Lovastatin-treated Cells Research Articles

Inhibition of the mevalonate pathway causes accumulation of CD14 in the endoplasmic reticulum of macrophages

Abstract Mevalonate kinase deficiency (MKD) is an autoinflammatory disease caused by mutations in the mevalonate kinase enzyme, which acts early in the mevalonate pathway to produce sterol and non-sterol isoprenoid compounds. Patients with MKD have episodes of inflammatory symptoms accompanied by increased plasma levels of pro-inflammatory cytokines. CD14 is a receptor that binds to lipopolysaccharide (LPS) and induces the release of cytokines. Isoprenoid depletion was previously shown to increase cellular and decrease extracellular levels of CD14 in macrophages after LPS stimulation. The objective of this study is to characterize the cellular localization of CD14 following isoprenoid depletion. RAW 264.7 macrophages were treated with lovastatin to deplete isoprenoids and CD14 localization was examined using immunofluorescence and western blotting after LPS stimulation. CD14 co-localized with the endoplasmic reticulum (ER) protein calnexin following lovastatin treatment. To quantitate ER CD14, cell lysates were digested with endoglycosidase (Endo) H, which removes high mannose glycans found on ER proteins, but not complex glycans added in the Golgi apparatus. Western blot results showed that the level of Endo H sensitive CD14 was higher in lovastatin as compared to carrier-treated cells. Co-incubation of lovastatin-treated cells with mevalonate or geranylgeranyl pyrophosphate (GGPP), but not cholesterol, prevented the ER accumulation of CD14 indicating that GGPP depletion may cause this effect. Understanding how mevalonate pathway products contribute to protein trafficking in macrophages will expand our understanding of inflammatory diseases.

Lovastatin Induces Apoptosis through Deprivation of Intracellular Geranylgeranyl Pyrophosphate and Induction of Puma Expression in C6 Glioma Cells

Lovastatin is widely used to decrease serum cholesterol levels and reduce the risk of cardiovascular diseases. Lovastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, an enzyme that converts HMG-CoA to mevalonate. Mevalonate is a precursor of various metabolic intermediates such as isopentenyl pyrophosphate, farnesyl pyrophosphate, geranylgeranyl pyrophosphate, and cholesterol. It has been reported that statins can induce apoptosis in various cell lines. In this study, we examined the mechanism of lovastatin-induced apoptosis in C6 and HCT116 cell lines. C6 is a rat glioma cell line and HCT116 is a human colon cancer cell line. Cells were maintained in Dulbecco's minimal essential medium containing 10% (v/v) fetal bovine serum. After treatment, the number of viable cells was estimated using a colorimetric assay based on tetrazolium salt. The percentage of dead cells was estimated using trypan blue cell viability assay or lactate dehydrogenase assay. The levels of Puma mRNA were estimated by reverse transcription quantitative polymerase chain reaction assay. The levels of PUMA were estimated by Western blot assay. Our data showed that lovastatin increased both mRNA and protein levels of Puma in C6 cells, which could be prevented in the presence of 100 μM geranylgeranyl pyrophosphate. Treatment with geranylgeranyl pyrophosphate, cycloheximide, and siPuma (siRNA against Puma) prevented the death of lovastatin-treated cells. Pifithrin-a, an inhibitor of tumor protein p53, neither prevented cell death nor decreased PUMA levels in lovastatin-treated cells. Pifithrin-a attenuated cell death and reduced PUMA levels in etoposide-treated C6 cells. Etoposide, an anticancer drug, has been known to induce p53-dependent apoptosis. To evaluate the role of p53 on the induction of Puma expression in lovastatin-treated cells, cytotoxic effects of lovastatin were examined in HCTm and HCTw cells. HCTm is a HCT116 cell line expressing inactive mutant p53 and HCTw is the wild-type. When cells were cultured in the presence of 25 μM lovastatin for 60 hours, the values of the percentage of cell death were 46% and 43% in HCTm and HCTw groups respectively: there was little difference in lovastatin-induced cell death between two groups. Etoposide-induced cell death was exacerbated in HCTw cells as compared to HCTm cells. When cells were cultured with 100 μM etoposide for 72 hours, the values of the percentage of cell death were 35% and 45% in HCTm and HCTw groups respectively. Pifithrin-a reduced etoposide-induced cell death in HCTw cells from 45% to 25%. Pifithrin-a did not attenuate etoposide-induced cell death in HCTm cells. Our data show that lovastatin depletes intracellular geranylgeranyl pyrophosphate to increase Puma expression in a p53-independent manner, resulting in apoptosis.

