Post-translational isoprenylation of cellular proteins is altered in response to mevalonate availability

Cells incorporate isoprenoid products derived from mevalonate (MVA) into several unique proteins. The aim of this study was to delineate the effects of blocking MVA synthesis on the covalent isoprenylation of these proteins in murine erythroleukemia cells. Inhibition of protein synthesis with cycloheximide prevented the incorporation of [3H]MVA into proteins, suggesting that isoprenylation normally occurs immediately after synthesis of the polypeptides. However, incubation of cells with lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, for as little as 1 h prior to addition of cycloheximide rendered the isoprenylation step insensitive to cycloheximide. Lovastatin had no apparent effect on the stability of the isoprenylated proteins, but the development of cycloheximide insensitivity during the lovastatin preincubation was dependent on synthesis of new protein during that period. Addition of 50-200 microM MVA to the culture medium eliminated the effects of preincubation with lovastatin. Preincubation of cells with 25-hydroxycholesterol, which suppresses the synthesis and enhances the degradation of HMG-CoA reductase but is not a competitive enzyme inhibitor, did not induce cycloheximide-insensitivity of the isoprenylation reaction. The results suggest that blocking MVA synthesis with lovastatin causes a rapid depletion of isoprenoid groups available for protein modification. Consequently, there is an accumulation of non-isoprenylated substrate proteins. Shifts in the ratio of modified vs. unmodified proteins in response to MVA availability may have implications for the changes in cell morphology, cell proliferation and HMG-CoA reductase gene expression that occur when cells are subjected to MVA deprivation.     

Lack of correlation between 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and lovastatin resistance in nerve growth factor treated PC-12 cells

Summary 1. The relationships among the mevalonic acid (MVA) forming enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (CoA) reductase, cell growth and differentiation, and the cytotoxic effects of the reductase inhibitor lovastatin were studied in PC-12 cells, exposed to growth factors.2. When added individually, nerve growth factor (NGF), basic fibroblast growth factor, and epidermal growth factor induce an increase in HMG-CoA reductase activity in cells grown in serum-containing medium. In the presence of serum, the effect of NGF on HMG-CoA reductase is persistent.3. Short-term serum starvation and long-term NGF treatment, in combination, have an additive effect, resulting in a high reductase activity.4. Unlike serum and MVA, which downregulate levels of HMG-CoA reductase by accelerating its degradation, NGF upregulates reductase by slowing the rate of its degradation. This mechanism, however, appears to operate only in the presence of serum, as after prolonged growth with NGF in serum-free medium, cells have a low reductase activity.5. PC-12 cells grown in the absence of NGF are highly sensitive to lovastatin (25 µM) and more than 70% of the cells die after 48 hr. NGF confers lovastatin resistance on cells grown in the presence or in the absence of serum (only 30–40% cell death after 48 hr with lovastatin).6. NGF-induced resistance on lovastatin develops with time and is apparent only in the well-differentiated PC-12 cells whether or not the cells express a high reductase activity.7. Thus, levels of HMG-CoA reductase activity and lovastatin resistance in PC-12 cells are not directly correlated, though clearly inversed lovastatin cytotoxicity and elevated reductase activities are expressed during the period of cell proliferation.8. These data suggest that fully differentiated neuronal cells may not be affected by prolonged high doses of lovastatin.     

Negative regulation of cell proliferation by mevalonate or one of the mevalonate phosphates

