaenoic acid (DHA), besides improving cardiovascular functions, are also known for their anticancer activities. However, use of these compounds for treating or preventing cancer is limited because of their efficacies. The approach pursued involved chemical linkage of these two chemotypes. A lovastatin–docosahexaenoate (LOV–DHA) conjugate was prepared and tested against selected breast tumor cells lines with differential expression of estrogen receptors (ER) and Heregulin-2 (Her-2). The LOV–DHA conjugate exhibited superior cytotoxic effects against ER⁻/Her-2⁻ cell lines (MDA-MB-231 and MDA-MB-468), which were not observed with DHA or lovastatin alone, or in combination. Lovastatin supplementation arrested cells in the G₀/G₁ phase and enhanced expression levels of p21, whereas the conjugate did not demonstrate cell cycle arrest nor increased p21 expression. The LOV–DHA conjugate induced significant (P < 0.05) apoptosis as low as 1 μM, whereas DHA and lovastatin were ineffective at this concentration. The growth inhibitory effects of lovastatin were reversed by the addition of mevalonate, whereas mevalonate had no effect on the LOV–DHA conjugate-induced growth inhibition in MDA-MB-231 cells. Furthermore, the LOV–DHA conjugates were stable in mouse serum and intracellularly in MDA-MB-231 cells. These data suggest that the LOV–DHA conjugate mediated its effects through a HMG-CoA reductase-independent pathway and exerted significantly (P < 0.05) higher anticancer effects in breast cancer cells than lovastatin or DHA alone.