Expression Of Farnesyl Diphosphate Synthase Research Articles

Farnesyl Diphosphate Synthase Gene Associated with Loss of Bone Mass Density and Alendronate Treatment Failure in Patients with Primary Osteoporosis.

Aminobisphosphonates (NBPs) are the first-choice medication for osteoporosis (OP); NBP treatment aims at increasing bone mineral density (BMD) by inhibiting the activity of farnesyl diphosphate synthase (FDPS) enzyme in osteoclasts. Despite its efficacy, inadequate response to the drug and side effects have been reported. The A allele of the rs2297480 (A > C) SNP, found in the regulatory region of the FDPS gene, is associated with reduced gene transcription. This study evaluates the FDPS variant rs2297480 (A > C) association with OP patients' response to alendronate sodium treatment. A total of 304 OP patients and 112 controls were enrolled; patients treated with alendronate sodium for two years were classified, according to BMD variations at specific regions (lumbar spine (L1-L4), femoral neck (FN) and total hip (TH), as responders (OP-R) (n = 20) and non-responders (OP-NR) (n = 40). We observed an association of CC genotype with treatment failure (p = 0.045), followed by a BMD decrease in the regions L1-L4 (CC = -2.21% ± 2.56; p = 0.026) and TH (CC = -2.06% ± 1.84; p = 0.015) after two years of alendronate sodium treatment. Relative expression of the FDPS gene was also evaluated in OP-R and OP-NR patients. Higher expression of the FDPS gene was also observed in OP-NR group (FC = 1.84 ± 0.77; p = 0.006) when compared to OP-R. In conclusion, the influence observed of FDPS expression and the rs2897480 variant on alendronate treatment highlights the importance of a genetic approach to improve the efficacy of treatment for primary osteoporosis.

Liberation of daidzein by gut microbial β-galactosidase suppresses acetaminophen-induced hepatotoxicity in mice

Acetaminophen (APAP) overdose is a leading cause of drug-induced liver injury (DILI). The impact of the gut microbiota and associated metabolites on APAP and liver function remains unclear. We show that APAP disturbance is associated with a distinct gut microbial community, with notable decreases in Lactobacillus vaginalis. Mice receiving L.vaginalis showed resistance to APAP hepatotoxicity due to the liberation of the isoflavone daidzein from the diet by bacterial β-galactosidase. The hepatoprotective effects of L.vaginalis in APAP-exposed germ-free mice were abolished with a β-galactosidase inhibitor. Similarly, β-galactosidase-deficient L.vaginalis produced poorer outcomes in APAP-treated mice than the wild-type strain, but these differences were overcome with daidzein administration. Mechanistically, daidzein prevented ferroptotic death, which was linked to decreased expression of farnesyl diphosphate synthase (Fdps) that activated a key ferroptosis pathway involving AKT-GSK3β-Nrf2. Thus, liberation of daidzein by L.vaginalis β-galactosidase inhibits Fdps-mediated hepatocyte ferroptosis, providing promising therapeutic approaches for DILI.

Farnesyl diphosphate synthase promotes cell proliferation by regulating gene expression and alternative splicing profiles in HeLa cells.

Farnesyl diphosphate synthase (FDPS), an essential enzyme involved in the mevalonate pathway, is implicated in various diseases, including multiple types of cancer. As an RNA-binding protein (RBP), FDPS is also involved in transcriptional and post-transcriptional regulation. However, to the best of our knowledge, transcriptome-wide targets of FDPS still remain unknown. In the present study, FDPS expression patterns in pan-cancer were analyzed. In addition, it was investigated how FDPS overexpression (FDPS-OE) regulates the transcriptome in HeLa cells. FDPS-OE increased the proliferation rate in HeLa cells by MTT assay. Using transcriptome-wide high throughput sequencing and bioinformatics analysis, it was found that FDPS upregulated the expression levels of genes enriched in cell proliferation and extracellular matrix organization, including the laminin subunit γ2, interferon-induced proteins with tetratricopeptide repeats 2 and matrix metallopeptidase 19 genes. According to alternative splicing (AS) analysis, FDPS modulated the splicing patterns of the bone morphogenic protein 1, semaphorin 4D, annexin A2 and sirtuin 2 genes, which are enriched in the cell cycle and DNA repair, and are related to cell proliferation. To corroborate the FDPS-regulated transcriptome findings, FDPS was overexpressed in human osteosarcoma cells. Differentially expressed genes and regulated AS genes in the cells were both validated by reverse transcription-quantitative PCR. The results suggested that, as an emerging RBP, FDPS may serve an important role in transcriptome profiles by altering gene expression and regulating AS. FDPS also affected the cell proliferation rate. These findings broaden the understanding of the molecular functions of FDPS, and the potential of FDPS as a target in therapy should be investigated.

