Addition Of Geranylgeranyl Pyrophosphate Research Articles

USP28 controls SREBP2 and the mevalonate pathway to drive tumour growth in squamous cancer

SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.

Statins Enhance Killing of Multiple Myeloma Cells By the BCL-2 Inhibitor Venetoclax and the MCL-1 Inhibitor S63845

The BCL-2 inhibitor venetoclax has begun to change the landscape of therapy for diverse blood cancers. Combining venetoclax with standard of care agents has led to impressive response rates, and increased depth and durability of response compared to monotherapy. However, there is a continuing need to develop safe and effective combinations in multiple myeloma (MM), where the BELLINI triple combination trial (venetoclax-dexamethasone-bortezomib) was closed to accrual due to adverse events. We reported that HMG-CoA-reductase inhibitors (statins), a safe and widely prescribed class of drugs, enhance the efficacy of venetoclax in human leukemia and lymphoma cell lines and in a retrospective study of patients with chronic lymphocytic leukemia (JS Lee et al., Sci. Transl. Med.2018, 10(445)). In MM, the subset with t11;14 translocation is BCL-2-dependent and sensitive to venetoclax, whereas other MM subtypes are more dependent on MCL-1. Here we show that statins sensitize MM cell lines and primary cells to killing by either venetoclax or the MCL-1 inhibitor S63845. The compound pitavastatin is particularly potent, active in vitro at a concentration (100 - 300 nM) that is clinically achievable. In OPM2 cells, pitavastatin synergizes with venetoclax and increases apoptosis in triple combination with dexamethasone. Pitavastatin also potentiates venetoclax killing in dexamethasone-resistant MM1R cells. In MCL-1-dependent MM cells, statins increase sensitivity to S63845. Notably, statins do not increase killing of normal human lymphocytes by either venetoclax or S63845. Statin responsiveness in MM cell lines correlates with p53-independent upregulation of PUMA, BAX and BAK, and gene editing experiments suggest that all three contribute to the apoptotic mechanism. Statin-induced apoptosis and PUMA expression are rescued by addition of geranylgeranyl-pyrophosphate, and recapitulated by an inhibitor of geranylgeranyl-transferase I. Together these findings establish a mechanistic framework to explain how statins promote apoptosis by BH3 mimetic drugs like venetoclax and S63845. Further, these results highlight the potential of statins to enhance therapeutic efficacy of BCL-2 and MCL-1 inhibitor regimens in MM, with possibly lesser toxicity than other combination strategies. Disclosures Boise: Genentech Inc.: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Research Funding. OffLabel Disclosure: Venetoclax is a BCL-2 inhibitor that is approved for some indications. I will discuss the use of venetoclax for multiple myeloma, a disease for which venetoclax is not approved.

FBXW7 regulates endothelial barrier function by suppression of the cholesterol synthesis pathway and prenylation of RhoB.

Rho GTPases control both the actin cytoskeleton and adherens junction stability and are recognized as essential regulators of endothelial barrier function. They act as molecular switches and are primarily regulated by the exchange of GDP and GTP. However, posttranslational modifications such as phosphorylation, prenylation, and ubiquitination can additionally alter their localization, stability, and activity. F-box proteins are involved in the recognition of substrate proteins predestined for ubiquitination and subsequent degradation. Given the importance of ubiquitination, we studied the effect of the loss of 62 members of the F-box protein family on endothelial barrier function in human umbilical vein endothelial cells. Endothelial barrier function was quantified by electrical cell impedance sensing and macromolecule passage assay. Our RNA interference–based screen identified FBXW7 as a key regulator of endothelial barrier function. Mechanistically, loss of FBXW7 induced the accumulation of the RhoB GTPase in endothelial cells, resulting in their increased contractility and permeability. FBXW7 knockdown induced activation of the cholesterol biosynthesis pathway and changed the prenylation of RhoB. This effect was reversed by farnesyl transferase inhibitors and by the addition of geranylgeranyl pyrophosphate. In summary, this study identifies FBXW7 as a novel regulator of endothelial barrier function in vitro. Loss of FBXW7 indirectly modulates RhoB activity via alteration of the cholesterol biosynthesis pathway and, consequently, of the prenylation status and activity of RhoB, resulting in increased contractility and disruption of the endothelial barrier.

