Mevalonate Pyrophosphate Research Articles

Phosphomevalonate kinase deficiency expands the genetic spectrum of systemic autoinflammatory diseases

In the isoprenoid biosynthesis pathway, mevalonate is phosphorylated in two subsequent enzyme steps by mevalonate kinase (MVK) and phosphomevalonate kinase (PMVK) to generate mevalonate pyrophosphate that is further metabolized to produce sterol and non-sterol isoprenoids. Biallelic pathogenic variants in the MVK gene result in the autoinflammatory metabolic disorder MVK Deficiency (MKD). So far, however, no patients with proven PMVK deficiency due to biallelic pathogenic variants in the PMVK gene have been reported. This study aims to report the first patient with functionally confirmed PMVK deficiency, including the clinical, biochemical and immunological consequences of a homozygous missense variant in the PMVK gene. We performed whole-exome sequencing and functional studies in cells from a patient who, upon clinical and immunological evaluation, was suspected of an autoinflammatory disease. We identified a homozygous PMVK p.Val131Ala (NM_006556.4: c.392T>C,) missense variant in our patient. Pathogenicity was supported by genetic algorithms and modeling analysis and confirmed in patient cells that revealed a markedly reduced PMVK enzyme activity due to a virtually complete absence of PMVK protein. Clinically, the patient showed various similarities, but also distinct features compared to MKD patients, and responded well to therapeutic IL-1 inhibition. In this study we report the first patient with proven PMVK deficiency due to a homozygous missense variant in PMVK leading to an autoinflammatory disease. PMVK deficiency expands the genetic spectrum of systemic autoinflammatory diseases (SAID), characterized by recurrent fevers, arthritis and cytopenia and thus should be included in the differential diagnosis and genetic testing for SAIDs.

Transcriptome Analysis of Antrodia cinnamomea Mycelia from Different Wood Substrates

Antrodia cinnamomea, an edible and medicinal fungus with significant economic value and application prospects, is rich in terpenoids, benzenoids, lignans, polysaccharides, and benzoquinone, succinic and maleic derivatives. In this study, the transcriptome of A. cinnamomea cultured on the wood substrates of Cinnamomum glanduliferum (YZM), C. camphora (XZM), and C. kanehirae (NZM) was sequenced using the high-throughput sequencing technology Illumina HiSeq 2000, and the data were assembled by de novo strategy to obtain 78,729 Unigenes with an N50 of 4,463 bp. Compared with public databases, about 11,435, 6,947, and 5,994 Unigenes were annotated to the Non-Redundant (NR), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genome (KEGG), respectively. The comprehensive analysis of the mycelium terpene biosynthesis-related genes in A. cinnamomea revealed that the expression of acetyl-CoA acetyltransferase (AACT), acyl-CoA dehydrogenase (MCAD), 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA), mevalonate pyrophosphate decarboxylase (MVD), and isopentenyl diphosphate isomerase (IDI) was significantly higher on NZM compared to the other two wood substrates. Similarly, the expression of geranylgeranyltransferase (GGT) was significantly higher on YZM compared to NZM and XZM, and the expression of farnesyl transferase (FTase) was significantly higher on XZM. Furthermore, the expressions of 2,3-oxidized squalene cyclase (OCS), squalene synthase (SQS), and squalene epoxidase (SE) were significantly higher on NZM. Overall, this study provides a potential approach to explore the molecular regulation mechanism of terpenoid biosynthesis in A. cinnamomea.

The Effect of Methylation Modification of MDD on the Expression of Key Genes in the Process of Saponin Synthesis in Eleutherococcus senticosus

