Geranylgeranyl pyrophosphate-mediated protein geranylgeranylation regulates endothelial cell proliferation and apoptosis during vasculogenesis in mouse embryo.

Vascular development is essential for the establishment of the circulatory system during embryonic development and requires the proliferation of endothelial cells. However, the underpinning regulatory mechanisms are not well understood. Here, we report that geranylgeranyl pyrophosphate (GGPP), a metabolite involved in protein geranylgeranylation, plays an indispensable role in embryonic vascular development. GGPP is synthesized by geranylgeranyl pyrophosphate synthase (GGPPS) in the mevalonate pathway. The selective knockout of Ggpps in endothelial cells led to aberrant vascular development and embryonic lethality, resulting from the decreased proliferation and enhanced apoptosis of endothelial cells during vasculogenesis. The defect in protein geranylgeranylation induced by GGPP depletion inhibited the membrane localization of RhoA and enhanced yes-associated protein (YAP) phosphorylation, thereby prohibiting the entry of YAP into the nucleus and the expression of YAP target genes related to cell proliferation and the antiapoptosis process. Moreover, inhibition of the mevalonate pathway by simvastatin induced endothelial cell proliferation defects and apoptosis, which were ameliorated by GGPP. Geranylgeraniol (GGOH), a precursor of GGPP, ameliorated the harmful effects of simvastatin on vascular development of developing fetuses in pregnant mice. These results indicate that GGPP-mediated protein geranylgeranylation is essential for endothelial cell proliferation and the antiapoptosis process during embryonic vascular development.

Farnesyl pyrophosphate is a new danger signal inducing acute cell death.

Cell death is a vital event in life. Infections and injuries cause lytic cell death, which gives rise to danger signals that can further induce cell death, inflammation, and tissue damage. The mevalonate (MVA) pathway is an essential, highly conserved and dynamic metabolic pathway. Here, we discover that farnesyl pyrophosphate (FPP), a metabolic intermediate of the MVA pathway, functions as a newly identified danger signal to trigger acute cell death leading to neuron loss in stroke. Harboring both a hydrophobic 15-carbon isoprenyl chain and a heavily charged pyrophosphate head, FPP leads to acute cell death independent of its downstream metabolic pathways. Mechanistically, extracellular calcium influx and the cation channel transient receptor potential melastatin 2 (TRPM2) exhibit essential roles in FPP-induced cell death. FPP activates TRPM2 opening for ion influx. Furthermore, in terms of a mouse model constructing by middle cerebral artery occlusion (MCAO), FPP accumulates in the brain, which indicates the function of the FPP and TRPM2 danger signal axis in ischemic injury. Overall, our data have revealed a novel function of the MVA pathway intermediate metabolite FPP as a danger signal via transient receptor potential cation channels.

New Approaches to the Prevention and Treatment of Viral Diseases.

The review discusses a new approach to the prevention and treatment of viral infections based on the use of pine needles polyprenyl phosphate (PPP) and associated with the infringement of prenylation process-the attachment of farnesol or geranyl geraniol to the viral protein. Currently, prenylation has been detected in type 1 adenovirus, hepatitis C virus, several herpes viruses, influenza virus, HIV. However, this list is far from complete, given that prenylated proteins play an extremely important role in the activity of the virus. We assume that the interferon produced in response to PPP may suppress expression of the SREBP2 transcription factor. As a result, the mevalonic acid pathway is violated and, as a result, the formation of early polyprenols precursors (geraniol, geranyl geraniol, farnesol), which are necessary for the prenylation of viral proteins, is blocked and the formation of mature, virulent virus particles is broken. As a consequence, the maturation of viral particles is inhibited, and defective particles are formed. Polyprenol was extracted from greenery (pine, fir and spruce needles, mulberry leaves, etc.), purified by chromatography, phosphorylated and identified by HPLC and NMR. Obtained PPP was used as antiviral in some experimental models in vitro and in vivo. During numerous studies, it was found that PPP manifested versatile antiviral effects, both in vitro and in vivo. The maximum effect was observed with viruses in which the presence of prenylated proteins was established, namely influenza A virus, HIV-1, tick-borne encephalitis virus, hepatitis A and C viruses, herpes simplex viruses type 1 and 2, some coronavirus. The available data obtained both in the experimental conditions and during clinical trials allow us to regard PPPs as safe and effective medicine for prevention and treatment of viral diseases.