Abstract 2054: Cerivastatin overcomes the acquired resistance to crizotinib in EML4-ALK positive non-small cell lung cancer by controlling YAP activation

Abstract Crizotinib is highly effective in patients with non-small cell lung cancer (NSCLC) harboring the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion. However, its efficacy has been limited by the development of acquired resistance, and the mechanisms of such resistance remain largely unknown. Herein, we investigated a possible candidate for circumventing the acquired resistance to crizotinib. We established a model of acquired resistance to crizotinib (H3122-CR) by exposing EML4-ALK-positive H3122 lung cancer cells to increasing doses of crizotinib, and performed MTT screening using a library of FDA (Food and Drug Administration)-approved drugs composed of a collection of 640 clinically used compounds. Our MTT screening identified that cerivastatin, a drug targeting 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, significantly inhibited the cell growth in H3122-CR cells, which was completely restored by addition of geranylgeranyl pyrophosphate (GGPP), a key metabolite of mevalonate pathway. We also found that yes-associated protein (YAP), a major effector of the Hippo tumor suppressor pathway, was robustly accumulated in nucleus with a concomitant decrement of YAP phosphorylation in H3122-CR cells compared to parental cells. Importantly, inhibition of geranylgeranylation (GGylation) by GGTI-298 or cerivastatin markedly increased YAP phosphorylation, led to cytoplasmic translocation of YAP, and subsequently induced YAP inactivation. Moreover, the enrichment of EGFR signaling pathway and cell cycle signature including transcriptional targets of YAP was enhanced in H3122-CR cells, whose induction contributed YAP-mediated resistance to crizotinib. Inhibition of YAP function with siRNA or verteporfin as a YAP inhibitor greatly abrogated cell growth of H3122-CR cells by modulating cell cycle regulators and EGFR activation. Finally, we confirmed up-regulation of nuclear YAP expression in crizotinib-acquired resistant patient derived tumor xenograft (PDTX) models established from EML4-ALK positive NSCLC patients. Collectively, our findings define the GGylation-mediated YAP transcriptional activation as a new mechanism of resistance to crizotinib, providing a rationale for further exploring statins as a possible anticancer agent to overcome the acquired resistance to crizotinib in EML4-ALK positive NSCLC cells. Citation Format: Miran Yun, Hun Mi Choi, Kyoung Ho Pyo, Byoung Chul Cho. Cerivastatin overcomes the acquired resistance to crizotinib in EML4-ALK positive non-small cell lung cancer by controlling YAP activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2054. doi:10.1158/1538-7445.AM2017-2054

Sodium ferulate lowers portal pressure in rats with secondary biliary cirrhosis through the RhoA/Rho-kinase signaling pathway: a preliminary study.

