Charles E. Elson

Studies of the Isoprenoid-Mediated Inhibition of Mevalonate Synthesis Applied to Cancer Chemotherapy and Chemoprevention

Pools of farnesyl diphosphate and other phosphorylated products of the mevalonate pathway are essential to the post-translational processing and physiological function of small G proteins, nuclear lamins, and growth factor receptors. Inhibitors of enzyme activities providing those pools, namely, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and mevalonic acid-pyrophosphate decarboxylase, and of activities requiring substrates from the pools, the prenyl protein transferases, have potential for development as novel chemotherapeutic agents. Their potentials as suggested by the clinical responses recorded in Phase I and II investigations of inhibitors of HMG CoA reductase (the statins), of mevalonic acid-pyrophosphate decarboxylase (sodium phenylacetate and sodium phenylbutyrate), and of farnesyl protein transferase (R115777, SCH66336, BMS-214662, Tipifarnib, L-778,123, and, prematurely, perillyl alcohol) are dimmed by dose-limiting toxicities. These nondlscriminant growth-suppresslve agents induce G1 arrest and initiate apoptosis and differentiation, effects attributed to modulation of cell signaling pathways either by modulating gene expression, suppressing the post-translational processing of signaling proteins and growth factor receptors, or altering dlacylglycerol signaling. Diverse isoprenoids and the HMG CoA reductase inhibitor, lovastatin, modulate cell growth, induce cell cycle arrest, initiate apoptosis, and suppress cellular signaling activities. Perillyl alcohol, the isoprenoid of greatest clinical interest, initially was considered to inhibit farnesyl protein transferase; follow-up studies revealed that perillyl alcohol suppresses the synthesis of small G proteins and HMG CoA reductase. In sterologenic tissues, sterol feedback control, mediated by sterol regulatory element binding proteins (SREBPs) 1a and 2, exerts the primary regulation on HMG CoA reductase activity at the transcriptional level. Secondary regulation, a nonsterol isoprenoid-mediated fine-tuning of reductase activity, occurs at the levels of reductase translation and degradation. HMG CoA reductase activity in tumors is elevated and resistant to sterol feedback regulation, possibly as a consequence of aberrant SREBP activities. Nonetheless, tumor reductase remains sensitive to isoprenoid-mediated post-transcrlptional downregulation. Farnesol, an acyclic sesquiterpene, and farnesyl homologs, γ-tocotrienol and various farnesyl derivatives, inhibit reductase synthesis and accelerate reductase degradation. Cyclic monoterpenes, d-limonene, menthol and perillyl alcohol and β-ionone, a carotenoid fragment, lower reductase mass; perillyl alcohol and d-limonene lower reductase mass by modulating translational efficiency. The elevated reductase expression and greater demand for nonsterol products to maintain growth amplify the susceptibility of tumor reductase to Isoprenoids, therein rendering tumor cells more responsive than normal cells to isoprenoid-mediated growth suppression. Blends of lovastatin, a potent nondiscrlminant Inhibitor of HMG CoA reductase, and γ-tocotrienol, a potent isoprenoid shown to post-transcriptlonally attenuate reductase activity with specificity for tumors, synergistically affect the growth of human DU145 and LNCaP prostate carcinoma cells and pending extensive preclinical evaluation, potentially offer a novel chemotherapeutic strategy free of the dose-limiting toxicity associated with high-dose lovastatin and other nondiscrlminant mevalonate pathway inhibitors.

Response of hypercholesterolemic subjects to administration of tocotrienols

The cholesterol-suppressive actions of Palmvitee and γ-tocotrienol were assessed in hypercholesterolemic subjects after acclimation to the American Heart Association Step 1 dietary regimen for four and eight weeks, respectively. The four-week dietary regimen alone elicited a 5% decrease (P<0.05) in the cholesterol level of the 36 subjects. Subjects continuing on the dietary regimen for a second four-week period experienced an additional 2% decrease in their cholesterol levels. Dietary assessments based on unanticipated recalls of 24-h food intake records suggest that significant reductions in energy and fat, predominantly in saturated fat, intakes are responsible. The subjects experienced significant Palmvitee- and γ-tocotrienol-mediated decreases in cholesterol. The group of subjects acclimated to the dietary regimen for four weeks responded to Palmvitee (a blend of tocols providing 40 mg α-tocopherol, 48 mg α-tocotrienol, 112 mg γ-tocotrienol, and 60 mg δ-tocotrienol/day for four weeks) with a 10% decrease in cholesterol (P<0.05). Dietary assessments showed no further change in energy and fat intakes. α-Tocopherol attenuates the cholesterol-suppressive action of the tocotrienols. The second group of subjects, acclimated to the dietary regimen for eight weeks, received 200 mg-γ-tocotrienol/d for four weeks. The cholesterol-suppressive potency of this α-tocopherol-free preparation was calculated to be equivalent to that of the mixture of tocotrienols (220 mg) used in the prior study. Cholesterol levels of the 16 subjects in the second group decreased 13% (P<0.05) during the four-week trial. Plasma apolipoprotein B andex vivo generation of thromboxane B2 were similarly responsive to the tocotrienol preparations, whereas neither preparation had an impact on high density lipoprotein cholesterol and apolipoprotein A-I levels.