Statins induce skeletal muscle atrophy via GGPP depletion-dependent myostatin overexpression in skeletal muscle and brown adipose tissue.

Myopathy is the major adverse effect of statins. However, the underlying mechanism of statin-induced skeletal muscle atrophy, one of statin-induced myopathy, remains to be elucidated. Myostatin is a negative regulator of skeletal muscle mass and functions. Whether myostatin is involved in statin-induced skeletal muscle atrophy remains unknown. In this study, we uncovered that simvastatin administration increased serum myostatin levels in mice. Inhibition of myostatin with follistatin, an antagonist of myostatin, improved simvastatin-induced skeletal muscle atrophy. Simvastatin induced myostatin expression not only in skeletal muscle but also in brown adipose tissue (BAT). Mechanistically, simvastatin inhibited the phosphorylation of forkhead box protein O1 (FOXO1) in C2C12 myotubes, promoting the nuclear translocation of FOXO1 and thereby stimulating the transcription of myostatin. In differentiated brown adipocytes, simvastatin promoted myostatin expression mainly by inhibiting the expression of interferon regulatory factor 4 (IRF4). Moreover, the stimulative effect of simvastatin on myostatin expression was blunted by geranylgeranyl diphosphate (GGPP) supplementation in both myotubes and brown adipocytes, suggesting that GGPP depletion was attributed to simvastatin-induced myostatin expression. Besides, the capacities of statins on stimulating myostatin expression were positively correlated with the lipophilicity of statins. Our findings provide new insights into statin-induced skeletal muscle atrophy. Graphical headlights 1. Simvastatin induces skeletal muscle atrophy via increasing serum myostatin levels in mice; 2. Simvastatin promotes myostatin expression in both skeletal muscle and brown adipose tissue through inhibiting GGPP production; 3. The stimulating effect of statins on myostatin expression is positively correlated with the lipophilicity of statins.

The mevalonate pathway is an actionable vulnerability of t(4;14)-positive multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy that is often driven by chromosomal translocations. In particular, patients with t(4;14)-positive disease have worse prognosis compared to other MM subtypes. Herein, we demonstrated that t(4;14)-positive cells are highly dependent on the mevalonate (MVA) pathway for survival. Moreover, we showed that this metabolic vulnerability is immediately actionable, as inhibiting the MVA pathway with a statin preferentially induced apoptosis in t(4;14)-positive cells. In response to statin treatment, t(4;14)-positive cells activated the integrated stress response (ISR), which was augmented by co-treatment with bortezomib, a proteasome inhibitor. We identified that t(4;14)-positive cells depend on the MVA pathway for the synthesis of geranylgeranyl pyrophosphate (GGPP), as exogenous GGPP fully rescued statin-induced ISR activation and apoptosis. Inhibiting protein geranylgeranylation similarly induced the ISR in t(4;14)-positive cells, suggesting that this subtype of MM depends on GGPP, at least in part, for protein geranylgeranylation. Notably, fluvastatin treatment synergized with bortezomib to induce apoptosis in t(4;14)-positive cells and potentiated the anti-tumor activity of bortezomib in vivo. Our data implicate the t(4;14) translocation as a biomarker of statin sensitivity and warrant further clinical evaluation of a statin in combination with bortezomib for the treatment of t(4;14)-positive disease.

Human Diploid Fibroblast Cell Lines - a Model System for Studying Immunodulatory Properties of Modern Immunobiological Drugs and Viruses.