The role of mevalonate and its products in the regulation of cellular proliferation was examined using 6-fluoromevalonate (Fmev), a compound that blocks the conversion of mevalonate pyrophosphate to isopentenyl pyrophosphate. Fmev suppressed DNA synthesis by a variety of transformed and malignant T cell, B cell, and myeloid cell lines. In contrast to results previously reported with mitogen-stimulated human peripheral blood T cell DNA synthesis, low concentrations of low density lipoprotein (LDL) alone could not restore proliferation to these cell lines. The same concentrations of LDL were able to provide sufficient cholesterol and support the growth of all cell lines when mevalonate synthesis was blocked with a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, lovastatin. Fmev-mediated inhibition was totally prevented in some but not all cell lines when the concentration of exogenous LDL was increased 5-10-fold above that required to permit proliferation of lovastatin-blocked cells. Residual HMG-CoA reductase activity of cells cultured with LDL inversely correlated with the restoration of growth to Fmev-blocked cultures. Confirmation of the critical role of HMG-CoA reductase activity and mevalonate synthesis in the inhibition of cellular proliferation by Fmev was obtained by demonstrating that the specific inhibitor of this enzyme, lovastatin, restored proliferation of Fmev-blocked cells. Furthermore, supplementation of cultures with mevalonate, the product of HMG-CoA reductase activity, markedly inhibited proliferation of Fmev-blocked cells. These findings indicate that mevalonate or one of the mevalonate phosphates, which accumulates in Fmev-blocked cells, is a critical negative regulator of cellular proliferation.     

Enhanced Anti-Tumor Effect of Zoledronic Acid Combined with Temozolomide against Human Malignant Glioma Cell Expressing O6-Methylguanine DNA Methyltransferase

Temozolomide (TMZ), a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O⁶-methylguanine-DNA methyltranferase (MGMT), a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL), which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance.     

A novel bisphosphonate inhibitor of squalene synthase combined with a statin or a nitrogenous bisphosphonate in vitro

Statins and nitrogenous bisphosphonates (NBP) inhibit 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR) and farnesyl diphosphate synthase (FDPS), respectively, leading to depletion of farnesyl diphosphate (FPP) and disruption of protein prenylation. Squalene synthase (SQS) utilizes FPP in the first committed step from the mevalonate pathway toward cholesterol biosynthesis. Herein, we have identified novel bisphosphonates as potent and specific inhibitors of SQS, including the tetrasodium salt of 9-biphenyl-4,8-dimethyl-nona-3,7-dienyl-1,1-bisphosphonic acid (compound 5). Compound 5 reduced cholesterol biosynthesis and lead to a substantial intracellular accumulation of FPP without reducing cell viability in HepG2 cells. At high concentrations, lovastatin and zoledronate impaired protein prenylation and decreased cell viability, which limits their potential use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced FPP depletion and impairment of protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented zoledronate-induced impairment of both protein farnesylation and geranylgeranylation. Cotreatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly prevent the reduction of cell viability caused by lovastatin or zoledronate alone. The combination of an SQS inhibitor with an HMGCR or FDPS inhibitor provides a rational approach for reducing cholesterol synthesis while preventing nonsterol isoprenoid depletion.     

Co-inhibition of epidermal growth factor receptor and type 1 insulin-like growth factor receptor synergistically sensitizes human malignant glioma cells to CD95L-induced apoptosis

Inhibition of epidermal growth factor receptor (EGFR) signaling sensitizes human malignant glioma cells to death ligand-induced apoptosis. However, tumor cells may compensate the loss of EGFR signaling by activation of the type 1 insulin-like growth factor receptor (IGF-1R). We here report that antagonism of the IGF-1R with the small-molecule inhibitor AG1024 in combination with inhibitors of the EGFR synergistically sensitizes human malignant glioma cells to CD95L-induced apoptosis. This cell death is p53-independent, but requires caspase 8 activity. The levels of the receptor, CD95, are not altered by the inhibitors alone or in combination. Analysis of the downstream signaling pathways reveals synergistic inhibition of ribosomal protein S6 phosphorylation by inhibitor co-treatment, suggesting an involvement of the mammalian target of rapamycin pathway. These findings suggest that adding inhibitors of IGF-1R may be a strategy to overcome escape from the anti-apoptotic effects of EGFR inhibition in malignant gliomas.     