Anti-apoptotic effects of dexamethasone and phosphoinositide 3-kinase inhibitors on PUMA-mediated apoptosis of lovastatin-treated cells

Anti-apoptotic effects of dexamethasone and phosphoinositide 3-kinase inhibitors on PUMA-mediated apoptosis of lovastatin-treated cells

Lovastatin treatment mitigates the pro-inflammatory cytokine response in respiratory syncytial virus infected macrophage cells

Disease severity following respiratory syncytial virus (RSV) infection is associated with inflammation due to enhanced pro-inflammatory cytokine secretion, and lung macrophage cells play a role in this process. In this study we evaluated the hydroxymethylglutaryl coenzyme A reductase inhibitor lovastatin as an anti-inflammatory drug to control RSV-induced cytokine secretion in the murine RAW 264.7 (RAW) macrophage cell line and in primary murine lung macrophages. These cells could be efficiently infected with RSV in vitro, and although no significant level of infectious virus particles were produced, the increased expression of several virus structural proteins could be detected. Virus infection and gene expression correlated with increased pro-inflammatory cytokine secretion by 24h post infection. Lovastatin treatment did not reduce the cellular cholesterol levels in RSV-infected cells, nor did it inhibit RSV infection. However, we observed a significant reduction in the pro-inflammatory cytokine levels in lovastatin-treated RSV-infected cells. Since enhanced pro-inflammatory cytokine secretion is a major factor in RSV-associated pathology our findings highlighted the potential use of statins to mitigate and control the inflammatory response due to RSV infection. Furthermore, our study suggested that RAW cells maybe a simple and cost-effective model cell system to screen small molecule libraries to identify compounds that are effective in reducing RSV-induced inflammation.

Lovastatin inhibits proliferation of anaplastic thyroid cancer cells through up-regulation of p27 by interfering with the Rho/ROCK-mediated pathway

Previously, we demonstrated that lovastatin, a HMG-CoA reductase inhibitor, induced apoptosis, differentiation, and inhibition of invasiveness of human anaplastic thyroid carcinoma cells (ATCs). Here, we further examined the effect of lovastatin on the growth of ARO cells. Lovastatin (0–20 μM) concentration-dependently decreased cell number in cultured ATC and arrested the cell at the G0/G1 phase of the cell cycle. Western blot analysis revealed that lovastatin caused an increase of the protein level of p27 and cyclin-dependent kinase (CDK)4 and a decrease of the protein level of cyclin A2, cyclin D3, and phosphorylated Rb (pRb), but did not significantly change the protein levels of p21, cyclins D1 and E, and CDK2, in ARO cells. The formation of the CDK2–p27 complex was increased and the CDK2 activity was decreased in the lovastatin-treated ARO cells. Pretreatment of ARO cells with a p27, but not p21, antisense oligonucleotide prevented the lovastatin-induced G0/G1 arrest in ARO cells. The lovastatin-induced growth inhibition and translocation of RhoA and Rac1 in ARO cells were completely prevented by mevalonate and partially by geranylgeranyl pyrophosphate. Treatment of ARO cells with Y27632, an inhibitor of Rho-associated kinase, abolished the GGPP-mediated prevention of lovastatin-induced anti-proliferation and up-regulation and prolonged degradation of p27. Taken together, these data suggest that lovastatin treatment caused a reduction of Rho geranylgeranylation, which in turn increased the expression and stability of p27, and then inhibited ARO cell proliferation. These data suggest that lovastatin merits further investigation as multipotent therapy for treatment ATC.