Mevalonate pyrophosphate decarboxylase is a kind of key enzyme in the terpenoid synthesis pathway in Eleutherococcus senticosus. The results of bisulfite sequencing showed that there were three kinds of samples with a low (0.68%), medium (0.72%) and high (0.79%) DNA methylation ratio in the promoter of MDD in E. senticosus, respectively. The transcriptome sequencing results showed that the expression of MDD in E. senticosus was significantly up-regulated in the types with low DNA methylation ratios of MDD (P < 0.05). There was a significant negative correlation between the saponin content in E. senticosus and the DNA methylation ratio of MDD promoter (P < 0.05). The screening results of differentially expressed transcription factors among the three groups with different DNA methylation ratio showed that a total of 4 transcription factors could bind to 6 CpG sites. Protein-protein interaction analysis showed that E. senticosus MDD could interacted with other key enzymes in the process of terpenoid synthesis. In addition, it was found that the DNA methylation of MDD promoter was mainly regulated by DNA methyltransferase. These results demonstrated that under the action of DNA methyltransferase, the changes of DNA methylation of MDD promoter could regulate its own expression level by affecting the combination ability of transcription factors, and then affected the expression of other genes in terpenoid synthesis pathway. The synthesis and accumulation of saponins in E. senticosus was also changed.

In silico/computational analysis of mevalonate pyrophosphate decarboxylase gene families in Campanulids.

Mevalonate pyrophosphate decarboxylase (MPD) is a key enzyme in terpenoid biosynthesis. MPD plays an important role in the upstream regulation of secondary plant metabolism. However, studies on the MPD gene are relatively very few despite its importance in plant metabolism. Currently, no systematic analysis has been conducted on the MPD gene in plants under the order Apiales, which comprises important medicinal plants such as Panax ginseng and Panax notoginseng. This study sought to explore the structural characteristics of the MPD gene and the effect of adaptive evolution on the gene by comparing and analyzing MPD gene sequences of different campanulids species. For that, phylogenetic and adaptive evolution analyses were carried out using sequences for 11 Campanulids species. MPD sequence characteristics of each species were then analyzed, and the collinearity analysis of the genes was performed. As a result, a total of 21 MPD proteins were identified in 11 Campanulids species through BLAST analysis. Phylogenetic analysis, physical and chemical properties prediction, gene family analysis, and gene structure prediction showed that the MPD gene has undergone purifying selection and exhibited highly conserved structure. Analysis of physicochemical properties further showed that the MPD protein was a hydrophilic protein without a transmembrane region. Moreover, collinearity analysis in Apiales showed that MPD gene on chromosome 2 of D. carota and chromosome 1 of C. sativum were collinear. The findings showed that MPD gene is highly conserved. This may be a common characteristic of all essential enzymes in the biosynthesis pathways of medicinal plants. Notably, MPD gene is significantly affected by environmental factors which subsequently modulate its expression. The current study’s findings provide a basis for follow-up studies on MPD gene and key enzymes in other medicinal plants.

Advanced Strategies for Production of Natural Products in Yeast.

Natural products account for more than 50% of all small-molecule pharmaceutical agents currently in clinical use. However, low availability often becomes problematic when a bioactive natural product is promising to become a pharmaceutical or leading compound. Advances in synthetic biology and metabolic engineering provide a feasible solution for sustainable supply of these compounds. In this review, we have summarized current progress in engineering yeast cell factories for production of natural products, including terpenoids, alkaloids, and phenylpropanoids. We then discuss advanced strategies in metabolic engineering at three different dimensions, including point, line, and plane (corresponding to the individual enzymes and cofactors, metabolic pathways, and the global cellular network). In particular, we comprehensively discuss how to engineer cofactor biosynthesis for enhancing the biosynthesis efficiency, other than the enzyme activity. Finally, current challenges and perspective are also discussed for future engineering direction.

Brain Regions with Reduced Amounts of Mevalonate Pyrophosphate Decarboxylase Correspond to Sites of Strokes in Stroke-Prone Spontaneously Hypertensive Rats

Brain Regions with Reduced Amounts of Mevalonate Pyrophosphate Decarboxylase Correspond to Sites of Strokes in Stroke-Prone Spontaneously Hypertensive Rats

Stroke-prone spontaneously hypertensive rats have reduced hydroxysteroid 17-β dehydrogenase 7 levels for low cholesterol biosynthesis.