Reducing farnesyl diphosphate synthase levels activates Vγ9Vδ2 T cells and improves tumor suppression in murine xenograft cancer models

Human Vγ9Vδ2 T cells are attractive candidates for cancer immunotherapy due to their potent capacity for tumor recognition and cytolysis of many tumor cell types. However, efforts to deploy clinical strategies for Vγ9Vδ2 T cell cancer therapy are hampered by insufficient potency. We are pursuing an alternate strategy of modifying tumors to increase the capacity for Vγ9Vδ2 T cell activation, as a means for strengthening the anti-tumor response by resident or ex vivo manufactured Vγ9Vδ2 T cells. Vγ9Vδ2 T cells are activated in vitro by non-peptidic antigens including isopentenyl pyrophosphate (IPP), a substrate of farnesyl diphosphate synthase (FDPS) in the pathway for biosynthesis of isoprenoids. In an effort to improve in vivo potency of Vγ9Vδ2 T cells, we reduced FDPS expression in tumor cells using a lentivirus vector encoding a short-hairpin RNA that targets FDPS mRNA (LV-shFDPS). Prostate (PC3) or hepatocellular carcinoma (Huh-7) cells transduced with LV-shFDPS induced Vγ9Vδ2 T cell stimulation in vitro, resulting in increased cytokine expression and tumor cell cytotoxicity. Immune deficient mice implanted with LV-shFDPS transduced tumor cells showed dramatic responses to intraperitoneal injection of Vγ9Vδ2 T cells with strong suppression of tumor growth. In vivo potency was increased by transducing tumor cells with a vector expressing both shFDPS and human IL-2. Tumor suppression by Vγ9Vδ2 T cells was dose-dependent with greater effects observed in mice injected with 100% LV-shFDPS transduced cells compared to mice injected with a mixture of 50% LV-shFDPS transduced cells and 50% control (no vector) tumor cells. Delivery of LV-shFDPS by intratumoral injection was insufficient to knockdown FDPS in the majority of tumor cells, resulting in insignificant tumor suppression by Vγ9Vδ2 T cells. Thus, Vγ9Vδ2 T cells efficiently targeted and suppressed tumors expressing shFDPS in mouse xenotransplant models. This proof-of-concept study demonstrates the potential for suppression of genetically modified tumors by human Vγ9Vδ2 T cells and indicates that co-expression of cytokines may boost the anti-tumor effect.

Engineered Saccharomyces cerevisiae for the De Novo Biosynthesis of (-)-Menthol.

Menthol, a high-value commodity monoterpenoid chemical, holds an important market share commercially because of its distinct functions. The menthol on the market mainly originates from plant extraction, which is facing challenges such as the seasonal fluctuations and long growth cycle of plants. Therefore, this study attempted to realize the de novo synthesis of menthol through microbial fermentation. First, through heterologous expression and subcellular localization observation, a synthetic route from glucose to (−)-menthol was successfully designed and constructed in Saccharomyces cerevisiae. Then, the mevalonate (MVA) pathway was enhanced, and the expression of farnesyl diphosphate synthase (ERG20) was dynamically regulated to improve the synthesis of D-limonene, a key precursor of (−)-menthol. Shake flask fermentation results showed that the D-limonene titer of the recombinant strain reached 459.59 mg/L. Next, the synthesis pathway from D-limonene to (−)-menthol was strengthened, and the fermentation medium was optimized. The (−)-menthol titer of 6.28 mg/L was obtained, implying that the de novo synthesis of menthol was successfully realized for the first time. This study provides a good foundation for the synthesis of menthol through microbial fermentation.