The immunomodulatory properties of immunobiological drugs Glutoxim and Phosprenyl we well as vesicular stomatitis virus and inactivated tick-borne encephalitis vaccine virus were studied using human diploid fibroblast cell line from the collection of M. P. Chumakov Federal Research Center for Research and Development of Immunobiological Products. All tested preparations exhibited immunomodulatory activity in human diploid fibroblast cell line. Glutoxim in doses of 0.1 and 0.25 µg/ml stimulated production of IL-6 and IL-10 during 24-48 h of culturing, but did not stimulate production of IL-1ß. Phosprenyl, on the contrary, increased production of IL-1ß and the levels of IL-6 and IL-10. Vesicular stomatitis virus stimulated the production of IL-1ß, IL-6, and IL-10, while inactivated tick-borne encephalitis vaccine virus stimulated the production of cytokines IL-8 and IL-18. Immunomodulatory activity of inactivated tick-borne encephalitis vaccine virus was first demonstrated in the in vitro system.

Anti-tumor effects of mevalonate pathway inhibition in ovarian cancer.

BACKGROUND: Ovarian cancer remains the most fatal gynecological malignancy. Current therapeutic options are limited due to late diagnosis in the majority of the cases, metastatic spread to the peritoneal cavity and the onset of chemo-resistance. Thus, novel therapeutic approaches are required. Statins and amino-bisphosphonates are inhibitors of the mevalonate pathway, which is a fundamental pathway of cellular metabolism, essential for cholesterol production and posttranslational protein farnesylation and geranylgeranylation. While this pathway has emerged as a promising treatment target in several human malignancies, its potential as a therapeutic approach in ovarian cancer is still not fully understood. METHODS: Human ovarian cancer cell lines (IGROV-1, A2780, A2780cis) were treated with increasing concentrations (0.5-100 µM) of statins (simvastatin, atorvastatin, rosuvastatin) and zoledronic acid. Effects on cell vitality and apoptosis were assessed using Cell Titer Blue®, Caspase 3/7 Glo®, clonogenic assays as well as cleaved poly (ADP-ribose) polymerase (cPARP) detection. The inhibition of the mevalonate pathway was confirmed using Western Blot of unprenylated Ras and Rap1a proteins. Quantitative real-time PCR and ELISA were used to analyze modulations on several key regulators of ovarian cancer tumorigenesis. RESULTS: The treatment of IGROV-1 and A2780 cells with statins and zoledronic acid reduced vitality (by up to 80%; p < 0.001) and induced apoptosis by up to 8-folds (p < 0.001) in a dose-dependent fashion. Rescue experiments using farnesyl pyrophosphate or geranylgeranyl pyrophosphate evidenced that blocked geranylgeranylation is the major underlying mechanism of the pro-apoptotic effects. Gene expression of the tumor-promoting cytokines and mediators, such as transforming growth factor (TGF)-ß1, vascular endothelial growth factor (VEGF), interleukin (IL)-8, and IL-6 were significantly suppressed by statins and zoledronic acid by up to 90% (p < 0.001). For all readouts, simvastatin was most potent of all agents used. Cisplatin-resistant A2780cis cells showed a relative resistance to statins and zoledronic acid. However, similar to the effects in A2780 cells, simvastatin and zoledronic acid significantly induced caspase 3/7 activation (6-folds; p < 0.001). CONCLUSION: Our in vitro findings point to promising anti-tumor effects of statins and zoledronic acid in ovarian cancer and warrant additional validation in preclinical and clinical settings.

The glucocorticoid-induced leucine zipper mediates statin-induced muscle damage.