Reduced serum cholesterol content was recently reported to be one of the factors responsible for cerebral haemorrhage. Stroke-prone spontaneously hypertensive rats (SHRSP) are known to have lower serum cholesterol content than normotensive Wistar-Kyoto rats (WKY). We previously reported that lower levels of mevalonate pyrophosphate decarboxylase (MPD) and squalene epoxidase (SQE), which are associated with cholesterol biosynthesis in the liver, are involved in the low serum cholesterol content in SHRSP. Here, we investigate the levels of sterol 14-demethylase (CYP51), methylsterol monooxygenase (SC4MOL), and hydroxysteroid 17-β dehydrogenase 7 (HSD17B7), which contribute to the cholesterol synthesis pathway in the conversion of lanosterol to zymosterol, in SHRSP and WKY. The HSD17B7 mRNA levels in the liver of SHRSP were markedly lower than those in WKY, whereas no significant differences were observed in CYP51 and SC4MOL levels in the two types of rats. The relative levels of protein, heteronuclear RNA, and mRNA of HSD17B7 were also significantly lower in SHRSP than in WKY. The degradation rates of HSD17B7 were the same in SHRSP and WKY. The protein levels of HSD17B7 were not significantly reduced in tissues other than the liver, including the brain, lung, heart, spleen, kidney, and testis, in SHRSP. Moreover, HSD17B7 activity was significantly lower in SHRSP than in WKY. Thus, our results indicated that low protein levels and activity of HSD17B7 are responsible for the reduced cholesterol content in SHRSP, indicating that HSD17B7, along with MPD and SQE, is involved in the decreased cholesterol synthesis in the liver of SHRSP.

Study of heterologous efficient synthesis of β-amyrin and high-density fermentation

In this study, the synthetic pathway of β-amyrin was constructed in the pre-constructed Saccharomyces cerevisiae chassis strain Y0 by introducing β-amyrin synthase from Glycyrrhiza uralensis, resulting strain Y1-C20-6, which successfully produced β-amyrin up to 5.97 mg·L~(-1). Then, the mevalonate pyrophosphate decarboxylase gene(ERG19), mevalonate kinase gene(ERG12), 3-hydroxy-3-methylglutaryl-CoA synthase gene(ERG13), phosphomevalonate kinase gene(ERG8) and IPP isomerase gene(IDI1)were overexpressed to promoted the metabolic fluxto the direction of β-amyrin synthesis for further improving β-amyrin production, resulting the strain Y2-C2-4 which produced β-amyrin of 10.3 mg·L~(-1)under the shake flask fermentation condition. This is 100% higher than that of strain Y1-C20-6, illustrating the positive effect of the metabolic engineering strategy applied in this study. The titer of β-amyrin was further improved up to 157.4 mg·L~(-1) in the fed-batch fermentation, which was almost 26 fold of that produced by strain Y1-C20-6. This study not only laid the foundation for the biosynthesis of β-amyrin but also provided a favorable chassis strain for elucidation of cytochrome oxidases and glycosyltransferases of β-amyrin-based triterpenoids.

An Adaptation To Life In Acid Through A Novel Mevalonate Pathway.

Extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previously identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments.

Abstract 4182: Statins induce apoptosis in osteosarcoma cells by activation of Ampk and p38-MAPK via suppression of mevalonate pathway