The inhibition of adenine nucleotide translocase activity by oleoyl CoA and its reversal in rat liver mitochondria.

The inhibitory effect of oleic acid on the 32Pi-ATP exchange activity and 14C-ADP translocation through the mitochondrial membrane can be reversed by substrates preferentially utilizing CoA and thereby preventing acylation of the fatty acid. By contrast the inhibition produced by oleoyl CoA can be reversed only when carnitine is added to augment oxidation of the CoA ester. The slow rate of respiration and sluggish response to ADP in oleoyl CoA treated mitochondria is overcome by the uncoupler, salicyl-anilide XIII. Inhibition of translocation of adenine nucleotides through the inner mitochondrial membrane by oleoyl CoA could regulate metabolism by inducing a transition from state 3 to state 4 respiration.

Inhibition of cholesterol and fatty acid biosynthesis in liver enzymes and chicken hepatocytes by polar fractions of garlic.

Different concentrations of polar fractions, methanol-soluble (MESF), or water-soluble (WASF), of 1–8% equivalent to fresh garlic paste were added to yellow corn-soybean based diets and fed to 5-week-old male broiler chickens for 3 weeks to measure the inhibition of hepatic β-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) reductase, cholesterol 7α-hydroxylase (7α-hydroxy) and fatty acid synthetase (FAS). Dose-related decreases in the activities of these enzymes were obtained. Decreases in serum total cholesterol and in low density lipoprotein (LDL) levels were also observed. There was no effect on the level of cholesterol in high density lipoprotein (HDL). The most effective dose for these decreases was found 0.54% (MESF) and 1.2% (WASF) equivalent to 6% of the fresh garlic. The inhibition of HMG-CoA reductase and FAS by 25–300 μm of MESF or WASF for 15 min was tested in vitro, in male and female chicken hepatocytes. Inhibitions of activity were dose-dependent and the degree of inhibition increased with duration of incubation (150 μg of MESF or WASF 5 to 60 min). Dietary supplementation of odorless WASF of garlic was found to be very effective in lowering the total and LDL cholesterol levels compared to control chickens.

Tropical oils: Nutritional and scientific issues

Individually and in combination with other oils, the tropical oils impart into manufactured foods functional properties that appeal to consumers. The use of and/or labeling in the ingredient lists give the impression that these oils are used extensively in commercially processed foods. The estimated daily intake of tropical oils by adult males is slightly more than one fourth of a tablespoon (3.8 g), 75% of which consists of saturated fatty acids. Dietary fats containing saturated fatty acids at the beta-position tend to raise plasma total and LDL-cholesterol, which, of course, contribute to atherosclerosis and coronary heart disease. Health professionals express concern that consumers who choose foods containing tropical oils unknowingly increase their intake of saturated fatty acids. The saturated fatty acid-rich tropical oils, coconut oil, hydrogenated coconut oil, and palm kernel oil, raise cholesterol levels; studies demonstrating this effect are often confounded by a developing essential fatty acid deficiency. Palm oil, an essential fatty acid-sufficient tropical oil, raises plasma cholesterol only when an excess of cholesterol is presented in the diet. The failure of palm oil to elevate blood cholesterol as predicted by the regression equations developed by Keys et al. and Hegsted et al. might be due to the dominant alpha-position location of its constituent saturated fatty acids. If so, the substitution of interesterified artificial fats for palm oil in food formulations, a recommendation of some health professionals, has the potential of raising cholesterol levels. A second rationale addresses prospective roles minor constituents of palm oil might play in health maintenance. This rationale is founded on the following observations. Dietary palm oil does not raise plasma cholesterol. Single fat studies suggests that oils richer in polyunsaturated fatty acid content tend to decrease thrombus formation. Anomalously, palm oil differs from other of the more saturated fats in tending to decrease thrombus formation. Finally, in studies comparing palm oil with other fats and oils, experimental carcinogenesis is enhanced both by vegetable oils richer in linoleic acid content and by more highly saturated animal fats. The carotenoid constituents of red palm oil are potent dietary anticarcinogens. A second group of antioxidants, the tocotrienols, are present in both palm olein and red palm oil. These vitamin E-active constituents are potent suppressors of cholesterol biosynthesis; emerging data point to their anticarcinogenic and antithrombotic activities. This review does not support claims that foods containing palm oil have no place in a prudent diet.