Farnesyl diphosphate synthase regulated endothelial proliferation and autophagy during rat pulmonary arterial hypertension induced by monocrotaline

BackgroundThe proliferation ability and autophagy level of pulmonary artery endothelial cells (PAECs) play an important role in promoting the development of pulmonary artery hypertension (PAH), and there is still no effective treatment for PAH. Farnesyl diphosphate synthase (FDPS) is a key enzyme in the mevalonate pathway. The intermediate metabolites of this pathway are closely related to the activity of autophagy-associated small G proteins, including Ras-related C3 botulinum toxin substrate 1 (Rac1). Studies have shown that the mevalonate pathway affects the activation levels of different small G proteins, autophagy signaling pathways, vascular endothelial function, and so on. However, the exact relationship between them is still unclear in PAH.MethodIn vitro, western blotting and mRFP-GFP-LC3 puncta formation assays were used to observe the expression of FDPS and the level of autophagy in PAECs treated with monocrotaline pyrrole (MCTP). In addition, cell proliferation and migration assays were used to assess the effect of FDPS on endothelial function, and Rac1 activity assays were used to evaluate the effect of Rac1 activation on PAEC autophagy via the PI3K/AKT/mTOR signaling pathway. In vivo, the right heart catheterization method, hematoxylin and eosin (H&E) staining and western blotting were used to determine the effect of FDPS on PAEC autophagy and monocrotaline (MCT)-induced PAH.ResultsWe show that the expression of FDPS is increased in the PAH module in vitro and in vivo, concomitant with the induction of autophagy and the activation of Rac1. Our data demonstrate that inhibition of FDPS ameliorates endothelial function and decreases MCT-induced autophagy levels. Mechanistically, we found that FDPS promotes autophagy, Rac1 activity and endothelial disfunction through the PI3K/AKT/mTOR signaling pathway.ConclusionOur study suggests that FDPS contributes to active small G protein-induced autophagy during MCT-induced PAH, which may serve as a potential therapeutic target against PAH.

Zoledronic Acid Is Not Equally Potent on Osteoclasts Generated From Different Individuals.

ABSTRACTZoledronic acid is a bisphosphonate commonly used to treat bone diseases such as osteoporosis and cancer‐induced bone disease. Patients exhibit a variable sensitivity to zoledronic acid; the underlying explanation for this remains unclear. The objective of this study was to obtain more knowledge in this regard. We hypothesized that osteoclasts generated from different individuals would show a variable sensitivity to zoledronic acid in vitro. Osteoclasts were generated using monocytes from 46 healthy female blood donors (40 to 66 years). Matured osteoclasts were reseeded onto bone slices precoated with different concentrations of zoledronic acid. IC50 values were determined based on total eroded bone surface after 3 days of resorption. The IC50 for inhibition of osteoclastic bone resorption varied from 0.06 to 12.57μM zoledronic acid; thus, a more than 200‐fold difference in sensitivity to zoledronic acid among osteoclasts from different individuals was observed. Multiple linear regression analyses showed that the determined IC50 correlated with smoking status, and the average number of nuclei per osteoclast in vitro. Further analyses showed that: (i) increasing protein levels of mature cathepsin K in osteoclast cultures rendered the osteoclasts less sensitive to zoledronic acid; (ii) surprisingly, neither the gene nor the protein expression of farnesyl diphosphate synthase was found to correlate with the IC50; and (iii) trench‐forming osteoclasts were found to be more sensitive to zoledronic acid than pit‐forming osteoclasts within the same cell culture. Thus, we conclude that there indeed is a high degree of variation in the potency of zoledronic acid on osteoclasts when generated from different individuals. We propose that our findings can explain some of the varying clinical efficacy of zoledronic acid therapy observed in patients, and may therefore be of clinical importance, which should be investigated in a clinical trial combining in vitro and in vivo investigations. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Molecular Cloning, Characterisation, and Heterologous Expression of Farnesyl Diphosphate Synthase from Sanghuangporus baumii.