Abstract Osteosarcoma (OS) is the most common, non-hematopoietic, primary malignant bone tumor. OS is characterized by its aggressive local growth and systemic dissemination. Although combination of surgical operation and adjuvant chemotherapy greatly improved the prognosis, more than 20% of patients still cannot get long-term survival. Therefore, novel therapeutic approaches should be expected to be developed. Previously, we developed an OS mouse model by overexpressing c-MYC in bone marrow stromal cells derived from Ink4a/Arf knockout mice. We isolated highly tumorigenic cells (designated AXT cells) from tumors after serial transplantation. Inoculation of AXT cells into syngeneic C57BL/6 mice results in the development of lethal OS with metastatic lesions including lung, which pathologically and clinically mimics human osteoblastic osteosarcoma. To obtain the novel therapeutic agents for OS, we performed drug screening using existing drug collections and found that statins strongly suppressed AXT cell growth and migration in vitro. Simvastatin induces caspase dependent apoptosis, which can be completely rescued by the supplement of mevalonate and geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate, suggesting that OS cells are susceptible to the inhibition of the mevalonate pathway. As the responsible effectors, we found treatment of simvastatin drives RhoA-GTP translocation from cell membrane to cytosol as well as the disruption of interaction between RhoA and Rho-GDI, indicating that, in spite of the accumulation of RhoA-GTP in OS cells, RhoA cannot work properly, which appears to affect cell motility and anchorage dependent survival. As downstream signaling of RhoA, p38-MAPK and AMPK are strongly activated by simvastatin treatment. Activation of p38-MAPK is not accompanied by the ROS production. The specific inhibitor for p38-MAPK or AMPK can rescue this activation as well as the simvastatin-induced cell death, respectively, suggesting that activation of both kinases is responsible for the anti-tumor effect of simvastatin. A single treatment of statins attenuated OS tumor growth in vivo. These findings suggest that the activation of p38-MAPK and AMPK by statins become a potential therapeutic option for OS. Citation Format: Walied Abd-Elghani Kamel, Eiji Sugihara, Sayaka Iwai Yamaguchi, Hiroyuki Nobusue, Kenta Maki, Akihiro Muto, Hideyuki Saya, Takatsune Shimizu. Statins induce apoptosis in osteosarcoma cells by activation of Ampk and p38-MAPK via suppression of mevalonate pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4182.

BJ-1108, a 6-Amino-2,4,5-Trimethylpyridin-3-ol Analog, Inhibits Serotonin-Induced Angiogenesis and Tumor Growth through PI3K/NOX Pathway.

5-Hydroxytryptamine (5-HT) induces proliferation of cancer cells and vascular cells. In addition to 5-HT production by several cancer cells including gastrointestinal and breast cancer, a significant level of 5-HT is released from activated platelets in the thrombotic environment of tumors, suggesting that inhibition of 5-HT signaling may constitute a new target for antiangiogenic anticancer drug discovery. In the current study we clearly demonstrate that 5-HT-induced angiogenesis was mediated through the 5-HT1 receptor-linked Gβγ/Src/PI3K pathway, but not through the MAPK/ERK/p38 pathway. In addition, 5-HT induced production of NADPH oxidase (NOX)-derived reactive oxygen species (ROS). In an effort to develop new molecularly targeted anticancer agents against 5-HT action in tumor growth, we demonstrate that BJ-1108, a derivative of 6-amino-2,4,5-trimethylpyridin-3-ol, significantly inhibited 5-HT-induced angiogenesis. In addition, BJ-1108 induced a significant reduction in the size and weight of excised tumors in breast cancer cell-inoculated CAM assay, showing proportionate suppression of tumor growth along with inhibition of angiogenesis. In human umbilical vein endothelial cells (HUVECs), BJ-1108 significantly suppressed 5-HT-induced ROS generation and phosphorylation of PI3K/Akt but not of Src. Unlike NOX inhibitors, BJ-1108, which showed better antioxidant activity than vitamin C, barely suppressed superoxide anion induced by mevalonate or geranylgeranyl pyrophosphate which directly activates NOX without help from other signaling molecules in HUVECs, implying that the anti-angiogenic action of BJ-1108 was not mediated through direct action on NOX activation, or free radical scavenging activity. In conclusion, BJ-1108 inhibited 5-HT-induced angiogenesis through PI3K/NOX signaling but not through Src, ERK, or p38.

Molecular cloning and expression analysis of mevalonate pyrophosphate decarboxylase inAntrodia cinnamomea

Mevalonate pyrophosphate decarboxylase (MVD) plays important roles in triterpenoid biosynthesis via the mevalonate pathway. A novel MVD gene was isolated and identified in Antrodia cinnamomea (Ac-mvd). The full-length cDNA contained an open reading frame with a length of 1,209 bp and encoded a 402-amino acid polypeptide with a molecular mass of 43.3 kDa and a theoretical pI of 8.23. As incubation time was prolonged for 28 days, the triterpenoid content in the mycelium gradually increased and reached 39.192 ± 2.025 mg/g; the triterpenoid content of the fruiting body grew in the hay of Cinnamomum kanehirae (ACFB-CK), was 49.391 ± 2.675 mg/g, which was significantly different from other samples ( P Antrodia cinnamomea were detected on the 7th day. The expression levels gradually decreased as culture time was extended from 14 days to 42 days.