Defective rotavirus particle assembly in lovastatin-treated MA104 cells

Rotavirus is a non-enveloped virus that depends on cellular lipids for cell entry and associates with lipid rafts during assembly. However, the effects of cellular lipids on rotavirus assembly are still not fully understood. The present study analyzes the effects of lovastatin, an inhibitor of cholesterol biosynthesis, during rotavirus infection in MA104 cells with regard to viral growth and particle assembly. Following viral infection, a 2-log relative reduction of viral titers was observed in drug-treated cells, while viral mRNA levels in infected cells remained unaltered in both groups. Furthermore, the levels of some viral proteins in drug-treated cells were elevated. The observed discordance between the viral RNA and protein levels and the decrease in infectivity titers of viral progeny in the drug-treated cells suggested that the drug affects viral assembly, the viral proteins not being properly incorporated into virions. Transmission electron microscopic (TEM) analysis revealed that in drug-treated cells there was an increase in “empty-looking” rotavirus particles devoid of an electron-dense core as compared to the normal, electron-dense particles seen in untreated infected cells. The present study thus provides visual evidence of defective rotavirus particle assembly as a result of cholesterol depletion.

Effects of lovastatin on Rho isoform expression, activity, and association with guanine nucleotide dissociation inhibitors

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC1.1.1.88) inhibitors (statins) reduce cholesterol synthesis and prevent cardiovascular disease; they can also inhibit prenylation of Ras and Rho proteins, and have anti-neoplastic effects. Rho proteins cycle between an active, GTP-bound, and an inactive, GDP-bound form, and Rho prenylation is important for Rho's interaction with upstream regulators and downstream effectors, but the effects of statins on Rho signaling are incompletely understood. We found that the HMG-CoA reductase inhibitor lovastatin markedly induced the expression of RhoA, B, and C in human erythroleukemia (HEL) cells. The drug increased RhoA and C only in their unprenylated forms, but it increased both prenylated and unprenylated RhoB and did not significantly affect N- and K-Ras prenylation, suggesting that it inhibited geranyl-geranylation more efficiently than farnesylation. Quantitative analysis of nucleotides bound to Rho demonstrated a 3.7-fold increase in Rho-GTP and a similar increase in Rho-GDP in lovastatin-treated cells, leaving the fraction of Rho in the active, GTP-bound form constant at 5.8%. Lovastatin reduced Rho association with Rho guanine dissociation inhibitor (RhoGDI)-α and -β, and prenylation-deficient Rho mutants did not associate with RhoGDI. siRNA inhibition of RhoGDIα expression increased Rho-GTP, suggesting that decreased Rho/RhoGDIα association explained an increase in unprenylated Rho-GTP in lovastatin-treated cells. Unprenylated Rho A, B, and C were partly functional in activating serum response element-dependent transcription. In conclusion, we quantified effects of lovastatin on RhoA, B, and C isoforms, and provide a molecular mechanism whereby statins cause accumulation of unprenylated Rho-GTP.

Lovastatin Enhances the Replication of the Oncolytic Adenovirus dl1520 and Its Antineoplastic Activity against Anaplastic Thyroid Carcinoma Cells

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive solid tumors and shows morphological features of a highly malignant, undifferentiated neoplasm. Patients with ATC have a poor prognosis with a mean survival time of 2-6 months; surgery, radiotherapy, and chemotherapy do not improve survival. Gene therapy approaches and oncolytic viruses have been tested for the treatment of ATC. To enhance the antineoplastic effects of the oncolytic adenovirus dl1520 (Onyx-015), we treated ATC cells with lovastatin (3-hydroxy-methylglutaryl-CoA reductase inhibitor), a drug used for the treatment of hypercholesterolemia, which has previously been reported to exert growth-inhibitory and apoptotic activity on ATC cells. Lovastatin treatment significantly increased the effects of dl1520 against ATC cells. The replication of dl1520 in ATC cells was enhanced by lovastatin treatment, and a significant increase of the expression of the early gene E1A 13 S and the late gene Penton was observed in lovastatin-treated cells. Furthermore, lovastatin treatment significantly enhanced the effects of dl1520 against ATC tumor xenografts. Lovastatin treatment could be exploited to increase the efficacy of oncolytic adenoviruses, and further studies are warranted to confirm the feasibility of the approach in ATC patients.