Identification in Haloferax volcanii of Phosphomevalonate Decarboxylase and Isopentenyl Phosphate Kinase as Catalysts of the Terminal Enzyme Reactions in an Archaeal Alternate Mevalonate Pathway

Mevalonate (MVA) metabolism provides the isoprenoids used in archaeal lipid biosynthesis. In synthesis of isopentenyl diphosphate, the classical MVA pathway involves decarboxylation of mevalonate diphosphate, while an alternate pathway has been proposed to involve decarboxylation of mevalonate monophosphate. To identify the enzymes responsible for metabolism of mevalonate 5-phosphate to isopentenyl diphosphate in Haloferax volcanii, two open reading frames (HVO_2762 and HVO_1412) were selected for expression and characterization. Characterization of these proteins indicated that one enzyme is an isopentenyl phosphate kinase that forms isopentenyl diphosphate (in a reaction analogous to that of Methanococcus jannaschii MJ0044). The second enzyme exhibits a decarboxylase activity that has never been directly attributed to this protein or any homologous protein. It catalyzes the synthesis of isopentenyl phosphate from mevalonate monophosphate, a reaction that has been proposed but never demonstrated by direct experimental proof, which is provided in this account. This enzyme, phosphomevalonate decarboxylase (PMD), exhibits strong inhibition by 6-fluoromevalonate monophosphate but negligible inhibition by 6-fluoromevalonate diphosphate (a potent inhibitor of the classical mevalonate pathway), reinforcing its selectivity for monophosphorylated ligands. Inhibition by the fluorinated analog also suggests that the PMD utilizes a reaction mechanism similar to that demonstrated for the classical MVA pathway decarboxylase. These observations represent the first experimental demonstration in H. volcanii of both the phosphomevalonate decarboxylase and isopentenyl phosphate kinase reactions that are required for an alternate mevalonate pathway in an archaeon. These results also represent, to our knowledge, the first identification and characterization of any phosphomevalonate decarboxylase.     

Lovastatin Inhibits VEGFR and AKT Activation: Synergistic Cytotoxicity in Combination with VEGFR Inhibitors

Background

In a recent study, we demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to inhibit the function of the epidermal growth factor receptor (EGFR). Lovastatin attenuated ligand-induced receptor activation and downstream signaling through the PI3K/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in a variety of tumor derived cell lines. The vascular endothelial growth factor receptor (VEGFR) and EGFR share similar activation, internalization and downstream signaling characteristics.

Methodology/Principal Findings

The VEGFRs, particularly VEGFR-2 (KDR, Flt-1), play important roles in regulating tumor angiogenesis by promoting endothelial cell proliferation, survival and migration. Certain tumors, such as malignant mesothelioma (MM), also express both the VEGF ligand and VEGFRs that act in an autocrine loop to directly stimulate tumor cell growth and survival. In this study, we have shown that lovastatin inhibits ligand-induced VEGFR-2 activation through inhibition of receptor internalization and also inhibits VEGF activation of AKT in human umbilical vein endothelial cells (HUVEC) and H28 MM cells employing immunofluorescence and Western blotting. Combinations of lovastatin and a VEGFR-2 inhibitor showed more robust AKT inhibition than either agent alone in the H28 MM cell line. Furthermore, combining 5 µM lovastatin treatment, a therapeutically relevant dose, with two different VEGFR-2 inhibitors in HUVEC and the H28 and H2052 mesothelioma derived cell lines demonstrated synergistic cytotoxicity as demonstrated by MTT cell viability and flow cytometric analyses.

Conclusions/Significance

These results highlight a novel mechanism by which lovastatin can regulate VEGFR-2 function and a potential therapeutic approach for MM through combining statins with VEGFR-2 inhibitors.     