Involvement of microRNA214 and transcriptional regulation in reductions in mevalonate pyrophosphate decarboxylase mRNA levels in stroke-prone spontaneously hypertensive rat livers.

Hypocholesterolemia has been epidemiologically identified as one of the causes of stroke (cerebral hemorrhage). We previously reported that lower protein levels of mevalonate pyrophosphate decarboxylase (MPD), which is responsible for reducing serum cholesterol levels in stroke-prone spontaneously hypertensive rats (SHRSP), in the liver were caused by a reduction in mRNA levels. However, the mechanism responsible for reducing MPD expression levels in the SHRSP liver remains unclear. Thus, we compared microRNA (miR)-214 combined with the 3'-untranslated region of MPD mRNA and heterogeneous nuclear RNA (hnRNA) between SHRSP and normotensive Wistar Kyoto rats (WKY). miR-214 levels in the liver were markedly higher in SHRSP than in WKY, whereas hnRNA levels were significantly lower. These results indicate that the upregulation of miR-214 and downregulation of MPD transcription in the liver both play a role in the development of hypocholesterolemia in SHRSP.

English

Organophosphorus compounds (OPs) are widely used as pesticides and chemical warfare agents (CWAs). These synthetic compounds reside in the environment and cause serious pollution problems. Organophosphorus compounds can be detoxified rapidly by hydrolysis on exposure to environment, which are liable to be influenced by abiotic and biotic factors. Microorganisms isolated from different niches are the predominant entities in the biosphere with an exceptional ability to metabolize various OPs for their growth. The focus of this work is organophosphorus hydrolase (OPH, E.C. 8.1.3.1), which catalyzes the hydrolysis of many organophosphorus compounds and greatly reduces the toxicity and even can completely mineralize them. Several OPH enzymes, including o-phenylenediamine dihydrochloride (OPD), methyl parathion hydrolase (MPH) mevalonate pyrophosphate decarboxylase (MPD) etc, have been identified which accomplish hydrolysis for specific classes of organophosphorus compounds. The functional gene encoding OPH protein have been cloned, expressed and purified in both prokaryotic and eukaryocyte expression vector. To increase the enzyme activity or enhance their broad-spectrum property, the wild activated OPH was used to modify activated sites by the chemical modification of specific amino acid residues with the use of appropriately designed coenzyme analogs. The applications of the functional strains and OPH enzymes in bioremediation of OPs pollutants included: (i) Bioremediation: the technologies can be generally classified as in situ or ex situ; (ii) immobilization: an immobilized enzyme is the OPH that is attached to an inert, insoluble material such as calcium alginate and agar; immobilized whole cell, the target cell is the functional strains that is capable of degrading special OPs; (iii) construction of genetically engineering bacteria. A versatile genetically engineering bacterium that gained more functional genes from different sources was constructed by gene engineering and enzyme engineering, showing a creative and promising application in OPs detoxification. Key words: Organophosphorus compounds, biodegradation, bioremediation, oph, genetically engineering.

Statins suppress glucose-induced plasminogen activator inhibitor-1 expression by regulating RhoA and nuclear factor-kB activities in cardiac microvascular endothelial cells

The aim of this study was to investigate the possible proinflammatory signaling pathways involved in statin inhibition of glucose-induced plasminogen activator inhibitor-1 (PAI-1) expression in cardiac microvascular endothelial cells (CMECs). Primary rat CMECs were grown in the presence of 5.7 or 23 mmol/L glucose. PAI-1 mRNA and protein expression levels were measured by realtime polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay, respectively. A pull-down assay was performed to determine RhoA activity. IκBα protein expression was measured by Western blotting, nuclear factor (NF)-κB activation was detected by electrophoretic mobility shift assay and its transcription activity was determined by a dual luciferase reporter gene assay. PAI-1 mRNA and protein expression levels were both increased with high glucose concentrations, but they were significantly suppressed by simvastatin and atorvastatin treatment (P < 0.01) and the effects were reversed by mevalonate (100 μmol/L) and geranylgeranyl pyrophosphate (10 μmol/L) but not farnesyl pyrophosphate (10 μmol/L). Such effects were similar to those of a RhoA inhibitor, C3 exoenzyme (5 μg/mL), inhibitors of RhoA kinase (ROCK), Y-27632 (10 μmol/L) and hydroxyfasudil (10 μmol/L) and an NF-κB inhibitor, BAY 11-7082 (5 μmol/L). High glucose-induced RhoA and NF-κB activations in CMECs were both significantly inhibited by statins (P < 0.01). Simvastatin and atorvastatin equally suppress high glucose-induced PAI-1 expression. These effects of statins may occur partly by regulating the RhoA/ROCK-NF-κB pathway. The multifunctional roles of statins may be particularly beneficial for patients with metabolic syndrome.