Cirrhotic rats show higher expression levels of hepatic RhoA and Rho-kinase than normal healthy rats, and the activation of this signaling pathway leads to portal hypertension. Sodium ferulate (SF) has been shown to decrease the production of geranylgeranyl pyrophosphate (GGPP), a substance essential for RhoA activation. In the present study, to investigate the effects of SF on fibrosis, portal hypertension and the RhoA/Rho-kinase pathway, hepatic cirrhosis was induced in rats by bile duct ligation. Liver function and fibrogenesis-related biochemical parameters, the hepatic hydroxyproline content, the pathological characteristics of the liver sections and the levels of hepatic α-smooth muscle actin (α-SMA; by immunohistochemistry) were analyzed to assess effects of SF on hepatic fibrosis. In addition, hepatic RhoA, Rho-kinase and endothelial nitric oxide synthase (eNOS) expression was examined by immunohistochemistry. Apoptosis in the SF-treated and SF + GGPP-treated rat primary hepatic stellate cells (HSCs) and a human stellate cell line (LX-2) was examined by flow cytometry. Intrahepatic resistance and responsiveness to the α1-adrenoceptor agonist, methoxamine, were investigated by in situ liver perfusion. Treatment with SF did not affect fibrosis-related biochemical parameters or the hydroxyproline content; however, SF reduced the histological evidence of fibrosis and hepatocyte damage. The SF-treated rats had a significantly lower expression of α-SMA and Rho-kinase, as well as an increased hepatic eNOS content; however, SF did not affect RhoA expression. The SF-treated HSCs had a significantly increased apoptotic rate compared to the untreated rats. Following the addition of GGPP, the rate apoptotic rate decreased. SF reduced basal intrahepatic resistance and the responsiveness of hepatic vascular smooth muscle to methoxamine. Therefore, our data demonstrate that SF reduces fibrogenesis, decreases portal pressure in cirrhotic rats and inhibits the activation of the RhoA/Rho-kinase signaling pathway.

The Impact of Statins on FGF–2—Stimulated Human Umbilical Vein Endothelial Cells

Aim: To determine the effects of different types of statins on proliferative and migrative behaviors of basic fibroblastic growth factor (FGF)–2–stimulated endothelial cells. Materials and Methods: Human umbilical vein endothelial cells (HUVECs) were isolated and cultured. Groups were arranged in order to observe the impact of each individual substance alone, or under stimulation with statin on FGF–2–stimulated endothelial cells. Endothelial cells were stimulated with human growth factor (HGF), statins, methyl–β–cyclodextrin (β–MCD), and either farnesyl pyrophosphate (FPP) ammonium salt, or geranylgeranyl–pyrophosphate (GGPP), respectively. Cell proliferation analyses were performed 48 hours after stimulation and gaps between migration borders were used in migration analyses. Results: The statins showed significant antiproliferative and anti–migrative effects and inhibited the proliferative behavior of FGF–2. However, endothelial cell proliferation and migration were significantly increased after mevalonate co–incubation. Experiments with β–MCD indicated that the destruction of lipid rafts had a negative impact on the action of FGF–2. Stimulation of statin–incubated cells with FPP had no additional effect on proliferation or migration. Notably, although FGF–2 exerted a pro–migrative effect, the effect was not shown in the FPP + FGF–2 group. The anti–migrative actions of statins along with disruption of membrane integrity were reversed by the addition of GGPP. Conclusion: The angiogenic effect of FGF–2 is suppressed through inhibition of the intracellular cholesterol biosynthesis via statins. Inhibitory effects of statins on FGF–2—stimulated HUVECs were observed to result from both the inhibition of isoprenylation and the destruction of lipid rafts on the cell membrane.

The differential effects of statins on the metastatic behaviour of prostate cancer

Background:Although statins do not affect the incidence of prostate cancer (CaP), usage reduces the risk of clinical progression and mortality. Although statins are known to downregulate the mevalonate pathway, the mechanism by which statins reduce CaP progression is unknown.Methods:Bone marrow stroma (BMS) was isolated with ethical approval from consenting patients undergoing surgery for non-malignant disease. PC-3 binding, invasion and colony formation within BMS was assessed by standardised in vitro co-culture assays in the presence of different statins.Results:Statins act directly on PC-3 cells with atorvastatin, mevastatin, simvastatin (1 μℳ) and rosuvastatin (5 μℳ), but not pravastatin, significantly reducing invasion towards BMS by an average of 66.68% (range 53.93–77.04% P<0.05) and significantly reducing both number (76.2±8.29 vs 122.9±2.48; P=0.0055) and size (0.2±0.0058 mm2 vs 0.27±0.012 mm2; P=0.0019) of colonies formed within BMS. Statin-treated colonies displayed a more compact morphology containing cells of a more epithelial phenotype, indicative of a reduction in the migrational ability of PC-3 cells. Normal PC-3 phenotype and invasive ability was recovered by the addition of geranylgeranyl pyrophosphate (GGPP).Conclusion:Lipophilic statins reduce the migration and colony formation of PC-3 cells in human BMS by inhibiting GGPP production, reducing the formation and the spread of metastatic prostate colonies.