A farnesyl diphosphate synthase (FPS) cDNA and promoter region was cloned from Sanghuangporus baumii. The gene contains a 150-bp 5'-untranslated region (UTR), a 154-bp 3'-UTR, and a 1062-bp open reading frame (ORF) encoding a 354 amino acid polypeptide. The FPS-DNA includes three exons (nucleotides 1 -123, 184-321, and 505-1305) and two introns (nucleotides 124-183 and 322-504). The FPS protein has a molecular weight of 40.73kDa, it is hydrophilic with a theoretical isoelectric point of 5.13, and the secondary and three-dimensional structure were analysed. There is a transcription start site at nucleotides 1318-1368 of the promoter, which includes typical eukaryotic promoter elements (TATA Box, CAAT Box, ARBE, AT-rich element, G-box, MBS, Sp1, LTR). FPS was expressed in Escherichia coli BL21, and the recombinant protein (63.41kDa) was subjected to dodecyl sulphate, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE). FPS transcription was measured during different developmental stages, and expression in 11 and 13days mycelia was upregulated 49.3-fold and 125.4-fold, respectively, compared with 9days mycelia controls. Through analysing, S. baumii triterpenoid content was correlated with the transcription level of FPS during different development stages, and the triterpenoid content peaked at day 15 (7.21mg/g).

FDPS cooperates with PTEN loss to promote prostate cancer progression through modulation of small GTPases/AKT axis.

Farnesyl Diphosphate Synthase (FDPS), a mevalonate pathway enzyme, is highly expressed in several cancers, including prostate cancer (PCa). To date, the mechanistic, functional and clinical significance of FDPS in cancer remains unexplored. We evaluated the FDPS expression and its cancer-associated phenotypes using in vitro and in vivo methods in PTEN deficient and sufficient human and mouse PCa cells and tumors. Interestingly, FDPS overexpression synergizes with PTEN deficiency in PTEN conditionally knockout mice (P<0.05) and expressed significantly higher in human (P<0.001) PCa tissues, cell lines and murine tumoroids compared to respective controls. In silico analysis revealed that FDPS is associated with increasing Gleason score, PTEN functionally deficient status and poor survival of PCa. Ectopic overexpression of FDPS promotes oncogenic phenotypes such as colony formation (P<0.01) and proliferation (P<0.01) through activation of AKT and ERK signaling by prenylating Rho A, Rho G and CDC42 small GTPases. Of interest, knockdown of FDPS in PCa cells exhibits decreased colony growth and proliferation (P<0.001) by modulating AKT and ERK pathways. Further, genetic and pharmacological inhibition of PI3K but not AKT reduced FDPS expression. Pharmacological targeting of FDPS by zoledronic acid (ZOL), which is already in clinics exhibit reduced growth and clonogenicity of human and murine PCa cells (P<0.01) and 3D tumoroids (P<0.02) by disrupting AKT and ERK signaling through direct interference of small GTPases protein prenylation. Thus, FDPS plays an oncogenic role in PTEN-deficient PCa through GTPase/AKT axis. Identifying mevalonate pathway proteins could serve as a therapeutic target in PTEN dysregulated tumors.

Targeting the phospholipase A2 receptor ameliorates premature aging phenotypes.

Hutchinson–Gilford progeria syndrome (HGPS) is a lethal premature aging that recapitulates many normal aging characteristics. This disorder is caused by mutation in the LMNA gene leading to the production of progerin which induces misshapen nuclei, cellular senescence, and aging. We previously showed that the phospholipase A2 receptor (PLA2R1) promotes senescence induced by replicative, oxidative, and oncogenic stress but its role during progerin‐induced senescence and in progeria is currently unknown. Here, we show that knockdown of PLA2R1 prevented senescence induced by progerin expression in human fibroblasts and markedly delayed senescence of HGPS patient‐derived fibroblasts. Whole‐body knockout of Pla2r1 in a mouse model of progeria decreased some premature aging phenotypes, such as rib fracture and decreased bone content, together with decreased senescence marker. Progerin‐expressing human fibroblasts exhibited a high frequency of misshapen nuclei and increased farnesyl diphosphate synthase (FDPS) expression compared to controls; knockdown of PLA2R1 reduced the frequency of misshapen nuclei and normalized FDPS expression. Pamidronate, a FDPS inhibitor, also reduced senescence and misshapen nuclei. Downstream of PLA2R1, we found that p53 mediated the progerin‐induced increase in FDPS expression and in misshapen nuclei. These results suggest that PLA2R1 mediates key premature aging phenotypes through a p53/FDPS pathway and might be a new therapeutic target.