Molecular cloning, characterization, and function analysis of a mevalonate pyrophosphate decarboxylase gene from Ganoderma lucidum

This study investigated the role of the mevalonate pyrophosphate decarboxylase gene in the triterpene biosynthetic pathway of Ganoderma lucidum. The mevalonate pyrophosphate decarboxylase gene (mvd) was isolated using a degenerate primer-PCR technique. An analysis of the Gl-mvd transcription profile revealed a positive correlation between the expression of the Gl-mvd gene and triterpene content changes in G. lucidum during development. Furthermore, a promoter deletion analysis was conducted in G. lucidum to investigate the promoter activity and the role of methyl jasmonate (MeJA) responsive elements in the mvd promoter under the MeJA elicitor. The overexpression of Gl-mvd increased triterpene accumulation compared with the wild-type strain and increased the expression of several genes involved in the triterpene biosynthetic pathway. The findings of this study suggest that mvd may play an important role in triterpene biosynthesis regulation. Moreover, there may be the interactions among the genes involved in the triterpene biosynthetic pathway in the G. lucidum. Additionally, this study provides an approach for improving triterpene content through the overexpression of a key gene.

Comparison of Receptors and Enzymes Regulating Cholesterol Levels in Liver between SHR/NDmcr-cp Rats and Normotensive Wistar Kyoto Rats at Ten Weeks of Age

The spontaneously hypertensive rat (SHR)/NDmcr-cp (SHR-cp), which is a metabolic syndrome model rat, was reported to show hypercholesteremia, as compared with lean littermates. The serum total cholesterol level in SHR-cp at 18 weeks of age is higher than that of normotensive Wistar Kyoto rat (WKY), but that in SHR-cp at 10 weeks of age is the same. The objective of this study is to clarify whether there are differences in the system regulating serum cholesterol levels between SHR-cp and WKY at 10 weeks of age. Total serum cholesterol levels, and cholesterol levels of high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) were similar in the two strains. However, the cholesterol levels in the liver of SHR-cp were lower than those of WKY. Next, mRNA levels of receptors (scavenger receptor class B type 1 [SRB1], LDL receptor [LDLR]) involved in uptake from serum to liver or enzymes of cholesterol catabolism (CYP7A1 and CYP8B1) and biosynthesis (mevalonate pyrophosphate decarboxylases [MPD]) in liver were compared between SHR-cp and WKY. High levels of MPD and LDLR and low levels of SRB1 were shown in SHR-cp, as compared with WKY. CYP7A1 and CYP8B1 levels were similar between SHR-cp and WKY. These results suggest that the serum cholesterol level in SHR-cp by the balance or regulation between the rise in cholesterol uptake and reduction in cholesterol biosynthesis in the liver is the same as that in WKY.