Lovastatin inhibits EGFR dimerization and AKT activation in squamous cell carcinoma cells: potential regulation by targeting rho proteins

We recently showed the ability of lovastatin to inhibit the function of the epidermal growth factor receptor (EGFR) and its downstream signaling of the phosphatidylinositol-3 kinase/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in various tumor-derived cell lines. In this study, lovastatin treatment was found to inhibit ligand-induced EGFR dimerization in squamous cell carcinoma cells and its activation of AKT and its downstream targets 4E-binding protein 1 and S6 kinase 1. This inhibition was associated with global protein translational inhibition shown by a decrease in RNA associated polysome fractions. The effects of lovastatin on EGFR function were reversed by the addition of geranylgeranyl pyrophosphate, which functions as a protein membrane anchor. Lovastatin treatment induced actin cytoskeletal disorganization and the expression of geranylgeranylated rho family proteins that regulate the actin cytoskeleton, including rhoA. Lovastatin-induced rhoA was inactive as EGF stimulation failed to activate rhoA and inhibition of the rho-associated kinase, a target and mediator of rhoA function, with Y-27632 also showed inhibitory effects on EGFR dimerization. The ability of lovastatin to inhibit EGFR dimerization is a novel exploitable mechanism regulating this therapeutically relevant target. To explore the potential clinical significance of this combination, we evaluated the effect of statin on the overall survival (OS) and disease-specific survival (DSS) of patients with advanced non-small-cell lung cancer enrolled in the NCIC Clinical Trials Group phase III clinical trials BR21 (EGFR tyrosine kinase inhibitor erlotinib versus placebo) and BR18 (carboplatin and paclitaxel with or without the metalloproteinase inhibitor BMS275291). In BR18, use of statin did not affect OS or DSS. In BR21, patients showed a trend for improvement in OS (HR: 0.69, P=0.098) and DSS (HR: 0.62, P=0.048), but there was no statin x treatment interaction effect (P=0.34 and P=0.51 for OS and DSS, respectively).

HMG-CoA reductase inhibitor lovastatin upregulates plasminogen activator production through RhoA-signaling in peritoneal cell line Met5A

This study was conducted to determine if hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor statin, known to protect postoperative adhesion in animal model, affect the expressing tissue-type plasminogen activator (tPA) in peritoneal cells in culture. Human peritoneal Met5A cells were used to examine the effects of hydrophobic statin lovastatin on the level of tPA. PA concentrations were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Active RhoA form was also examined. Lovastatin caused concentration-dependent tPA expression associated with fall of RhoA active level in Met5A cells. These lovastatin-induced changes were significantly overcome by the addition of geranylgeranyl pyrophosphate (intermediate of HMG-CoA pathway). A RhoA protein inhibitor C3 transferase mimicked the effects of lovastatin on the Met5A cells. These results suggest that lovastatin may be an effective stimulator of local peritoneal fibrinolytic activity, as it upregulates tPA expression in peritoneal Met5A cells through the reduction of RhoA geranylgeranylation. The extra-cholesterol lowering action of statin provides a new rationale to prevent peritoneal adhesion in postoperative patient.