Effect of inhibition of cholesterol synthetic pathway on the activation of Ras and MAP kinase in mesangial cells

Intermediary metabolites of cholesterol synthetic pathway are involved in cell proliferation. Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, blocks mevalonate synthesis, and has been shown to inhibit mesangial cell proliferation associated with diverse glomerular diseases. Since inhibition of farnesylation and plasma membrane anchorage of the Ras proteins is one suggested mechanism by which lovastatin prevents cellular proliferation, we investigated the effect of lovastatin and key mevalonate metabolites on the activation of mitogen-activated protein kinase (MAP kinase) and Ras in murine glomerular mesangial cells. The preincubation of mesangial cells with lovastatin inhibited the activation of MAP kinase stimulated by either FBS, PDGF, or EGF. Mevalonic acid and farnesyl-pyrophosphate, but not cholesterol or LDL, significantly prevented lovastatin-induced inhibition of agonist-stimulated MAP kinase. Lovastatin inhibited agonist-induced activation of Ras, and mevalonic acid and farnesylpyrophosphate antagonized this effect. Parallel to the MAP kinase and Ras data, lovastatin suppressed cell growth stimulated by serum, and mevalonic acid and farnesylpyrophosphate prevented lovastatin-mediated inhibition of cellular growth. These results suggest that lovastatin, by inhibiting the synthesis of farnesol, a key isoprenoid metabolite of mevalonate, modulates Ras-mediated cell signaling events associated with mesangial cell proliferation.     

Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate, the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytoplasmic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of Tobacco Bright Yellow-2 (TBY-2) cells by the HMGR-specific inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein representing two HMGR isozymes. Maximum induction was observed at 24 h. 1-Deoxy-d-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, complemented growth inhibition by mevinolin in the low millimolar concentration range. Furthermore, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-2H4]DX showed that sterols, normally derived from MVA, in the presence of mevinolin are synthesized via the MEP pathway. Fosmidomycin, an inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, was utilized to study the reverse complementation. Growth inhibition by fosmidomycin of TBY-2 cells could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-13C]MVA into plastoquinone, representative of plastidial isoprenoids. Best rates of incorporation of exogenous stably labeled precursors were observed in the presence of both inhibitors, thereby avoiding internal isotope dilution.     

Lovastatin does not accentuate but is rather additive to palmitate-induced apoptosis in cardiomyocytes

Fatty acids such as palmitate have been observed to induce apoptosis in cardiomyocytes but the mechanism of this cytotoxicity is unresolved. The present study sought to determine whether an aspect of fatty acid metabolism is responsible for palmitate-induced apoptosis in cardiomyocytes. As palmitate metabolism increases acetyl CoA production via increased beta oxidation within the mitochondria, we hypothesized that increased acetyl CoA entering the cholesterol biosynthesis pathway might produce intermediates or end products that would be toxic to the cell. To test this hypothesis, cardiomyocytes from embryonic chick cardiomyocytes were treated with the 3-hydroxy-3-methylgutaryl CoA (HMG-CoA) reductase inhibitor lovastatin that inhibits the cholesterol biosynthesis pathway downstream of the acetyl CoA trimerization into HMG-CoA. Lovastatin did not inhibit palmitate-induced apoptosis. Rather, lovastatin induced significant apoptosis itself and when combined with palmitate, the level of apoptosis was equal to the sum of palmitate alone and lovastatin alone. This observation suggests that palmitate and lovastatin are inducing apoptosis by two independent mechanisms. A role for mitochondrial metabolism via carnitine palmitoyl transferase (CPT) in palmitate-induced apoptosis was suggested since capric acid, a fatty acid that is metabolized within the mitochondria but does not utilize CPT-1, did not induce apoptosis. Palmitate-induced apoptosis was further related to the metabolism of saturated fatty acids as the unsaturated fatty acid oleic acid did not induce apoptosis. These data suggest that a unique feature about palmitate metabolism independent of its role in cholesterol biosynthesis is responsible for palmitate-induced apoptosis and the effects of palmitate are additive to those of lovastatin to induce cardiac apoptosis.     