Statins, the most prescribed class of drugs for the treatment of hypercholesterolemia, can cause muscle-related adverse effects. It has been shown that the glucocorticoid-induced leucine zipper (GILZ) plays a key role in the anti-myogenic action of dexamethasone. In the present study, we aimed to evaluate the role of GILZ in statin-induced myopathy. Statins induced GILZ expression in C2C12 cells, primary murine myoblasts/myotubes, primary human myoblasts, and in vivo in zebrafish embryos and human quadriceps femoris muscle. Gilz induction was mediated by FOXO3 activation and binding to the Gilz promoter, and could be reversed by the addition of geranylgeranyl, but not farnesyl, pyrophosphate. Atorvastatin decreased Akt phosphorylation and increased cleaved caspase-3 levels in myoblasts. This effect was reversed in myoblasts from GILZ knockout mice. Similarly, myofibers isolated from knockout animals were more resistant toward statin-induced cell death than their wild-type counterparts. Statins also impaired myoblast differentiation, and this effect was accompanied by GILZ induction. The in vivo relevance of our findings was supported by the observation that gilz overexpression in zebrafish embryos led to impaired embryonic muscle development. Taken together, our data point toward GILZ as an essential mediator of the molecular mechanisms leading to statin-induced muscle damage.

Atorvastatin Targets the Islet Mevalonate Pathway to Dysregulate mTOR Signaling and Reduce ß-Cell Functional Mass.

Statins are cholesterol-lowering agents that increase the incidence of diabetes and impair glucose tolerance via their detrimental effects on nonhepatic tissues, such as pancreatic islets, but the underlying mechanism has not been determined. In atorvastatin (ator)-treated high-fat diet-fed mice, we found reduced pancreatic ß-cell size and ß-cell mass, fewer mature insulin granules, and reduced insulin secretion and glucose tolerance. Transcriptome profiling of primary pancreatic islets showed that ator inhibited the expression of pancreatic transcription factor, mechanistic target of rapamycin (mTOR) signaling, and small G protein (sGP) genes. Supplementation of the mevalonate pathway intermediate geranylgeranyl pyrophosphate (GGPP), which is produced by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, significantly restored the attenuated mTOR activity, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) expression, and ß-cell function after ator, lovastatin, rosuvastatin, and fluvastatin treatment; this effect was potentially mediated by sGP prenylation. Rab5a, the sGP in pancreatic islets most affected by ator treatment, was found to positively regulate mTOR signaling and ß-cell function. Rab5a knockdown mimicked the effect of ator treatment on ß-cells. Thus, ator impairs ß-cell function by regulating sGPs, for example, Rab5a, which subsequently attenuates islet mTOR signaling and reduces functional ß-cell mass. GGPP supplementation could constitute a new approach for preventing statin-induced hyperglycemia.

The novel high-frequency variant of TRPV3 p.A628T in East Asians showing faster sensitization in response to chemical agonists.

TRPV3, a member of the thermosensitive Ca2+-permeable TRPV channel subfamily expressed in skin and sensory nerves, is also activated by chemical agonists such as 2-aminoethyl diphenylborinate (2-APB). Repetitive stimuli induce sensitization of TRPV3 activation, characterized by the cumulative increase in current amplitude and linearization of current-voltage relation (I/V curve). Through genomic analysis of various populations, we found non-rare TRPV3 mutation (p.A628T) in East Asian people with an allele frequency of 0.249 while 0.007 in Caucasian. Slope conductance of unitary channel was not different between WT and p.A628T. Whole-cell patch clamp study of wildtype TRPV3 (WT) and p.A628T overexpressed in HEK293T cells showed similar sensitization by the repetitive increase in temperature from 23 to 37 °C, while slightly higher sensitization to 43 °C in p.A628T. In contrast, the repetitive application of 2-APB (10 µM) or carvacrol (100 µM) induced faster sensitization in p.A628T than WT. However, 1 µM farnesyl pyrophosphate, an intrinsic lipid metabolite agonist, induced similar level of slow activations in WT and p.A628T. In Fura-2 microspectrofluorimetry, the 2-APB pulses induced a faster increase of [Ca2+]c in p.A628T than WT. In terms of ionic selectivity of channels, WT and p.A628T showed similar Ca2+ permeability (PCa/PNa) calculated from the reversal potential of I/V curves. Taken together, p.A628T shows faster sensitization to chemical agonists that are reflected as higher [Ca2+]c signaling. Based on the intriguing pharmacological sensitivity, the physiological implications of p.A628T in the East Asian population require further investigation.