Atorvastatin-induced cell toxicity in yeast is linked to disruption of protein isoprenylation

Statins, used to treat hypercholesterolemia, are one of the most frequently prescribed drug classes in the developed world. However, a significant proportion of users suffer symptoms of myotoxicity, and currently, the molecular mechanisms underlying myotoxicity remain ambiguous. In this study, Saccharomyces cerevisiae was exploited as a model system to gain further insight into the molecular mechanisms of atorvastatin toxicity. Atorvastatin-treated yeast cells display marked morphological deformities, have reduced cell viability and are highly vulnerable to perturbed mitochondrial function. Supplementation assays of atorvastatin-treated cells reveal that both loss of viability and mitochondrial dysfunction occur as a consequence of perturbation of the sterol synthesis pathway. This was further investigated by supplementing statin-treated cells with various metabolites of the sterol synthesis pathway that are believed to be essential for cell function. Ergosterol, coenzyme Q and a heme precursor were all ineffective in the prevention of statin-induced mitochondrial disruption and cell death. However, the addition of geranylgeranyl pyrophosphate and farnesyl pyrophosphate significantly restored cell viability, although these did not overcome petite induction. This highlights the pleiotropic nature of statin toxicity, but has established protein prenylation disruption as one of the principal mechanisms underlying statin-induced cell death in yeast.

Simvastatin suppresses LPS-induced MMP-1 expression in U937 mononuclear cells by inhibiting protein isoprenylation-mediated ERK activation.

Matrix metalloproteinase (MMP) plays a crucial role in periodontal disease and is up-regulated by oral Gram-negative, pathogen-derived LPS. In this study, we reported that simvastatin, a 3-hydroxyl-3-methylglutaryl-CoA reductase inhibitor, effectively inhibited LPS-stimulated MMP-1 as well as MMP-8 and MMP-9 expression by U937 mononuclear cells. Our studies showed that the geranylgeranyl transferase inhibitor inhibited LPS-stimulated MMP-1 expression, and addition of isoprenoid intermediate geranylgeranyl pyrophosphate (GGPP) reduced the inhibitory effect of simvastatin on LPS-stimulated MMP-1 expression. We also demonstrated that simvastatin inhibited the activation of Ras and Rac, and the inhibition was abolished by addition of GGPP. The above results indicate that protein isoprenylation is involved in the regulation of MMP-1 expression by LPS and simvastatin. Moreover, we showed that simvastatin inhibited LPS-stimulated nuclear AP-1, but not NF-kappaB activity, and the inhibition was reversed by addition of GGPP. Simvastatin also inhibited LPS-stimulated ERK but not p38 MAPK and JNK. Finally, we showed that the inhibition of LPS-stimulated ERK activation by simvastatin was reversed by GGPP. Taken together, this study showed that simvastatin suppresses LPS-induced MMP-1 expression in U937 mononuclear cells by targeting protein isoprenylation-mediated ERK activation.

HMG-CoA reductase inhibitor simvastatin overcomes bortezomib-induced apoptosis resistance by disrupting a geranylgeranyl pyrophosphate-dependent survival pathway

Simvastatin is a competitive inhibitor of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway required for the biosynthesis of cholesterol and higher isoprenoids such as geranylgeranyl pyrophosphate (GGPP). Apart from its capacity to lower cholesterol plasma levels and to protect against cardiovascular disease, simvastatin induces apoptosis in various cancer cells. We have generated human Namalwa Burkitt lymphoma cells that display general apoptosis resistance and hyperproliferation due to increased expression and proteolytic activity of 26S proteasomes in response to continuous treatment of the cells with the proteasome inhibitor bortezomib. In these cells, simvastatin does not inhibit proteasome activity, but induces apoptosis, G2/M cell cycle arrest and accumulation of p21 Waf1/Cip1, and effectively inhibits hyperproliferation. These effects are reversed by the addition of GGPP. GGPP-dependent plasma membrane localization of the small GTPase RhoA that is required for RhoA-mediated oncogenic signaling is completely inhibited by simvastatin. Finally, bortezomib but not simvastatin induces accumulation and stabilization of the anti-apoptotic protein Mcl-1, which is known to confer resistance to apoptosis in cancer cells. Thus, simvastatin overcomes bortezomib-induced apoptosis resistance by inhibiting synthesis of GGPP and disrupting a GGPP-dependent survival pathway.