Survivin down-regulation plays a crucial role in 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor-induced apoptosis in cancer

3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (HRIs) are widely used to reduce serum cholesterol in patients with hypercholesterolemia. Previous studies have shown that HRIs can induce apoptosis in colon cancer cells. In this study, we investigated the mechanisms underlying the apoptosis-inducing effect of HRIs in greater detail. The HRI lovastatin induced apoptosis in the human colon cancer cell line SW480 by blocking the cholesterol synthesis pathway. Immunoblot analysis of antiapoptotic molecules, including survivin, XIAP, cIAP-1, cIAP-2, Bcl-2, and Bcl-X(L), revealed that only survivin expression was decreased by lovastatin. Survivin down-regulation by RNA interference induced apoptosis, and survivin overexpression rendered the cells resistant to lovastatin-induced growth inhibition. These results indicate that survivin down-regulation contributes substantially to the proapoptotic properties of lovastatin. Farnesyl pyrophosphate and geranylgeranyl pyrophosphate, two downstream intermediates in the cholesterol synthesis pathway, simultaneously reversed survivin down-regulation and the blocking of Ras isoprenylation by lovastatin. Ras isoprenylation is important for the activation of Ras-mediated signaling, including the activation of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway. The PI3-kinase inhibitor down-regulated survivin in SW480 cells. In addition, lovastatin blocked Ras activation and Akt phosphorylation. We conclude that survivin down-regulation is crucial in lovastatin-induced apoptosis in cancer cells and that lovastatin decreases survivin expression by inhibiting Ras-mediated PI3-kinase activation via the blocking of Ras isoprenylation.     

甲羟戊酸通路对细胞生长调控作用的研究进展

甲羟戊酸(MVA)通路对细胞生长具有重要的调节作用,MVA及其衍生物通过对蛋白质异戊烯化和N糖基化修饰而影响Ras蛋白、生长因子及受体的功能、细胞内信号转导和细胞的生长。MVA通路参与血管活性物质生成的调节是其调节细胞生长的另一机制。MVA生成的限速酶羟甲基戊二酸单酰辅酶A(HMGCoA)则受MVA通路衍生物的反馈抑制。HMGCoA还原酶抑制剂通过抑制MVA及其衍生物的生成而抑制细胞的生长和增殖。     

Butyrate metabolism upstream and downstream acetyl-CoA synthesis and growth control of human colon carcinoma cells.

Butyrate is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibers in the colon lumen which severely affects the proliferation of colon cancer cells in in vitro experiments. Although butyrate is able to interfere with numerous cellular targets including cell cycle regulator expression, little is known about butyrate metabolism and its possible involvement in its effect upon colon carcinoma cell growth. In this study, we found that HT-29 Glc-/+ cells strongly accumulated and oxidized sodium butyrate without producing ketone bodies, nor modifying oxygen consumption nor mitochondrial ATP synthesis. HT-29 cells accumulated and oxidized sodium acetate at a higher level than butyrate. However, sodium butyrate, but not sodium acetate, reduced cell growth and increased the expression of the cell cycle effector cyclin D3 and the inhibitor of the G1/S cdk-cyclin complexes p21/WAF1/Cip1, demonstrating that butyrate metabolism downstream of acetyl-CoA synthesis is not required for the growth-restraining effect of this SCFA. Furthermore, HT-29 cells modestly incorporated the 14C-labelled carbon from sodium butyrate into cellular triacylglycerols and phospholipids. This incorporation was greatly increased when D-glucose was present in the incubation medium, corresponding to the capacity of hexose to circulate in the pentose phosphate pathway allowing NADPH synthesis required for lipogenesis. Interestingly, when HT-29 cells were cultured in the presence of sodium butyrate, their capacity to incorporate 14C-labelled sodium butyrate into triacylglycerols and phospholipids was increased more than twofold. In such experimental conditions, HT-29 cells when observed under an electronic microscope, were found to be characterized by an accumulation of lipid droplets in the cytosol. Our data strongly suggest that butyrate acts upon colon carcinoma cells upstream of acetyl-CoA synthesis. In contrast, the metabolism downstream of acetyl-CoA [i.e. oxidation in the tricarboxylic acid (TCA) cycle and lipid synthesis] likely acts as a regulator of butyrate intracellular concentration.     