Lovastatin suppresses erythropoietin receptor surface expression through dual inhibition of glycosylation and geranylgeranylation

Erythropoietin (Epo) is a cytokine that is required for the survival of erythroid progenitors through interaction with its receptor on the surface of these cells. Recent studies showed that erythropoietin receptor (EpoR) is expressed on many cancer cells. The factors that govern EpoR expression on the cell surface are poorly understood. Using both biotinlyation and radiolabeled Epo binding experiments, we show here that Epo starvation of the Epo-dependent erythroleukemia cell line, ASE2, leads to a time-dependent increase in both forms of EpoR, the maturing 64 kDa and the mature 66 kDa proteins. Mevalonate depletion inhibits the formation of the highly glycosylated mature form of EpoR without affecting the other form. Treatment of cells with lovastatin, a selective inhibitor of the rate-limiting enzyme in the mevalonate pathway leads to inhibition of cell surface EpoR that is induced by Epo starvation. The effect of lovastatin appears to be the consequence of inhibition of two processes, glycosylation and geranylgeranylation. Adding back geranylgeranyl pyrophosphate to lovastatin-treated cells completely prevents the lovastatin effect on EpoR expression. Dolichol, the sugar carrier in N-linked glycosylation that is derived from the mevalonate pathway, partially reverses lovastatin's effect. The glycosylation inhibitor tunicamycin also partially suppresses EpoR surface expression. Inhibiting protein geranylgeranylation mimics the effect of lovastatin and inhibits EpoR surface expression in a concentration-dependent manner. Finally, lovastatin inhibits Epo's stimulatory effects on cell proliferation. These results indicate that mevalonate derivatives are required for normal EpoR expression on the cell surface through two pathways, glycosylation and geranylgeranylation.

Influences of Lovastatin on membrane ion flow and intracellular signaling in breast cancer cells

Lovastatin, an inhibitor of cellular cholesterol synthesis, has an apparent anti-cancer property, but the detailed mechanisms of its anti-cancer effects remain poorly understood. We investigated the molecular mechanism of Lovastatin anti-tumor function through the study of its effect on membrane ion flow, gap junctional intercellular communication (GJIC), and the pathways of related signals in MCF-7 mammary cancer cells. After treatment for 24–72 h with 4, 8 or 16 μmol/L Lovastatin, cellular proliferation was examined via the MTT assay, and changes in membrane potential and cellular [Ca2+]i were monitored using confocal laser microscopy. In addition, the expression of plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA was analyzed via RT-PCR, the GJIC function was examined using the scrape-loading dye transfer (SLDT) technique, and MAPK phosphorylation levels were tested with the kinase activity assay. The results showed that Lovastatin treatment significantly inhibited the growth of MCF-7 breast cancer cells. It also increased the negative value of the membrane potential, leading to the hyperpolarization of cells. Moreover, Lovastatin treatment continuously enhanced [Ca2+]i, although the levels of PMCA1 mRNA were unchanged. GJIC was also upregulated in MCF-7 cells, with transfer of LY Fluorescence reaching 4 to 5 rows of cells from the scraped line after treatment with 16 μmol/L Lovastatin for 72 h. Finally, downregulation of ERK1 and p38MAPK phosphorylation were found in Lovastatin-treated MCF-7 cells. It could be deduced that Lovastatin can induce changes in cellular hyperpolarization and intracellular Ca2+ distributions, and increase GJIC function. These effects may result in changes in the downstream signal cascade, inhibiting the growth of MCF-7 cells.