Simvastatin inhibits growth via apoptosis and the induction of cell cycle arrest in human melanoma cells

Competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (the statins) that inhibit the synthesis of mevalonic acid are in wide use for treatment of hypercholesterolemia. Although antitumor effects on a variety of cell types have been reported for statins, the effect of simvastatin (one of the statins) on human melanoma cell lines is not known. Here, we report antitumor effects of simvastatin on human melanoma cell lines. We treated human melanoma cell lines, A375M, G361, C8161, GAK, and MMAc with simvastatin in various concentrations for 1 to 3 days. To investigate the antitumor effect of simvastatin, we analyzed cell viability, morphologic changes, reversibility of inhibition by geranylgeranyl pyrophosphate and farnesyl pyrophosphate, apoptosis and the cell cycle. Simvastatin treatment reduced cell viability in all five melanoma cell lines. The different melanoma cell lines, however, displayed different sensitivities to simvastatin. The addition of geranylgeranyl pyrophosphate to A375M and G361 cells in the presence of simvastatin completely restored the viability of cells, but the addition of farnesyl pyrophosphate did not. DNA fragmentation assay showed that simvastatin induced apoptosis in A375M and G361 cells. Simvastatin caused a G1 arrest in G361 and MMAc cells. Consistent with the cell cycle arrest, simvastatin caused an increase in the mRNA levels of p21 and p27 on G361 and MMAc cells. We conclude that simvastatin has an antitumor effect on human melanoma cells in vitro via apoptosis and cell cycle arrest. These results suggest that simvastatin may be an effective anticancer drug for malignant melanoma.

Manipulation of the mevalonate pathway in glial‐derived cells

Intermediates in the mevalonate pathway are critical for cell growth and survival. Statin‐induced inhibition of hydroxymethylglutaryl coenzyme A reductase prevents the formation of mevalonate and its derivatives, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). These isoprenoids post‐translationally modify signal transduction proteins. Glial (C6) and glioblastoma (A172) cells were incubated with 0.1 – 10 μM lovastatin without and with 10 μM mevalonate, FPP, or GGPP for 24 hours. Lovastatin alters RAS expression in A172, but not C6 cells. In both lines unmodified RAP1A does accumulate in a dose‐dependent manner from 1 – 10 μM lovastatin. The addition of GGPP, but not FPP, prevents this effect. Rheb protein levels increase with 10 μM lovastatin. This increase is enhanced with 10 μM GGPP. Selective inhibition of geranylgeranyl (10 μM GGTI‐286), but not farnesyl protein transferase (10 μM FTI‐277) results in an upregulation of Rheb in A172 but not C6 cells. The basis for these differences may lie in distinct regulation for the mevalonate pathway between these cell lines. Ongoing investigations will determine this regulatory difference.

Statin-induced inhibition of HIV-1 release from latently infected U1 cells reveals a critical role for protein prenylation in HIV-1 replication

Latent infection of human immunodeficiency virus type 1 (HIV-1) represents a major hurdle in the treatment of acquired immunodeficiency syndrome (AIDS) patients. Statins were recently reported to suppress acute HIV-1 infection and reduce infectious virion production, but the precise mechanism of inhibition has remained elusive. Here we demonstrate that lypophilic statins suppress HIV-1 virion release from tumor necrosis factor alpha-stimulated latently infected U1 cells through inhibition of protein geranylgeranylation, but not by cholesterol depletion. Indeed, this suppression was reversed by the addition of geranylgeranylpyrophosphate, and a geranylgeranyltransferase-1 inhibitor reduced HIV-1 production. Notably, silencing of the endogenous Rab11a GTPase expression in U1 cells by RNA interference destabilized Gag and reduced virion production both in vitro and in NOD/SCID/γcnull mice. Our findings thus suggest that small GTPase proteins play an important role in HIV-1 replication, and therefore could be attractive molecular targets for anti-HIV-1 therapy.