Fatty acid desaturase 2 is up-regulated by the treatment with statin through geranylgeranyl pyrophosphate-dependent Rho kinase pathway in HepG2 cells.

Statins have been reported to increase the plasma concentration of arachidonic acid (AA), an omega-6 long chain polyunsaturated fatty acid (LCPUFA) in several clinical studies indicating that statins affect the endogenous synthesis of LCUFAs. In the present study, we investigated the roles of the intrinsic mevalonate cascade and Rho-dependent pathway in LCPUFA synthesis, especially focusing on fatty acid desaturases (Fads) 2, using the human hepatocellular carcinoma cell line HepG2. Cell number and the activity of caspase-3 and 7 (caspase-3/7) was measured using a commercial kit. Gene expression was analyzed by quantitative real-time PCR. Protein expression was detected by Western blot analysis. Atorvastatin decreased cell viability and increased caspase-3/7 activity in a dose-dependent manner. At lower concentrations, atorvastatin stimulated both mRNA and protein expression of Fads2, and increased mRNA expression of FADS1 and ELVOL5. Both mevalonate and geranylgeranyl-pyrophosphate (GGPP), but not cholesterol, fully reversed atorvastatin-induced upregulation of Fads2, and mevalonate-effected reversal was inhibited by treatment with the Rho-associated protein kinase inhibitor Y-27632. These data clearly demonstrated that in human HepG2 cells, statins affect the endogenous synthesis of LCPUFAs by regulation of not only Fads2, but also Fads1 and Elovl5, through the GGPP-dependent Rho kinase pathway.

UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras.

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a key role in biosynthesis of vitamin K2 and coenzyme Q10 using geranylgeranyl diphosphate (GGPP). However, the mechanism by which UBIAD1 participates in tumorigenesis remains unknown. This study show that UBIAD1 interacts with H-Ras, retains H-Ras in the Golgi apparatus, prevents H-Ras trafficking from the Golgi apparatus to the plasma membrane, blocks the aberrant activation of Ras/MAPK signaling, and inhibits the proliferation of bladder cancer cells. In addition, GGPP was required to maintain the function of UBIAD1 in regulating the Ras/ERK signaling pathway. A Drosophila model was employed to confirm the function of UBIAD1/HEIX in vivo. The activation of Ras/ERK signaling at the plasma membrane induced melanotic masses in Drosophila larvae. Our study suggests that UBIAD1 serves as a tumor suppressor in cancer and tentatively reveals the underlying mechanism of melanotic mass formation in Drosophila.

Polyprenyl Phosphates Induce a High Humoral and Cellular Response to Immunization with Recombinant Proteins of the Replicative Complex of the Hepatitis C Virus.

The search for new adjuvants remains the critical task for the creation of hepatitis C vaccines due to the weak immunogenicity of biotechnological products. When immunizing mice with the recombinant proteins NS3 and NS5B of the hepatitis C virus (HCV), the adjuvant activity of three immunomodulators was compared. Phosprenyl® on the basis of polyprenyl phosphate (PPP), chemically synthesized analogue of the bacterial cell wall glucosaminyl muramyl dipeptide (GMDP), and IFN-α recombinant protein were tested. GMDP increased the activity of IgG1 antibodies 4-6 times but did not stimulate the production of IFN-γ; IFN-α has not shown any adjuvant properties. The introduction of recombinant HCV proteins together with PPP in low doses increased the activity of IgG2a isotype antibodies 4-7 times and increased IFN-γ secretion 3 times. Thus, it was first shown that PPP polarizes the immune response to Th1-type and is a promising adjuvant for the development of a vaccine against hepatitis C.

Inhibition of YAP reverses primary resistance to EGFR inhibitors in colorectal cancer cells.