Effects of Cholesterol-Lowering Statins on the Aqueous Humor Outflow Pathway

To investigate the effects of cholesterol-lowering statin drugs on trabecular meshwork cellular properties and aqueous humor outflow. Primary cell cultures of porcine trabecular meshwork (PTM) and ciliary body (PCB) were treated with either lovastatin or compactin, to determine the effects of statins on cell shape, actin cytoskeletal organization, and cell-extracellular matrix interactions (focal adhesions) by immunofluorescence staining. Changes in myosin light-chain (MLC) phosphorylation were evaluated by Western blot analysis. Changes in Rho GTPase content of membrane fractions from lovastatin-treated PTM cells were assessed by Western blot analysis. A constant-flow, organ-culture perfusion system was used to measure the effects of statins on aqueous humor outflow facility in the anterior segments of porcine eyes. PTM and PCB cells treated with lovastatin or compactin exhibited dramatic changes in cell shape and cytoskeletal organization within 24 hours, consisting of cell rounding, actin depolymerization, and decreased focal adhesions. These effects were found to be reversible on supplementation with geranylgeranyl pyrophosphate. Both lovastatin and compactin decreased MLC phosphorylation in PTM and PCB cells. PTM cells treated with lovastatin exhibited marked decreases in membrane-bound Rho GTPase. In addition, perfusion of organ-cultured porcine eye anterior segments with 100 microM lovastatin for 96 hours caused a significant increase in aqueous humor outflow facility (110%) compared with control eyes, in a reversible manner. This study demonstrates that the statin drugs lovastatin and compactin induce changes in cell shape and actin cytoskeletal organization and decrease MLC phosphorylation in PTM and PCB cells, all of which are events that are likely to lead to cellular and tissue relaxation. In addition, these effects of the statins appear to be mediated by inhibition of isoprenylation of the small GTP-binding proteins such as Rho GTPase. An important finding is that statins exert an ocular hypotensive response in an organ-culture perfusion model, indicating the potential for this class of drugs in glaucoma therapy.

Lovastatin inhibits tumor growth and lung metastasis in mouse mammary carcinoma model: a p53-independent mitochondrial-mediated apoptotic mechanism.

The effects of lovastatin, a potent inhibitor of hydroxymethylglutaryl-coenzyme A reductase, were studied in a mouse model of metastatic mammary cancer carrying a p53 mutation. Mice bearing mammary tumors, induced by inoculation of syngeneic BALB/c mice with BJMC3879 cells, were treated with lovastatin at 0, 25 and 50 mg/kg three times a week. Tumor volumes were significantly reduced in a dose-dependent manner throughout the 6 week study and were associated with both a decrease in DNA synthesis and an increase in apoptosis. The high dose of lovastatin also inhibited lung metastasis. In a corollary in vitro study, flow cytometric analyses of lovastatin-treated mammary cancer cells additionally showed cell cycle arrest at G1 phase and decreases in S and G2/M phases. Laser scanning cytometric analyses further demonstrated that cancer cells in S and G2/M were particularly susceptible to the effects of lovastatin. Transmission electron microscopic evaluation of TUNEL-confirmed apoptotic bodies in lovastatin-treated mammary carcinoma cells revealed many free 3'-OH ends of DNA in condensed chromatin within fragmented nuclei that occasionally assumed a characteristic half-moon shape. Consistent with initiation of apoptosis, cellular caspase-8, caspase-9 and caspase-3 activities were elevated in lovastatin-treated cells. The mitochondrial membrane potential was also decreased, with subsequent release of cytochrome c. However, lovastatin-induced cell death was significantly reduced by the broad spectrum caspase inhibitor z-VAD-fmk, as well as the caspase-9 inhibitor z-LEHD-fmk and the caspase-3 inhibitor z-DEVD-fmk, but not by the specific caspase-8 inhibitor z-IETD-fmk. Since immunoelectron microscopy showed translocation of Bax to the mitochondria in lovastatin-treated cells, lovastatin-induced apoptosis may, therefore, be ultimately dependent on Bax induction of cytochrome c release. These results suggest that lovastatin may be useful as an adjuvant therapy in breast cancers containing p53 mutations due to its ability to both suppress DNA synthesis and induce p53-independent mitochondria-mediated apoptosis.