Transcriptome analysis of Hevea brasiliensis in response to exogenous methyl jasmonate provides novel insights into regulation of jasmonate-elicited rubber biosynthesis.

The phytohomorne methyl jasmonate (MeJA) is known to trigger extensive reprogramming of gene expression leading to transcriptional activation of many secondary metabolic pathways. However, natural rubber is a commercially important secondary metabolite and little is known about the genetic and genomic basis of jasmonate-elicited rubber biosynthesis in rubber tree (Hevea brasiliensis). RNA sequencing (RNA-seq) of H. brasiliensis bark treated with 1g lanolin paste containing 0.02% w/w MeJA for 24h (M2) and 0.04% w/w MeJA for 24h (M4) was performed. A total of 2950 and 2850 differentially expressed genes in M2 and M4 compared with control (C) were respectively detected. Key genes involved in 2-C-methyl-D-erythritol 4-phosphate, rubber biosynthesis, glycolysis and carbon fixation (Calvin cycle) pathway were found to be up-regulated by MeJA treatment. Particularly, the expression of 3-hydroxy-3-metylglutaryl coenzyme A reductase in MVA pathway was down-regulated by MeJA treatment, but the expression of farnesyl diphosphate synthase (FPS) and cis-prenyltransferase (CPT, or rubber transferase) in rubber biosynthesis pathway were up-regulated by MeJA treatment. Up-regulation of critical genes in JA biosynthesis in response to MeJA treatment exhibited the self-activation of JA biosynthesis. In addition, up-regulated genes of great regulatory importance in cross-talk between JA and other hormone signaling, and of transcriptional regulation were identified. The increased expression levels of FPS and CPT in rubber biosynthesis pathway possibly resulted in an increased latex production in rubber tree treated with MeJA. The present results provide insights into the mechanism by which MeJA activates the rubber biosynthesis and the transcriptomedata can also serve as the foundation for future research into the molecular basis for MeJA regulation of other cellular processes.

Effect of salicylic acid and yeast extract on the accumulation of jasmonic acid and sesquiterpenoids in Panax ginseng adventitious roots

In different plant species, secondary metabolite biosynthesis is regulated by the phytohormone jasmonic acid (JA), which is derived by the action of lipoxygenase. In this study, we examined mono- and sesquiterpenoid accumulation and the related signal transduction pathways and biosynthetic genes in adventitious root cultures of Panax ginseng C.A. Meyer as induced by yeast extract (YE, 3 g/L), a biotic elicitor, and salicylic acid (SA, 200 μM), a signaling elicitor. The lipoxygenase (LOX) gene was highly expressed in 24 and 12 h after treatment with SA and YE. JA content was significantly increased in 24 h after SA treatment. The H2O2 content was the highest in 24 and 72 h after the onset of SA and YE treatment, respectively. RNA blot analysis showed that farnesyl diphosphate synthase (FPS) and isopentenyl pyrophosphate isomerase (IPPI) genes encoding enzymes of the biosynthesis of mono- and sesquiterpenoids were up-regulated by both elicitors. Farensol, isochiapin B sesquiterpenoids, champhor, and cineole monoterpenoids were highly accumulated after 24 h of SA treatment, while YE treatment induced bacchotricuneatin C, guaiazulene, isochiapin B, and p-benzoquinone sesquiterpenoid production. These results suggest that mono- and sesquiterpenoid accumulation induced by SA and YE occurs due to the IPPI and FPS expression and may be mediated by reactive oxygen species signaling and jasmonic acid signal transduction.