Reduction of Mevalonate Pyrophosphate Decarboxylase in Mouse Melanoma Cells Treated with &amp;delta;-Tocotrienol Is Not Associated with Reduction of Cholesterol Content or Release of Lysosomes and Melanosomes

We previously reported that a decrease in the melanin content of mouse melanoma cells (B16 cells) treated with δ-tocotrienol was the result of a decrease in the level of tyrosinase activity and protein. Use of δ-tocotrienol as a whitening agent, may therefore have side effects. In the present study, we examined whether δ-tocotrienol caused side effects (the release of lysosomes from and a decrease in the cholesterol content of cells). We also examined the release of melanosomes (lysosome-related organella). Neither of lysosomes nor melanosomes were released from cells treated with δ-tocotrienol, since βglucuronidase (melanosomal and lysosomal enzyme) activity, melanin content (melanosomal marker), and tyrosinase (melanosomal enzyme) activity did not increase in the cell culture medium. Although mevalonate pyrophosphate decarboxylase (MPD; an enzyme of cholesterol biosynthesis) was significantly reduced in the cells treated with δ-tocotrienol, cholesterol content was not. Thus, δ-tocotrienol might be useful as a therapeutic or preventive drug for hyperpigmentation and as a component of whitening and/or lightening cosmetics not causing severe side effect (reduction of cholesterol content and release of lysosomes/melanosomes), although δ-tocotrienol cause a decrease of MPD.

Comparison of the Gene Expression Levels of Mevalonate Pyrophosphate Decarboxylase between Stroke-prone Spontaneously Hhypertensive and Wistar Kyoto Rats

The relationship between protein and mRNA levels of mevalonate pyrophosphate decarboxylase (MPD) in rat tissues remains to be clarified. In this study, we examined the distribution of the mRNA in Wistar rat tissues by real-time PCR. When the relative expression of MPD in 1 mg of tissue was quantified using glyceraldehyde-3-phosphatedehydrogenase (GAPDH) as an internal control, the mRNA level was found to be markedly higher in the spleen and liver than in other organs. The correlation coefficient between protein and mRNA levels of MPD was 0.847, indicating that the protein level of MPD in Wistar rat is distributed in the tissues almost entirely dependent on the mRNA level of MPD. We previously reported that the protein levels of MPD in the liver and brain of spontaneously hypertensive rats, stroke-prone (SHRSP) are reduced. Thus, we compared mRNA levels of MPD in the liver and brain between Wistar Kyoto rats (WKY) and SHRSP. The levels in liver of SHRSP were significantly decreased, but not in brain, as compared with WKY. Also, mRNA levels of sterol regulatory element binding protein-2, which is transcription factor of cholesterol, in liver of SHRSP was similar to those of WKY. These findings indicate that the reduced protein level of MPD in the liver of SHRSP is caused by a decrease in mRNA level of MPD, and that in the brain of SHRSP is caused by increased degradation of the MPD protein.

D-δ-Tocotrienol-Mediated Suppression of the Proliferation of Human PANC-1, MIA PaCa-2, and BxPC-3 Pancreatic Carcinoma Cells

The rate-limiting activity of the mevalonate pathway, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, provides intermediates essential for growth. Competitive inhibitors of HMG CoA reductase, such as the statins, and down-regulators of reductase, such as the tocotrienols, suppress tumor growth. We evaluated the impact of d-delta-tocotrienol, the most potent vitamin E isomer, on human MIA PaCa-2 and PANC-1 pancreatic carcinoma cells and BxPC-3 pancreatic ductal adenocarcinoma cells. Cell proliferation was measured by using CellTiter 96 Aqueous One Solution (Promega, Madison, Wis). Cell cycle distribution was determined by flow cytometry. Apoptosis was evaluated by Annexin V staining and fluorescence microscopy after dual staining with acridine orange and ethidium bromide. d-delta-Tocotrienol induced concentration-dependent suppression of cell proliferation with 50% inhibitory concentrations of 28 (6) micromol/L (MIA PaCa-2), 35 (7) micromol/L (PANC-1), and 35 (8) microL (BxPC-3), respectively. These effects are attributable to cell cycle arrest at the G1 phase and apoptosis. Mevalonate attenuated d-delta-tocotrienol-mediated growth inhibition. A physiologically attainable blend of d-delta-tocotrienol and lovastatin synergistically suppressed the proliferation of MIA PaCa-2 cells. Suppression of mevalonate pathway activities, be it by modulators of HMG CoA reductase (statins, tocotrienols, and farnesol), farnesyl transferase (farnesyl transferase inhibitors), and/or mevalonate pyrophosphate decarboxylase (phenylacetate) activity, may have a potential in pancreatic cancer chemotherapy.