Mutant KRAS and BRAF are associated with primary EGFR inhibitor resistance in colorectal cancer (CRC). However, other biomarkers that could predict EGFR inhibitor resistance remain elusive. In the present study, immunoblotting and cell proliferation results revealed that yes­associated protein (YAP), a downstream effector of the Hippo pathway, was positively associated with primary cetuximab resistance in CRC cells. YAP knockdown enhanced the cytotoxicity of cetuximab in CRC cells. Simvastatin, a 3­hydroxy­3­methylglutaryl­coenzyme A (HMG­CoA) reductase inhibitor of the mevalonate pathway that inhibits YAP bioactivity through nuclear translocation and total YAP expression, increased the cytotoxicity of EGFR inhibitors (cetuximab and gefitinib) against CRC cells. The combination of simvastatin and EGFR inhibitors inhibited YAP and EGFR signaling more markedly than each agent alone. Adding back geranylgeranyl pyrophosphate (GGPP), a key product of the mevalonate pathway, reversed the YAP bioactivity inhibition induced by simvastatin and the cell proliferation inhibition induced by the combination of simvastatin and EGFR inhibitors. Collectively, these results revealed that YAP may be useful in identifying cetuximab resistance in CRC and indicated that targeting of both YAP and EGFR signals may present a promising therapeutic approach for CRC.

Curcumin regulates endogenous and exogenous metabolism via Nrf2-FXR-LXR pathway in NAFLD mice.

BACKGROUND: Curcumin is a natural polyphenol with beneficial effects on NAFLD patients and NAFLD is accompanied by metabolism decompensation. METHODS: This study was focused on the effect of curcumin on the relationship between endogenous bile acids metabolism pathway and exogenous xenobiotics metabolism pathway in C57BL/6 mice of non-alcoholic fatty liver disease induced by high-fat and high-fructose diet (HFHFr) and in cultured mice hepatocytes. RESULTS: Our results showed curcumin treatment apparently attenuated the hepatic steatosis and reversed the abnormalities of serum biochemical parameters in HFHFr-fed mice. Curcumin effectively reversed the expression of CYP3A and CYP7A in fatty liver status to restore metabolism capability. In the meantime, lipid synthesis has been controlled by curcumin, evidenced by the expression of CD36, SREBP-1c and FAS. Further, FXR, SHP and Nrf2 expressions were remarkably dropped in HFHFr-fed mice and LXRα expression was significantly enhanced, while curcumin treatment was quite effective to restore this pathway. In addition, LXRα antagonist GGPP pretreatment weakened the curcumin effects on CYP3A, CYP7A and SREBP-1c. CONCLUSIONS: These findings indicate that the Nrf2/FXR/LXRα pathway might synergistically regulate both endogenous and exogenous metabolism in NAFLD mice and LXRα may be a novel therapeutic target of curcumin for the prevention and treatment of NAFLD.

Statins impact primary embryonic mouse neural stem cell survival, cell death, and fate through distinct mechanisms.

Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway (CBP), and are used for the prevention of cardiovascular disease. The anti-inflammatory effects of statins may also provide therapeutic benefits and have led to their use in clinical trials for preeclampsia, a pregnancy-associated inflammatory condition, despite their current classification as category X (i.e. contraindicated during pregnancy). In the developing neocortex, products of the CBP play essential roles in proliferation and differentiation of neural stem-progenitor cells (NSPCs). To understand how statins could impact the developing brain, we studied effects of pravastatin and simvastatin on primary embryonic NSPC survival, proliferation, global transcription, and cell fate in vitro. We found that statins dose dependently decrease NSPC expansion by promoting cell death and autophagy of NSPCs progressing through the G1 phase of the cell cycle. Genome-wide transcriptome analysis demonstrates an increase in expression of CBP genes following pravastatin treatment, through activation of the SREBP2 transcription factor. Co-treatment with farnesyl pyrophosphate (FPP), a CBP metabolite downstream of HMG-CoA reductase, reduces SREBP2 activation and pravastatin-induced PARP cleavage. Finally, pravastatin and simvastatin differentially alter NSPC cell fate and mRNA expression during differentiation, through a non-CBP dependent pathway.