Statins decrease levels of the estrogen receptor in human breast cancers

The estrogen receptor (ER-α) is a nuclear transcription factor important in the regulation of estrogen-responsive genes and is a target for breast cancer therapies. Alternates to pharmacological estrogen depletion with aromatase inhibitors or ER-α blockade with tamoxifen includes agents that alter the levels of ER-α Fulvestrant is an ER-α downregulator that is of therapeutic benefit in the treatment of ER-α positive breast cancer. We have investigated the effects of mevalonic acid depletion by the statins on the expression of ER-α in human breast cancer MCF-7 cells. These cells were maintained in culture and incubated with combinations of lovastatin, tamoxifen, and fulvestrant. ER-α was detected by Western blot analysis (mouse anti-ER-α antibody 6F11, Novocastra). Lovastatin resulted in a time-dependent decrease in expression of ER-α, with partial effect seen at 15 hrs and maximal effect by 48 hrs. This effect was also seen in cells co-cultured in the presence of tamoxifen. This effect was comparable to that observed with treatment by fulvastrant. Mevalonic acid addition to lovastatin-treated cells restored the levels of ER-α. Northern blot analyses of cell lysates indicate that the statin effect on ER-α is at the level of decreasing of ER-α mRNA. These studies report the novel finding of the statins to decrease the level of the estrogen receptors and may lead to new approaches to the treatment of human breast cancers. Clinical Pharmacology & Therapeutics (2004) 75, P45–P45; doi: 10.1016/j.clpt.2003.11.170

Overdependence on the host-an Achilles' heel of HCV?

Hepatitis C virus (HCV) RNA replication depends on viral protein association with intracellular membranes, but the influence of membrane composition on viral replication is unclear. We report that HCV RNA replication and assembly of the viral replication complex require geranylgeranylation of one or more host proteins. In cultured hepatoma cells, HCV RNA replication was disrupted by treatment with lovastatin, an inhibitor of 3-hydroxy-3-methyglutaryl CoA reductase, or with an inhibitor of protein geranylgeranyl transferase I, each of which induced the dissolution of the HCV replication complex. Viral replication was not affected by treatment of cells with an inhibitor of farnesyl transferase. When added to lovastatin-treated cells, geranylgeraniol, but not farnesol, restored replication complex assembly and viral replication. Inasmuch as the HCV genome does not encode a canonical geranylgeranylated protein, the data suggest the involvement of a geranylgeranylated host protein in HCV replication. Inhibition of its geranylgeranylation affords a therapeutic strategy for treatment of HCV infection.

Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation.

Hepatitis C virus (HCV) RNA replication depends on viral protein association with intracellular membranes, but the influence of membrane composition on viral replication is unclear. We report that HCV RNA replication and assembly of the viral replication complex require geranylgeranylation of one or more host proteins. In cultured hepatoma cells, HCV RNA replication was disrupted by treatment with lovastatin, an inhibitor of 3-hydroxy-3-methyglutaryl CoA reductase, or with an inhibitor of protein geranylgeranyl transferase I, each of which induced the dissolution of the HCV replication complex. Viral replication was not affected by treatment of cells with an inhibitor of farnesyl transferase. When added to lovastatin-treated cells, geranylgeraniol, but not farnesol, restored replication complex assembly and viral replication. Inasmuch as the HCV genome does not encode a canonical geranylgeranylated protein, the data suggest the involvement of a geranylgeranylated host protein in HCV replication. Inhibition of its geranylgeranylation affords a therapeutic strategy for treatment of HCV infection.

Lovastatin induces apoptosis of anaplastic thyroid cancer cells via inhibition of protein geranylgeranylation and de novo protein synthesis.

Lovastatin has been used to treat hypercholesterolemia through blocking the mevalonate biosynthesis pathway. Inhibition of mevalonate synthesis may result in antiproliferation and cell apoptosis. The aim of the present study was to examine the apoptotic effect of lovastatin in human ARO cells and delineate its underlying molecular mechanism. Our results showed that lovastatin dose- and time-dependently induced apoptosis in ARO cells. Pretreatment with cycloheximide dose-dependently suppressed lovastatin-induced apoptosis, suggesting that de novo protein synthesis is required for lovastatin effect on the induction of apoptosis in ARO cells. Treatment of the cells with 50 microM lovastatin induced cytochrome c translocation from mitochondria to cytosol; increases in caspase-2, -3, and -9 activity; and poly (ADP-ribose) polymerase degradation in a time-dependent manner. However, administration of mevalonate or geranylgeraniol, but not farnesol, dose-dependently prevented lovastatin-induced poly (ADP-ribose) polymerase degradation and the occurrence of apoptosis, but treatment with geranylgeranyl transferase inhibitor, GGTI-298, which blocks the geranylgeranylation, induced an increase in the percentage of the apoptotic cells. These data suggest that geranylgeranylation is required for survival of the lovastatin-treated ARO cells. To support this notion, we demonstrate that lovastatin dose-dependently decreased the translocation of RhoA and Rac1, but not Ras, from cytosol to membrane fraction. Moreover, the lovastatin-induced translocation inhibitions in RhoA and Rac1 were prevented by mevalonate and geranylgeraniol but not farnesol. In conclusion, our data suggest that lovastatin induced apoptosis in ARO cells by inhibiting protein geranylgeranylation of the Rho family but not farnesylation of the Ras family.