Fluvastatin Induces Apoptosis on Human Tongue Carcinoma Cell Line HSC-3

Statins, which are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, suppress cell proliferation and induce apoptosis in various cancer cell lines. However, the effects of statins in head and neck carcinoma have not been reported. In this study, we investigated the mechanism by which fluvastatin induces apoptosis in HSC-3 cells. An increase in caspase-3 activity was observed. The apoptosis induced by fluvastatin was inhibited by the addition of geranylgeranyl pyrophosphate (GGPP) to the cell culture. When we examined the survival signals at the time of apoptotic induction, we also found that fluvastatin had caused a remarkable decrease in the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Moreover, we also found that U0126, a MEK1/2 inhibitor, induces apoptosis in HSC-3 cells. These results suggested that fluvastatin induces apoptosis by inhibiting GGPP biosynthesis and consequently decreasing the level of phosphorylated ERK1/2. The results of this study also indicate that fluvastatin may be used as an anticancer agent for tongue carcinoma.

Simvastatin Suppresses Self‐Renewal of Mouse Embryonic Stem Cells by Inhibiting RhoA Geranylgeranylation

Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, were originally developed to lower cholesterol. Their pleiotropic (or cholesterol-independent) effects at the cellular and molecular levels are highly related to numerous cellular functions, such as proliferation and differentiation. However, they are hardly studied in embryonic stem cells. In this study, we evaluated the effects of statins on mouse ESCs (J1, D3, and RW.4) to enhance our understanding of the molecular basis of ESC self-renewal. Treatment of ESCs with simvastatin, mevastatin, atorvastatin, or pravastatin induced morphological change and decreased cell proliferation. We observed that the use of simvastatin was most effective in all three ESCs. Loss of ESC self-renewal by simvastatin was determined by marked downregulation of ESC markers alkaline phosphatase, Oct4, Nanog, Rex-1, and SSEA-1. Simvastatin effects were selectively reversed by either mevalonate or its metabolite geranylgeranyl pyrophosphate (GGPP) but not by cholesterol or farnesyl pyrophosphate. These results suggest that simvastatin effects were mainly derived from depletion of intracellular pools of GGPP, the substrate required for the geranylgeranylation. Using this approach, we found that GGPP, a derivative of the mevalonate pathway, is critical for ESC self-renewal. Furthermore, we identified that simvastatin selectively blocked cytosol-to-membrane translocalization of RhoA small guanosine triphosphate-binding protein, known to be the major target for geranylgeranylation, and lowered the levels of Rho-kinase (ROCK)2 protein in ESCs. In addition, simvastatin downregulated the ROCK activity, and this effect was reversed by addition of GGPP. Our data suggest that simvastatin, independently of its cholesterol-lowering properties, impairs the ESC self-renewal by modulating RhoA/ROCK-dependent cell-signaling.

Cardioprotective effect of rosuvastatin in vivo is dependent on inhibition of geranylgeranyl pyrophosphate and altered RhoA membrane translocation

