Journal of the American College of Nutrition, Vol. 20, No. 6, 628-636 (2001)
Published by the American College of Nutrition
Alpha Tocopheryl Succinate, Retinoic Acid and Polar Carotenoids Enhanced the Growth-Inhibitory Effect of a Cholesterol-Lowering Drug on Immortalized and Transformed Nerve Cells in Culture
Bipin Kumar, MD,
William C. Cole, PhD and
Kedar N. Prasad, PhD
Center for Vitamins and Cancer Research, Department of Radiology, University of Colorado Health Sciences Center, Denver, Colorado
Address reprint requests to: Kedar N. Prasad, Ph.D., Campus Box C-278, Department of Radiology, University of Colorado Health Science Center, 4200 East 9th Ave., Denver, CO 80262. E-mail: kedar.prasad{at}uchsc.edu.
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ABSTRACT
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Objective: Statins (cholesterol lowering drugs) with a closed-ring structure (lovastatin, simvastatin and mevastatin) and an open-ring structure (pravastatin and fluvastatin) are currently used in the management of cardiac disease. Lovastatin and simvastatin inhibit the growth of tumor cells; however, the studies on the effect of a statin in combination with micronutrients such as 
-tocopheryl succinate (-TS), 13-cis retinoic acid (RA) and polar carotenoids (PC) have never been performed. The primary objective of this study was to investigate the effect of mevastatin alone and in combination with the above micronutrients on the growth of mouse neuroblastoma (NB) cells and rat immortalized dopamine (DA) neurons in culture. In addition, a comparative efficacy of mevastatin and pravastatin on the growth of NB cells was studied.
Methods: Cells were treated with mevastatin in combination with individual antioxidants, -TS, RA and polar carotenoids, 24 hours after plating. Fresh growth medium and agents were changed at two days after treatment, and the viability in control and experimental groups was determined at three days after treatment by MTT assay. Each experiment was repeated three times with triplicate samples per treatment. Growth in experimental groups was expressed as % of untreated cells.
Results: Mevastatin inhibited the growth of neuroblastoma (NB) cells and immortalized, non-tumorigenic dopamine (DA) neurons in culture in a dose-dependent manner. Immortalized DA neurons were more sensitive to mevastatin than NB cells. Pravastatin at similar concentrations was ineffective in inhibiting the growth of NB cells. Mevastatin in combination with -TS, RA or PC was more effective in reducing the growth of NB and DA neurons than the individual agents.
Conclusions: Statins with a closed-ring structure can inhibit the growth of established cancer cells as well as immortalized cells (equivalent to pre-malignant lesion), whereas statins with an open-ring structure may be ineffective. A combination of a statin having a closed-ring structure with -TS, RA and PC may be one of the potentially useful anti-cancer agents for prevention and treatment strategies.
Key words: statins, neuroblastoma cells, immortalized dopamine neurons, vitamin E, retinoic acid, polar carotenoids, growth
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INTRODUCTION
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Statins (cholesterol lowering drugs) and micronutrients such as vitamin E are currently being used in the management of cardiac disease. Statins can be divided into two groups, those containing a closed-ring structure (lovastatin, simvastatin and mevastatin) and those (pravastatin and fluvastatin) having an open-ring structure [1]. The open-ring structure is responsible for inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, whereas the closed ring structure is responsible for the inhibition of proteasome activity [2]. Statins with a closed-ring structure are converted to an open-ring structure in vivo by liver enzymes for their cholesterol-lowering effects; however, part of them remain as a closed-ring structure. The anti-cancer activities of lovastatin and simvastatin [3–19] and micronutrients such as vitamin E, vitamin A and carotenoids [20–25] in vitro and in vivo have been reported. However, the effects of a statin with a closed-ring structure in combination with these micronutrients on the growth of tumor cells have not been investigated. Therefore, we have investigated the effect of mevastatin alone or in combination with antioxidants on the growth of NB cells and immortalized non-tumorigenic dopamine (DA) neurons, using MTT assay technique. We report here that mevastatin with a closed-ring structure inhibited the growth of NB cells and DA neurons in culture in a dose-dependent manner; however, DA neurons were more sensitive than NB cells. Mevastatin in combination with retinoic acid (RA), d--tocopheryl succinate (-TS) or polar carotenoids (PC) was more effective than the individual agents. Pravastatin with an open ring structure was ineffective in reducing the growth of NB cells in culture.