Clusterin inhibition using OGX-011 synergistically enhances zoledronic acid activity in osteosarcoma.

Despite recent improvements in therapeutic management of osteosarcoma, ongoing challenges in improving the response to chemotherapy warrants new strategies still needed to improve overall patient survival. Among new therapeutic approaches, zoledronic acid (ZOL) represents a promising adjuvant molecule to chemotherapy to limit the osteolytic component of bone tumors. However, ZOL triggers the elevation of heat shock proteins (Hsp), including Hsp27 and clusterin (CLU), which could enhance tumor cell survival and treatment resistance. We hypothesized that targeting CLU using siRNA or the antisense drug, OGX-011, will suppress treatment-induced CLU induction and enhance ZOL-induced cell death in osteosarcoma (OS) cells. The combined effects of OGX-011 and ZOL were investigated in vitro on cell growth, viability, apoptosis and cell cycle repartition of ZOL-sensitive or -resistant human OS cell lines (SaOS2, U2OS, MG63 and MNNG/HOS). In OS cell lines, ZOL increased levels of HSPs, especially CLU, in a dose- and time-dependent manner by mechanism including increased HSF1 transcription activity. The OS resistant cells to ZOL exhibited higher CLU expression level than the sensitive cells. Moreover, CLU overexpression protects OS sensitive cells to ZOL-induced cell death by modulating the MDR1 and farnesyl diphosphate synthase expression. OGX-011 suppressed treatment-induced increases in CLU and synergistically enhanced the activity of ZOL on cell growth and apoptosis. These biologic events were accompanied by decreased expression of HSPs, MDR1 and HSF1 transcriptional activity. In vivo, OGX-011, administered 3 times a week (IP, 20mg/kg), potentiated the effect of ZOL (s.c; 50µg/kg), significantly inhibiting tumor growth by 50% and prolonging survival in MNNG/HOS xenograft model compared to ZOL alone. These results indicate that ZOL-mediated induction of CLU can be attenuated by OGX-011, with synergistic effects on delaying progression of osteosarcoma.

Unbiased analysis of pancreatic cancer radiation resistance reveals cholesterol biosynthesis as a novel target for radiosensitisation

Background:Despite its promise as a highly useful therapy for pancreatic cancer (PC), the addition of external beam radiation therapy to PC treatment has shown varying success in clinical trials. Understanding PC radioresistance and discovery of methods to sensitise PC to radiation will increase patient survival and improve quality of life. In this study, we identified PC radioresistance-associated pathways using global, unbiased techniques.Methods:Radioresistant cells were generated by sequential irradiation and recovery, and global genome cDNA microarray analysis was performed to identify differentially expressed genes in radiosensitive and radioresistant cells. Ingenuity pathway analysis was performed to discover cellular pathways and functions associated with differential radioresponse and identify potential small-molecule inhibitors for radiosensitisation. The expression of FDPS, one of the most differentially expressed genes, was determined in human PC tissues by IHC and the impact of its pharmacological inhibition with zoledronic acid (ZOL, Zometa) on radiosensitivity was determined by colony-forming assays. The radiosensitising effect of Zol in vivo was determined using allograft transplantation mouse model.Results:Microarray analysis indicated that 11 genes (FDPS, ACAT2, AG2, CLDN7, DHCR7, ELFN2, FASN, SC4MOL, SIX6, SLC12A2, and SQLE) were consistently associated with radioresistance in the cell lines, a majority of which are involved in cholesterol biosynthesis. We demonstrated that knockdown of farnesyl diphosphate synthase (FDPS), a branchpoint enzyme of the cholesterol synthesis pathway, radiosensitised PC cells. FDPS was significantly overexpressed in human PC tumour tissues compared with healthy pancreas samples. Also, pharmacologic inhibition of FDPS by ZOL radiosensitised PC cell lines, with a radiation enhancement ratio between 1.26 and 1.5. Further, ZOL treatment resulted in radiosensitisation of PC tumours in an allograft mouse model.Conclusions:Unbiased pathway analysis of radioresistance allowed for the discovery of novel pathways associated with resistance to ionising radiation in PC. Specifically, our analysis indicates the importance of the cholesterol synthesis pathway in PC radioresistance. Further, a novel radiosensitiser, ZOL, showed promising results and warrants further study into the universality of these findings in PC, as well as the true potential of this drug as a clinical radiosensitiser.