Hydroxymethyl glutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) protect the myocardium against ischemia-reperfusion injury via a mechanism unrelated to cholesterol lowering. Statins may inhibit isoprenylation and thereby prevent activation of proteins such as RhoA. We hypothesized that statins protect the myocardium against ischemia-reperfusion injury via a mechanism involving inhibition of geranylgeranyl pyrophosphate synthesis and translocation of RhoA to the plasma membrane. Sprague-Dawley rats were given either the HMG-CoA reductase inhibitor rosuvastatin, geranylgeranyl pyrophosphate dissolved in methanol, the combination of rosuvastatin and geranylgeranyl pyrophosphate, rosuvastatin and methanol, or distilled water (control) by intraperitoneal injection for 48 h before ischemia-reperfusion. Animals were anesthetized and either subjected to 30 min of coronary artery occlusion followed by 2 h of reperfusion where at infarct size was determined, or the expression of RhoA protein was determined in cytosolic and membrane fractions of nonischemic myocardium. There were no significant differences in hemodynamics between the control group and the other groups before ischemia or during ischemia and reperfusion. The infarct size was 80 +/- 3% of the area at risk in the control group. Rosuvastatin reduced infarct size to 64 +/- 2% (P<0.001 vs. control). Addition of geranylgeranyl pyrophosphate (77 +/- 2%, P<0.01 vs. rosuvastatin) but not methanol (65 +/- 2%, not significant vs. rosuvastatin) abolished the cardioprotective effect of rosuvastatin. Geranylgeranyl pyrophosphate alone did not affect infarct size per se (84 +/- 2%). Rosuvastatin increased the cytosol-to-membrane ratio of RhoA protein in the myocardium (P<0.05 vs. control). These changes were abolished by addition of geranylgeranyl pyrophosphate. We conclude that the cardioprotection and the increase of the RhoA cytosol-to-membrane ratio induced by rosuvastatin in vivo are blocked by geranylgeranyl pyrophosphate. The inhibition of geranylgeranyl pyrophosphate formation and subsequent modulation of cytosol/membrane-bound RhoA are of importance for the protective effect of statins against myocardial ischemia-reperfusion injury.

A novel mechanism of action for statins against diabetes-induced oxidative stress

Atorvastatin exerts beneficial vascular effects in diabetes, but the underlying mechanisms are yet to be elucidated. The aim of the present study was to determine whether Rac-1 is involved in the effect of atorvastatin on oxidative stress and vascular dysfunction. Using human aortic endothelial cells (HAECs) we evaluated the effect of high glucose levels on peroxide production by dihydrodichlorofluorescein and on Rac-1 activity using immunocytochemistry to detect Rac-1 translocation to the membrane. We evaluated vascular function, peroxide production by dihydroethidium and NADPH oxidase activity in vessels from atorvastatin-treated mice. Rac-1 activity was also assessed, both by immunoprecipitation of the Rac-p21-activated kinase complex and by analysis of Rac-1 translocation to the membrane. These experiments were also conducted in vessels infected with an adenoviral vector carrying a constitutively active mutant of Rac-1. In HAECs exposed to high glucose levels, atorvastatin prevented oxidative stress, and this protection was associated with impaired Rac-1 activation. This effect was also observed in a murine model of diabetes mellitus. More importantly, the addition of geranylgeranyl pyrophosphate (GGPP) blocked the effects of atorvastatin in both glucose-exposed HAECs and diabetic vessels. Atorvastatin failed to afford protection against vascular abnormalities in the presence of a constitutively active mutant of Rac-1. The results of this study demonstrate that the vascular antioxidant effect of atorvastatin in diabetes is mediated through inhibition of Rac-1 via a reduction in GGPP. Thus, selective Rac-1 inhibition should be considered in the design of novel pharmacological strategies to reduce the impact of diabetes mellitus on vascular function.

Lovastatin-induced RhoA modulation and its effect on senescence in prostate cancer cells

Lovastatin inhibits a 3-hydroxy 3-methylglutaryl coenzyme A reductase and prevents the synthesis of cholesterol precursors, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), responsible for important cell signaling in cell proliferation and migration. Recently, the anti-cancer effect of lovastatin has been suggested in various tumor types. In this study, we showed that a low dose lovastatin induced senescence and G1 cell cycle arrest in human prostate cancer cells. Addition of GGPP or mevalonate, but not FPP, prevented the lovastatin-induced G1 phase cell cycle arrest and cell senescence. We found that constitutively active RhoA (caRhoA) reversed lovastatin-induced senescence in caRhoA-transfected PC-3 cells. Thus, we postulate that modulation of RhoA may be critical in lovastatin-induced senescence in PC-3 cells.