Plant-Derived Monoterpenes Suppress Hamster Kidney Cell 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Synthesis at the Post-Transcriptional Level

The rate-limiting enzyme for mevalonate and cholesterol synthesis in mammalian cells is 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Control occurs through both transcriptional and post-transcriptional actions signaled by the end product, cholesterol, and by isoprenoid intermediates. End products of plant mevalonate metabolism, i.e., plant-derived isoprenoids, also suppress mammalian HMG-CoA reductase. Previous studies reported that isoprenoids suppress reductase synthesis at a post-transcriptional level. We tested the hypothesis that plant-derived isoprenoids also regulate mammalian HMG-CoA reductase synthesis at a post-transcriptional level by incubating lovastatin-treated C100 cells with mevalonate or a plant-derived isoprenoid (the monoterpenes, limonene, perillyl alcohol or geraniol) either alone or combined with the oxysterol, 25-hydroxycholesterol (25-OH C). Mevalonate decreased HMG-CoA reductase synthesis and mRNA levels by 65 and 66%, respectively (P < 0.05). The cyclic monoterpenes, limonene and perillyl alcohol, lowered HMG-CoA reductase synthesis by 70 and 89%, respectively (P < 0.05); although neither reduced HMG-CoA reductase mRNA levels (P = 0.88). Geraniol, an acyclic monoterpene, suppressed HMG-CoA reductase synthesis by 98% and lowered mRNA levels by 66% (P < 0.05). A combination of 25-OH C and either mevalonate or any three monoterpenes reduced HMG-CoA reductase mRNA levels (P < 0.05) compared with lovastatin-only treated cells. However, the dual combination of 25-OH C and either mevalonate or a monoterpene resulted in a greater decrease in HMG-CoA reductase synthesis than in mRNA levels. The difference between changes in HMG-CoA reductase synthesis and mRNA levels reflects a specific effect of isoprenoids on HMG-CoA reductase synthesis at the translational level. Mevalonate enhanced HMG-CoA reductase degradation, but no such effect was observed for the monoterpenes. These results indicate that the three plant-derived isoprenoids primarily suppress HMG-CoA reductase synthesis at a post-transcriptional level by attenuating HMG-CoA reductase mRNA translational efficiency.

Lovastatin alters cytoskeleton organization and inhibits experimental metastasis of mammary carcinoma cells.

Lovastatin is a competitive inhibitor of 3-hydroxy 3-methylglutaryl coenzyme A reductase, the key regulatory enzyme of cholesterol biosynthesis. This enzyme catalyzes the formation of mevalonate, which is also the precursor of isoprenoid moieties, such as farnesol and geraniol, that are incorporated into several molecules essential for tumor cell signaling. Here, we describe that pretreatment with a non-cytotoxic concentration of lovastatin (10 microM) dramatically inhibited the metastatic ability of F311 mammary carcinoma cells in syngeneic BALB/c mice. Similarly, daily i.p. treatment of animals with a well-tolerated dose of lovastatin (10 mg/kg/day) significantly reduced the number of experimental lung metastases. In vitro, incubation of F3II monolayers in the presence of lovastatin caused a rounded-cell morphology. Immunofluorescence analysis revealed a lack of cortical actin organization, micrutubule disruption and inhibition of integrin-mediated focal contacts in lovastatin-treated cells. Exposure of F3II cells to lovastatin significantly inhibited tumor cell adhesion and migration, and coincubation with the cholesterol precursor mevalonate prevented these effects. Lovastatin reduced membrane localization of Rho protein, a signaling molecule involved in the regulation of actin-based cell motility that needs geranylation for membrane association and activation. In addition, lovastatin induced a dose-dependent inhibition in the secretion of urokinase, a key proteolytic enzyme during tumor invasion and metastasis, and a significant increase of tissue-type plasminogen activator, a marker of good prognosis in mammary cancer. These data suggest that antimetastatic properties of lovastatin are strongly associated with alterations in cytoskeleton organization and the consequent modulation of adhesion, motility and proteolysis.