Induction of geranyl pyrophosphate pyrophosphatase activity by cholesterol-suppressive isoprenoids

Diets supplemented (1 mmol/kg) with thymol, carvacrol, and β-ionone significantly decreased the serum cholesterol levels of cockerels. These mevalonate-derived end products of plant secondary metabolism (isoprenoids) had no impact on two cytosolic prenyl alcohol (and ethanol) dehydrogenase activities; each treatment increased microsomal geranyl pyrophosphate pyrophosphatase activity by greater than twofold. The structural diversity of the isoprenoids which suppress cholesterol synthesis may be reconciled by their ability to increase pyrophosphatase activity, thus leading to the production of the endogenous, post-transcriptional regulator of 3-hydroxy-3-methyglutaryl coenzyme A reductase activity.

Human metabolism of the experimental cancer therapeutic agentd-limonene

Abstractd-Limonene has efficacy in preclinical models of breast cancer, causing >80% of carcinomas to regress with little host toxicity. We performed a pilot study on healthy human volunteers to identify plasma metabolites of limonene and to assess the toxicity of supradietary quantities ofd-limonene. Seven subjects ingested 100 mg/kg limonene in a custard. Blood was drawn at 0 and 24 h for chemistry-panel analysis and at 0, 4, and 24 h for limonenemetabolite analysis. On-line capillary gas chromatography/mass spectrometry (GC/MS) analysis indicated that at least five compounds were present at 4 h that were not present at time zero. Two major peaks were identified as the rat limonene metabolites dihydroperillic acid and perillic acid, and two minor peaks were found to be the respective methyl esters of these acids. A third major peak was identified as limonene-1,2-diol. Limonene was a minor component. At a dose of 100 mg/kg, limonene caused no gradable toxicity. Limonene is metabolized by humans and rats in a similar manner. These observations and the high therapeutic ratio of limonene in the chemotherapy of rodent cancers suggest that limonene may be an efficacious chemotherapeutic agent for human malignancies.

A comparison of tocopherol and tocotrienol for the chemoprevention of chemically induced rat mammary tumors

Two forms of vitamin E, tocopherol and tocotrienol, were tested for chemopreventive activity in two chemically induced rat mammary-tumor models. When mammary tumors were induced by 7,12-dimethylbenz(a)anthracene (DMBA, 50 mg/kg), only the tocotrienol group had a statistically significant increase in tumor latency. There was no effect of either compound on tumor multiplicity. When tumors were induced by N-nitrosomethylurea (NMU, 30 mg/kg), neither analogue of vitamin E modified latency, whereas tocotrienol increased tumor multiplicity. In summary, neither vitamin analog had a major impact on mammary-tumor development after tumor induction with either DMBA or NMU.

Inhibition of type I and type II geranylgeranyl-protein transferases by the monoterpene perillyl alcohol in NIH3T3 cells

The monoterpene perillyl alcohol has anticancer activities that include both prevention and treatment of a wide variety of cancers in animal models. In purified enzyme studies, perillyl alcohol inhibited farnesyl-protein transferase and type I geranylgeranyl-protein transferase. However, whether and which of the polyprenyl-protein transferases is inhibited by perillyl alcohol in vivo is not known. The previously reported monoterpene-induced inhibition of the incorporation of [14C]mevalonolactone into proteins in cultured cells could be due to an inhibition of one or several enzymes in the mevalonate pathway or to changes in the levels of protein substrates for isoprenylation. In the current study, we first analyzed the levels of individual phosphorylated isoprenoid intermediates between mevalonate and geranylgeranyl pyrophosphate in NIH3T3 cells labeled for 4 hr with [14C]mevalonolactone and found that perillyl alcohol did not inhibit the synthesis of these intermediates. Next, proteins including Ras, RhoA, and Rab6 were immunoprecipitated from NIH3T3 cells. Perillyl alcohol was found to inhibit the incorporation of [14C]mevalonolactone into RhoA and Rab6 but not Ras protein. The cellular levels of these three proteins were constant over the 4-hr treatment period. Finally, the distribution of Ras, Rap1, and Rab6 proteins between the aqueous and the detergent-enriched phases was measured. Rap1 and Rab6 but not Ras from perillyl alcohol-treated NIH3T3 cells accumulated in the aqueous phase. Thus, we conclude that perillyl alcohol can inhibit the in vivo prenylation of specific proteins by type I and type II geranylgeranyl-protein transferases but not farnesyl-protein transferase in NIH3T3 cells.

The role of fatty acid binding protein on the metabolism of fatty acids in isolated rat hepatocytes

In isolated rat hepatocytes flavaspidic acid, a competitor with free fatty acids for the fatty-acid-binding-protein, decreased the uptake of oleic acid and triglyceride synthesis but stimulated the formation of CO2 and ketone bodies from oleic acid. Flavaspidic acid had no effect on the utilization of octanoic acid. Stimulation of the microsomal fatty-acid-activating enzyme by the fatty-acid-binding protein was reversed by flavaspidic acid. In contrast, the binding protein inhibited the mitochondrial fatty-acid-activating enzyme. Flavaspidic acid not only prevented this inhibition but actually stimulated the enzyme activity. The results indicate that the cytosol fatty-acid-binding protein directs the metabolism of long chain fatty acids toward esterification as well as enhancing their cellular uptake.