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MATERIALS AND METHODS
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Cell Culture
Murine neuroblastoma clone (NBP2), which has both tyrosine hydroxylase and choline transferase [26], was used in this study. These cells were grown in F12 medium containing 10% agammaglobulin bovine new born serum (inactivated at 60°C for 30 min.), penicillin (100 units/mL) and streptomycin (100 µg/mL). Rat immortalized clone of DA neurons (1RB3AN27), which has DA transporter protein [27], was used in this study. These cells were immortalized by transfecting fetal rat mesencephalic cells with a plasmid vector, pSV3neo which contains large T-antigen gene of SV40 virus under the control of SV40 early promoter and a neomycin-resistant gene. These cells were grown in RPMI-1640 medium containing 10% fetal bovine serum and the same amounts of antibiotics as those added for NB growth medium. All cells were maintained at 37°C in a humidified atmosphere of 5% CO2. The doubling times for NB cells and immortalized DA neurons were 18 hours and 27 hours, respectively [26,27].
Solution Preparations
Mevastatin and pravastatin (Sigma, St. Louis, MO) were dissolved in ethanol and water, respectively, at a concentration of 5 mg/mL and were further diluted as needed. D--tocopheryl succinate (Henkel Corporation, Chicago, IL) and RRR-13-cis-retinoic acid (Sigma) were dissolved in ethanol at a concentration of 5 mg/mL and were further diluted as needed. Synthetic ß-carotene (Hoffman La Roche) was dissolved in DMSO:ethanol mixture (1:9) at a concentration of 5 mg/mL. This suspension was further diluted in the same solvent at a concentration of 1 mg/mL. This preparation contains unidentified peaks without any peak of beta-carotene. These unidentified peaks were referred to as polar carotenoids (PC) because of a higher degree of solubility. All solutions were protected from the light and were stored at 4°C until use.
Assay of Growth
MTT assay was used to determine the viability of cells, and the growth of experimental groups was determined as % of untreated controls. The CellTiter 96R Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI) is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. The CellTiter 96R Aqueous One Solution reagent contains a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-fophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES). PES has enhanced chemical stability, which allows it to be combined with MTS to form a stable solution. The MTS tetrazolium compound (Owen’s reagent) is bio-reduced by cells into a colored formazan product that is soluble in RPMI-1640 without phenol red (Sigma) medium.
Murine NB and immortalized DA neurons (20,000) were plated in 24-well chambers (Falcon) in their respective growth medium. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2. Twenty-four hours after plating, mevastatin alone or in combination with an antioxidant was added at different concentrations. After 48 hours of treatment, the fresh medium and drugs were changed. After 72 hours of treatment, the medium was removed and 400 µL of RPMI-1640 medium without phenol red was added with 20 µg/100 µL CellTiter 96R Aqueous One Solution Cell Proliferation Assay solution. Cells were incubated for 4 hours. The quantity of formazan product, as measured by the absorbance at 490 nm by fluorometry 1420 VICTOR multi-label counter (Wallac EG and G Company), is directly proportional to the number of living cells in culture. The rate of expression of damage produced by micronutrients is rather slow; therefore, a treatment time of three days (equivalent to three generation times) is needed in order to produce a significant effect on growth inhibition. Each experiment was performed at least three times in triplicate.
HPLC Profiles of Statins
Cells were incubated in the presence of mevastatin (10 µg/mL) for a period of 4 and 24 hours at 37°C. After incubation cells were removed from the dishes by a standard procedure, pellets were washed, and then a pellet of 10 million cells was prepared. Mevastatin was extracted by adding 0.5 mL of extraction solvent (acetonitrile and THF in the ratio of 3:2) vortexed frequently and then centrifuged at 3000 RPM. A 50 µL aliquot of supernatant was injected into a C-18 column using mobile phase solvent (70% acetonitrile, 30% water and 0.1% trifluoroacetic acid). The HPLC profile was determined at wavelength 238 nm, sensitivity 2 and flow rate 1 mL/mL. The extraction efficiency under the above experimental condition was 98.9%. To determine the purity of mevastatin and pravastatin, a solution of these compounds was mixed in equal volume, and HPLC profile was determined by directly injecting 50 µL of this solution into the column as described above.
Statistical Analysis
Data variability about the mean for growth was expressed as the 95% confidence interval (95% CI = (1.96 x SD)/square root of sample size. The additive and synergistic effect was determined by the following formula: surviving fraction in treatment A times surviving fraction in treatment B equals surviving fraction C times 100. If the observed value of combined treatment of A and B is similar to that of C, the effect is considered additive; if the observed value of combined treatment A and B is lower than that of C, the effect is considered synergistic; if the observed value of combined treatment is less than that of C, the effect is considered protective.
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RESULTS
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Effect of Mevastatin Alone on Growth
Mevastatin inhibited the growth of NB cells and non-tumorigenic DA neurons in a dose-dependent manner (Fig. 1A); however, DA neurons were more sensitive than NB cells. A 50% growth inhibition in NB cells required about 25.6 µM of mevastatin, whereas it needed only about 5.12 µM of mevastatin for DA neurons, a difference in sensitivity of about fivefold. Pravastatin, which contains an open-ring structure, at a concentration up to 22.4 µM was ineffective in reducing the growth of NB cells in culture (Fig. 1A). Therefore, the effect of pravastatin on the growth of dopamine neurons was not studied. Pravastatin was also found to be ineffective on PC-12 cells in culture [14].