Lipoprotein lipase- and hepatic triglyceride lipase-promoted very low density lipoprotein degradation proceeds via an apolipoprotein E-dependent mechanism

Apolipoprotein E (apoE) is the primary recognition signal on triglyceride-rich lipoproteins responsible for interacting with low density lipoprotein (LDL) receptors and LDL receptor-related protein (LRP). It has been shown that lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) promote receptor-mediated uptake and degradation of very low density lipoproteins (VLDL) and remnant particles, possibly by directly binding to lipoprotein receptors. In this study we have investigated the requirement for apoE in lipase-stimulated VLDL degradation. We compared binding and degradation of normal and apoE-depleted human VLDL and apoE knockout mouse VLDL in human foreskin fibroblasts. Surface binding at 37°C of apoE knockout VLDL was greater than that of normal VLDL by 3- and 40-fold, respectively, in the presence of LPL and HTGL. In spite of the greater stimulation of surface binding, lipase-stimulated degradation of apoE knockout mouse VLDL was significantly lower than that of normal VLDL (30, 30, and 80%, respectively, for control, LPL, and HTGL treatments). In the presence of LPL and HTGL, surface binding of apoE-depleted human VLDL was, respectively, 40 and 200% of normal VLDL whereas degradation was, respectively, 25 and 50% of normal VLDL. LPL and HTGL stimulated degradation of normal VLDL in a dose-dependent manner and by a LDL receptor-mediated pathway. Maximum stimulation (4-fold) was seen in the presence LPL (1 μg/ml) or HTGL (3 μg/ml) in lovastatin-treated cells. On the other hand, degradation of apoE-depleted VLDL was not significantly increased by the presence of lipases even in lovastatin-treated cells. Surface binding of apoE-depleted VLDL to metabolically inactive cells at 4°C was higher in control and HTGL-treated cells, but unchanged in the presence of LPL. Degradation of prebound apoE-depleted VLDL was only 35% as efficient as that of normal VLDL. Surface binding of apoE knockout or apoE-depleted VLDL was to heparin sulfate proteoglycans because it was completely abolished by heparinase treatment. However, apoE appears to be a primary determinant for receptor-mediated VLDL degradation. Our studies suggest that overexpression of LPL or HTGL may not protect against lipoprotein accumulation seen in apoE deficiency.—Medh, J. D., G. L. Fry, S. L. Bowen, S. Ruben, H. Wong, and D. A. Chappell. Lipoprotein lipase- and hepatic triglyceride lipase-promoted very low density lipoprotein degradation proceeds via an apolipoprotein E-dependent mechanism. J. Lipid Res. 2000. 41: 1858–1871.

A U-rich element in the 5' untranslated region is necessary for the translation of p27 mRNA.

Increased translation of p27 mRNA correlates with withdrawal of cells from the cell cycle. This raised the possibility that antimitogenic signals might mediate their effects on p27 expression by altering complexes that formed on p27 mRNA, regulating its translation. In this report, we identify a U-rich sequence in the 5' untranslated region (5'UTR) of p27 mRNA that is necessary for efficient translation in proliferating and nonproliferating cells. We show that a number of factors bind to the 5'UTR in vitro in a manner dependent on the U-rich element, and their availability in the cytosol is controlled in a growth- and cell cycle-dependent fashion. One of these factors is HuR, a protein previously implicated in mRNA stability, transport, and translation. Another is hnRNP C1 and C2, proteins implicated in mRNA processing and the translation of a specific subset of mRNAs expressed in differentiated cells. In lovastatin-treated MDA468 cells, the mobility of the associated hnRNP C1 and C2 proteins changed, and this correlated with increased p27 expression. Together, these data suggest that the U-rich dependent RNP complex on the 5'UTR may regulate the translation of p27 mRNA and may be a target of antimitogenic signals.