5-Aza-2′-deoxycytidine Induced Growth Inhibition of Leukemia Cells through Modulating Endogenous Cholesterol Biosynthesis

5-Aza-2'-deoxycytidine (5-Aza-CdR), a nucleoside analog that can inhibit DNA cytosine methylation, possesses potent antitumorigenic activities for myeloid disorders. Although 5-Aza-CdR is known to be incorporated into DNA and inhibit DNA (cytosine-5)-methyltransferases, the precise mechanisms underlying the drug's antineoplastic activity remain unclear. Here we utilized a mass spectrometry-based quantitative proteomic method to analyze the 5-Aza-CdR-induced perturbation of protein expression in Jurkat-T cells at the global proteome scale. Among the ≈ 2780 quantified proteins, 188 exhibited significant alteration in expression levels upon a 24-hr treatment with 5 μm 5-Aza-CdR. In particular, we found that drug treatment led to substantially reduced expression of farnesyl diphosphate synthase (FDPS) and farnesyl diphosphate farnesyltransferase (FDFT1), two important enzymes involved in de novo cholesterol synthesis. Consistent with this finding, 5-Aza-CdR treatment of leukemia (Jurkat-T, K562 and HL60) and melanoma (WM-266-4) cells led to a marked decrease in cellular cholesterol content and pronounced growth inhibition, which could be rescued by externally added cholesterol. Exposure of these cells to 5-Aza-CdR also led to epigenetic reactivation of dipeptidyl peptidase 4 (DPP4) gene. Additionally, suppression of DPP4 expression with siRNA induced elevated protein levels of FDPS and FDFT1, and increased cholesterol biosynthesis in WM-266-4 cells. Together, the results from the present study revealed, for the first time, that 5-Aza-CdR exerts its cytotoxic effects in leukemia and melanoma cells through epigenetic reactivation of DPP4 gene and the resultant inhibition of cholesterol biosynthesis in these cells.

Molecular cloning of farnesyl diphosphate synthase from Eleutherococcus senticosus and its bioinformatics and expression analysis

To clone farnesyl diphosphate synthase (FPS) gene from Eleutherococcus senticosus and analyze the bioinformatics and expression of the gene. The FPS full length cDNA was cloned by rapid amplification of cDNA ends (RACE). The data was analyzed by bioinformatics method, the structure and function of FPS was deduced. The expression of FPS in different organ of E. senticosus was detected by RT-PCR. The full length of FPS cDNA was 1 499 bp containing a 1 029 bp ORF that encoded 342 amino acids. The deduced protein sequence exhibited two Asp riches conserved motifs (DDXXD). Without transmembrane domain, FPS was located in cytoplasm. RT-PCR result showed that FPS gene expressed in different organs of E. senticosus. The expression amounts of FPS in different organs were different significantly (P < 0.05). The FPS gene of E. senticosus was successfully cloned for the first time, and provided a stable foundation for studying on its effect and expression control on E. senticosus saponins biosynthesis.

Sterol-regulatory-element-binding protein 2 and nuclear factor Y control human farnesyl diphosphate synthase expression and affect cell proliferation in hepatoblastoma cells

FDPS (farnesyl diphosphate synthase) catalyses the formation of farnesyl diphosphate, a key intermediate in the synthesis of cholesterol and isoprenylated cellular metabolites. FDPS is also the molecular target of nitrogen-containing bisphosphonates, which are used as bone-antiresorptive drugs in various disorders. In the present study, we characterized the sterol-response element and NF-Y (nuclear factor Y)-binding site in the human FDPS promoter. Using a luciferase assay, electrophoretic mobility-shift assay and chromatin immunoprecipitation assay, we demonstrated that these elements are responsible for the transcription of the FDPS gene, and that its transcriptional activation is mediated by SREBP-2 (sterol-regulatory-element-binding protein 2) and NF-Y. We also investigated whether sterol-mediated FDPS expression is involved in the cell proliferation induced by zoledronic acid, an FDPS inhibitor. We show that the SREBP-2- and NF-Y-mediated regulation of FDPS gene transcription modulates cell proliferation. These results suggest that SREBP-2 and NF-Y are required to trigger cell proliferation through the induction of FDPS expression and that the pharmacological action of zoledronic acid is involved in this pathway.