Metabolism of intracerebrally injected mevalonate in brain of suckling and young adult rats

We have investigated the in vivo metabolism via sterol and nonsterol pathways of intracerebrally injected mevalonate (MVA) in brains from suckling (10-day-old) and young adult (60-day-old) rats. Results of our study indicated that increasing the amounts of MVA injected increased MVA incorporation into all the lipid fractions examined. The incorporation of MVA into nonsaponiable lipids (NSF) and digitonin precipitable sterols (DPS) was similar in brains from adult and suckling rats. In brain tissue from both suckling and young adult rats the synthesis of dolichol from MVA varied with the amounts of MVA injected. Significant amounts of MVA were recovered in phosphorylated and free polyprenols (farnesol and geraniol) in brain tissue from rats of both ages. Also in both groups of animals, the amounts of MVA incorporated in phosphorylated and free farnesol were higher than the amounts recovered in either, phosphorylated or free geraniol. The amounts of MVA incorporated into the prenoic/fatty acid fraction by brain tissue from both suckling and young adult rats were less than 1% of the total MVA incorporated (nonsaponifiable and saponifiable lipids). Incorporation of MVA into the prenoic/fatty acid fraction by brain tissue was higher in suckling than in young adult rats. These data indicate that the brain tissue from suckling and young adult rats do not differ in their capacity to metabolize MVA into squalene and sterols and that in brain, metabolism of MVA by a shunt pathway is minimal. This suggests that in vivo regulation of cholesterol synthesis during brain development must occur at a step(s) in the sterol synthetic pathway prior to mevalonate, and that metabolism of mevalonate by shunt pathway did not play a role in the developmental regulation of brain sterol synthesis. The data also suggest that in both groups of animals the synthesis of squalene by synthetase may in part control brain sterol synthesis and the synthesis of dolichol is regulated by MVA concentration in the tissue.     

Combination Chemotherapy of BCNU and Didox Acts Synergystically in 9L Glioma Cells

Compounds inhibiting DNA repair and synthesis are expected to act synergistically with BCNU, a standard agent in the therapy of glioblastoma multiforme, and improve survival of patients with malignant gliomas. Ribonucleotide reductase (EC1.17.4.1; RR) catalyzes the rate‐limiting step in DNA synthesis and plays a critical role in maintaining crucial substrates for DNA repair. We have studied the effects of Didox, an inhibitor of RR on 9L glioma cells in combination with BCNU². We analyzed intracellular dNTP pools and found that Didox significantly depleted the intracellular dNTP concentrations. Experiments using cytotoxicity, growth inhibition and clonogenic assays showed significant synergism of Didox and BCNU. Combination regimens using synchronous administration demonstrated highest cytotoxicity. We have also identified altered gene expression in a number of DNA repair related enzymes after BCNU treatment using large‐scale cDNA arrays. The coadministration with Didox could reverse the expression of some of the overexpressed repair gene suggesting possible pathways to circumvent the developing resistance in 9L glioma cells against BCNU. These results introduce the combination of Didox and BCNU as a viable alternative for the treatment of malignant gliomas.     

Evidence for isopentenyladenine modification on a cell cycle-regulated protein

We have prepared antibodies that recognize isopentenyladenosine (i6A), a modified nucleoside derived from mevalonic acid (MVA). In immunoblot assays, affinity-purified anti-i6 A antibodies specifically bound to a 26-kDa protein (i6A26) in Chinese hamster ovary cells. Anti-i6A recognition of i6A26 was blocked with i6A but not adenosine or isopentenol. Employing immunoblot analysis we have quantitated the level of i6A26 in cells expressing various rates of DNA synthesis. The cellular content of i6A26 was reduced 4-fold in quiescent cells cultured in the absence of serum. When serum-deprived cells were stimulated to enter the cell cycle, the amount of i6A26 increased in the cells during the G1 phase. However, when synchronized cells were stimulated with serum-containing medium in the presence of mevinolin (an inhibitor of cellular MVA synthesis), we observed impaired G1 expression of i6A26 and delayed onset of S phase DNA synthesis. Mevinolin addition to asynchronously growing cells resulted in low rates of cellular DNA synthesis and suppressed levels of i6A26 which were reversed by coincubation with MVA. The ability of MVA to restore DNA synthesis and the cellular content of i6A26 in mevinolin-treated cells showed similar MVA concentration and time dependences. Regenerating liver tissue also exhibited elevated levels of i6A26. Thus, the expression of i6A26 correlates with cellular proliferation and growth. We speculate that i6A26 contains isopentenyladenine moieties and mediates isoprenoid regulation of DNA synthesis. Isopentenyladenylated proteins may also function in cytokinin regulation of proliferation and differentiation in plants.     