Isoprenoids increase bovine endometrial stromal cell tolerance to the cholesterol-dependent cytolysin from Trueperella pyogenes.

Preventing postpartum uterine disease depends on the ability of endometrial cells to tolerate the presence of the bacteria that invade the uterus after parturition. Postpartum uterine disease and endometrial pathology in cattle are most associated with the pathogen Trueperella pyogenes. Trueperella pyogenes secretes a cholesterol-dependent cytolysin, pyolysin, which causes cytolysis by forming pores in the plasma membrane of endometrial stromal cells. The aim of the present study was to identify cell-intrinsic pathways that increase bovine endometrial stromal cell tolerance to pyolysin. Pyolysin caused dose-dependent cytolysis of bovine endometrial stromal cells and leakage of lactate dehydrogenase into supernatants. Cell tolerance to pyolysin was increased by inhibitors that target the mevalonate and cholesterol synthesis pathway, but not the mitogen-activated protein kinase, cell cycle, or metabolic pathways. Cellular cholesterol was reduced and cell tolerance to pyolysin was increased by supplying the mevalonate-derived isoprenoid farnesyl pyrophosphate, or by inhibiting farnesyl-diphosphate farnesyltransferase 1 or geranylgeranyl diphosphate synthase 1 to increase the abundance of farnesyl pyrophosphate. Supplying the mevalonate-derived isoprenoid geranylgeranyl pyrophosphate also increased cell tolerance to pyolysin, but independent of changes in cellular cholesterol. However, geranylgeranyl pyrophosphate inhibits nuclear receptor subfamily 1 group H receptors (NR1H, also known as liver X receptors), and reducing the expression of the genes encoding NR1H3 or NR1H2 increased stromal cell tolerance to pyolysin. In conclusion, mevalonate-derived isoprenoids increased bovine endometrial stromal cell tolerance to pyolysin, which was associated with reducing cellular cholesterol and inhibiting NR1H receptors.

Simvastatin exerts anticancer effects in osteosarcoma cell lines via geranylgeranylation and c-Jun activation.

Osteosarcoma is the leading primary bone cancer in young adults and exhibits high chemoresistance rates. Therefore, characterization of both alternative treatment options and the underlying mechanisms is essential. Simvastatin, a cholesterol-lowering drug, has among its pleiotropic effects anticancer potential. Characterizing this potential and the underlying mechanisms in osteosarcoma is the subject of the present study. Human osteosarcoma cells (SaOS-2 and U2OS) were treated with simvastatin (4-66 µM) for 48 or 72 h. The effects of downstream substrate mevalonate (MA) or substrates for isoprenylation farnesyl pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP) were evaluated using add-back experiments. Tumour growth using MTT assay, apoptosis, cell cycle and signalling cascades involved in simvastatin-induced manipulation were analysed. The results revealed that simvastatin dose-dependently inhibited cell growth. Simvastatin significantly induced apoptosis, increased the Bax/Bcl-2 ratio, and cleavage of caspase-3 and PARP protein. Simvastatin impaired cell cycle progression as shown by significantly increased percentages of cells in the G0/G1 phase and lower percentages of cells in the S phase. Gene expression levels of cell cycle-regulating genes (TP53, CDKN1A and CDK1) were markedly altered. These effects were not completely abolished by FPP, but were reversed by MA and GGPP. JNK and c-Jun phosphorylation was enhanced after simvastatin treatment, while those were abolished when either MA or GGPP were added. In conclusion, simvastatin acts primarily by reducing prenylation to induce apoptosis and reduce osteosarcoma cell growth. Particularly enhanced activation of c-Jun seems to play a pivotal role in osteosarcoma cell death.

The role of atorvastatin in suppressing tumor growth of uterine fibroids.