Farnesyl diphosphate synthase attenuates paclitaxel-induced apoptotic cell death in human glioblastoma U87MG cells

Increased expression of farnesyl diphosphate synthase (FPPS) by stable transfection appeared to attenuate paclitaxel-induced apoptotic cell death in human glioblastoma U87MG cells. The present results suggest that the apoptotic functions of p53 and c-Jun N-terminal kinase (JNK) are affected by FPPS. Farnesyl diphosphate, a catalytic product of FPPS, also attenuated mentioned paclitaxel-induced apoptotic cell death. As expected, the FPPS inhibitor, pamidronate, enhanced paclitaxel-induced apoptotic cell death. The present results suggest that FPPS plays an important role in apoptotic cell death of cancer cells by blocking the JNK signaling cascade and activating mevalonate metabolism in paclitaxel-treated glioblastoma cells.

Rb Regulates DNA Damage Response and Cellular Senescence through E2F-Dependent Suppression of N-Ras Isoprenylation

Oncogene-induced cellular senescence is well documented, but little is known about how infinite cell proliferation induced by loss of tumor suppressor genes is antagonized by cellular functions. Rb heterozygous mice generate Rb-deficient C cell adenomas that progress to adenocarcinomas following biallelic loss of N-ras. Here, we demonstrate that pRb inactivation induces aberrant expression of farnesyl diphosphate synthase, many prenyltransferases, and their upstream regulators sterol regulatory element-binding proteins (SREBPs) in an E2F-dependent manner, leading to enhanced isoprenylation and activation of N-Ras. Consequently, elevated N-Ras activity induces DNA damage response and p130-dependent cellular senescence in Rb-deficient cells. Furthermore, Rb heterozygous mice additionally lacking any of Ink4a, Arf, or Suv39h1 generated C cell adenocarcinomas, suggesting that cellular senescence antagonizes Rb-deficient carcinogenesis.

Farnesyl diphosphate synthase is involved in the resistance to zoledronic acid of osteosarcoma cells

We recently demonstrated original anti-tumor effects of zoledronic acid (Zol) on osteosarcoma cell lines independently of their p53 and Rb status. The present study investigated the potential Zol-resistance acquired by osteosarcoma cells after prolonged treatment. After 12 weeks of culture in the presence of 1 μm Zol, the effects of high doses of Zol (10–100 μm) were compared between the untreated rat (OSRGA, ROS) and human (MG63, SAOS2) osteosarcoma cells and Zol-pretreated cells in terms of cell proliferation, cell cycle analysis, migration assay and cytoskeleton organization. Long-term treatment with 1 μm Zol reduced the sensitivity of osteosarcoma cells to high concentrations of Zol. Furthermore, the Zol-resistant cells were sensitive to conventional anti-cancer agents demonstrating that this resistance process is independent of the multidrug resistance phenotype. However, as similar experiments performed in the presence of clodronate and pamidronate evidenced that this drug resistance was restricted to the nitrogen-containing bisphosphonates, we then hypothesized that this resistance could be associated with a differential expression of farnesyl diphos-phate synthase (FPPS) also observed in human osteosarcoma samples. The transfection of Zol-resistant cells with FPPS siRNA strongly increased their sensitivity to Zol. This study demonstrates for the first time the induction of metabolic resistance after prolonged Zol treatment of osteosarcoma cells confirming the therapeutic potential of Zol for the treatment of bone malignant pathologies, but points out the importance of the treatment regimen may be important in terms of duration and dose to avoid the development of drug metabolic resistance.