SCD1 inhibition causes cancer cell death by depleting mono-unsaturated fatty acids

Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway.     

Cyclosporine a induces growth arrest or programmed cell death of human glioma cells

Human malignant gliomas are highly resistant to current therapeutic approaches. We previously demonstrated that cyclosporine A (CsA) induces an apoptotic cell death in rat C6 glioma cells. In the present study, we found the induction of growth arrest or cell death of human malignant glioma cells exposed to CsA. In studied glioma cells, an accumulation of p21Cip1/Waf1 protein, a cell cycle inhibitor, was observed following CsA treatment, even in the absence of functional p53 tumour suppressor. CsA induced a senescence-associated growth arrest, in U87-MG glioma cells with functional p53, while in U373 and T98G glioma cells with mutated p53, CsA treatment triggered cell death associated with alterations of cell morphology, cytoplasm vacuolation, and condensation of chromatin. In T98G cells this effect was completely abolished by simultaneous treatment with an inhibitor of protein synthesis, cycloheximide (CHX). Moreover, CsA-induced cell death was accompanied by activation of executory caspases followed by PARP cleavage. CsA treatment did not elevate fasL expression and had no effect on mitochondrial membrane potential. We conclude that CsA triggers either growth arrest or non-apoptotic, programmed cell death in human malignant glioma cells. Moreover, CsA employs mechanisms different to those in the action of radio- and chemotherapeutics, and operating even in cells resistant to conventional treatments. Thus, CsA or related drugs may be an effective novel strategy to treat drug-resistant gliomas or complement apoptosis-based therapies.     

Characterization of lovastatin–docosahexaenoate anticancer properties against breast cancer cells

Lovastatin (LOV) and docosahexaenoic acid (DHA), besides improving cardiovascular functions, are also known for their anticancer activities. However, use of these compounds for treating or preventing cancer is limited because of their efficacies. The approach pursued involved chemical linkage of these two chemotypes. A lovastatin–docosahexaenoate (LOV–DHA) conjugate was prepared and tested against selected breast tumor cells lines with differential expression of estrogen receptors (ER) and Heregulin-2 (Her-2). The LOV–DHA conjugate exhibited superior cytotoxic effects against ER⁻/Her-2⁻ cell lines (MDA-MB-231 and MDA-MB-468), which were not observed with DHA or lovastatin alone, or in combination. Lovastatin supplementation arrested cells in the G₀/G₁ phase and enhanced expression levels of p21, whereas the conjugate did not demonstrate cell cycle arrest nor increased p21 expression. The LOV–DHA conjugate induced significant (P < 0.05) apoptosis as low as 1 μM, whereas DHA and lovastatin were ineffective at this concentration. The growth inhibitory effects of lovastatin were reversed by the addition of mevalonate, whereas mevalonate had no effect on the LOV–DHA conjugate-induced growth inhibition in MDA-MB-231 cells. Furthermore, the LOV–DHA conjugates were stable in mouse serum and intracellularly in MDA-MB-231 cells. These data suggest that the LOV–DHA conjugate mediated its effects through a HMG-CoA reductase-independent pathway and exerted significantly (P < 0.05) higher anticancer effects in breast cancer cells than lovastatin or DHA alone.