BACKGROUND: Medical therapeutic options remain quite limited for uterine fibroids treatment. Statins, competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, have anti-tumoral effects on multiple cancer types, however, little is known about their effects on uterine fibroids. METHODS: Initially, we conducted a retrospective study of 120 patients with uterine fibroids and hyperlipidemia from the Second Affiliated Hospital of Wenzhou Medical University. Then, we evaluated the effect of atorvastatin on proliferation and apoptosis both in immortalized uterine fibroids cells and primary uterine fibroids cells. Furthermore, the molecular mechanism by which atorvastatin suppressed uterine fibroids cell growth was explored. RESULTS: Our results showed that atorvastatin use for 1 or 2 years significantly suppressed growth of uterine fibroids. Atorvastatin inhibited the proliferation of immortalized and primary uterine fibroids cells in a dose and time-dependent manner and stimulated apoptosis of uterine fibroids cells by inducing caspase-3 activation, up-regulating Bim and down-regulating Bcl-2. Additionally, atorvastatin treatment suppressed phosphorylation of ERK1/2 and JNK. Furthermore, GGPP, a downstream lipid isoprenoid intermediate, significantly rescued the effect of atorvastatin. CONCLUSIONS: These results suggest that atorvastatin exerts anti-tumoral effects on uterine fibroids through inhibition of cell proliferation and induction of apoptosis in HMG-CoA-dependent pathway. Our results provide the first clinical and preclinical data on the use of atorvastatin as a promising nonsurgical treatment option for uterine fibroids.

Inhibition of the RhoA/Rho-associated, coiled-coil-containing protein kinase-1 pathway is involved in the therapeutic effects of simvastatin on pulmonary arterial hypertension.

BACKGROUND: Recent research has shown that statins improve pulmonary arterial hypertension (PAH), but their mechanisms of action are not fully understood. This study aimed to investigate the role of RhoA/ROCK1 regulation in the therapeutic effects of simvastatin on PAH. METHODS: For in vivo experiments, rats (N = 40) were randomly assigned to four groups: control, simvastatin, monocrotaline (MCT), and MCT + simvastatin. The MCT group and MCT + simvastatin groups received proline dithiocarbamate (50 mg/kg, i.p.) on the first day of the study. The MCT + simvastatin group received simvastatin (2 mg/kg) daily for 4 weeks, after which pulmonary arterial pressure was measured by right heart catheterization. The protein and mRNA levels of Rho and ROCK1 were measured by immunohistochemistry, Western blot, and PCR. For in vitro experiments, human pulmonary endothelial cells were divided into seven groups: control, simvastatin, monocrotaline pyrrole (MCTP), MCTP + simvastatin, MCTP + simvastatin + mevalonate, MCTP + simvastatin + farnesyl pyrophosphate (FPP), and MCTP + simvastatin + FPP + geranylgeranyl pyrophosphate (GGPP). After 72 h exposed to the drugs, the protein and mRNA levels of RhoA and ROCK1 were measured by Western blot and PCR. RESULTS: The MCT group showed increased mean pulmonary arterial pressure, marked vascular remodeling, and increased protein and mRNA levels of RhoA and ROCK1 compared to the other groups (P < 0.05). In vitro, the MCTP group showed a marked proliferation of vascular endothelial cells, as well as increased protein and mRNA levels of RhoA and ROCK1 compared to the MCTP + simvastatin group. The MCTP + simvastatin + mevalonate group, MCTP + simvastatin+ FPP group, and MCTP + simvastatin + FPP + GGPP group showed increased mRNA levels of RhoA and ROCK1, as well as increased protein levels of RhoA, compared to the MCTP + simvastatin group. CONCLUSIONS: Simvastatin improved vascular remodeling and inhibited the development of PAH. The effects of simvastatin were mediated by inhibition of RhoA/ROCK1. Simvastatin decreased RhoA/ROCK1 overexpression by inhibition of mevalonate, FPP, and GGPP synthesis.