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Fig. 1A. Effect of mevastatin on the growth of mouse neuroblastoma (NBP2) cells and rat immortalized non-tumorigenic dopamine (DA) neurons, 1 RB3AN27 (N27) in culture. Cells were plated in a 24-well chamber and compounds were added 24 hours later. Cell viability was determined after three days of treatment. Each experiment was repeated three times with triplicate samples. Error bars are 95% confidence intervals. 1B. High performance liquid chromatography (HPLC) profile of pravastatin (P, open-ring structure) and mevastatin (M, closed-ring structure) indicating distinct peaks.
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The HPLC profile revealed that the pravastatin (P) peak appeared earlier than the mevastatin (M) peak and that there was no impurity in any of these preparations (Fig. 1B). In order to investigate whether any portion of mevastatin is converted to an open-ring structure by NB cells, cells were incubated in the presence of mevastatin for 4 and 24 hours. Mevastatin was extracted, and the HPLC profile was determined. Data show that NB cells did not accumulate any detectable level of mevastatin after 4 hours of incubation; however, after 24 hours significant amounts of this statin accumulated in the cells (Table 1). Results also showed that NB cells failed to convert any portion of mevastatin into an open-ring structure.
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Table 1. Uptake and Metabolism of Mevastatin with a Close-Ring Structure in Undifferentiated Neuroblastoma (NBP2) Cells in Culture
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Effect of Micronutrients Alone on Growth
Micronutrients, such as RA, -TS and PC at high doses inhibited the growth of NB cells in a dose-dependent manner (Fig. 2A). NB cells were most sensitive to PC and least sensitive to -TS. Similar observations were made on immortalized DA neurons except that -TS produced no growth inhibition at doses up to 28.3 µM (Fig. 2B). Solvent (ethanol or DMSO and ethanol mixture) treatment did not alter the growth of NB cells or DA neurons.
Effect of Mevastatin in Combination with Various Concentrations of Micronutrients
Mevastatin doses of 25.6 µM for NB cells and 5.12 µM for DA neurons, which inhibited the growth by about 50%, were selected for this study. Mevastatin in combination with retinoic acid, -TS or polar carotenoids was more effective in reducing the growth of NB cells (Fig. 3A) and DA neurons (Fig. 3B) than the individual agents. Mevastatin in combination with -TS produced a synergistic effect on growth inhibition at all -TS concentrations in NB cells; however, in combination with PC, it produced an additive effect at all PC concentrations. On the other hand, mevastatin, in combination with RA, produced an additive effect at lower concentrations (8.3 and 16.6 µM) and a synergistic effect at higher concentrations (33.3 and 49.9 µM). In dopamine neurons, mevastatin in combination with a low dose of -TS increased the survival more than that produced by mevastatin alone (Fig. 3B). The synergistic effect of the combined treatment was observed only at a high concentration of -TS (28.3 µM). This concentration of -TS by itself had no effect on the growth of dopamine neurons. Mevastatin, in combination with RA, produced a synergistic effect on growth inhibition only at a high RA concentration (66.8 µM), whereas in combination with PC, it produced a synergistic effect at all PC concentrations. Solvent treatment did not alter the growth of NB cells or DA neurons.
Effect of Micronutrients in Combination with Various Concentrations of Mevastatin
Generally, micronutrients such as -TS, RA and PC in combination with high doses of mevastatin were more effective in reducing the growth of NB cells (Table 2) and dopamine neurons (Table 3). In NB cells, all micronutrients in combination with a low dose (5.1 µM) of mevastatin produced an additive effect on growth inhibition, whereas at higher concentrations of mevastatin, they produced a synergistic effect (Table 2). In dopamine neurons, micronutrients (-TS and RA) in combination with a low dose of mevastatin (1.3 µM) produced growth inhibition which was similar to that caused by individual agents; however, mevastatin at this dose protected dopamine neurons from the growth-inhibitory effects of PC (Table 3). -TS in combination with a mevastatin dose of 2.6 µM produced no additional effect on growth inhibition, whereas RA and PC in combination with this dose of mevastatin produced an additive effect. All micronutrients in combination with a high dose of mevastatin (5.1 µM) produced a synergistic effect (Table 3). Solvent treatment did not alter the growth of NB cells or dopamine neurons.
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Table 2. Effect of -Tocopheryl Succinate (-TS), Retinoic Acid (RA) and Polar Carotenoids (PC) in Combination with Various Concentrations of Mevastatin on the Growth of Neuroblastoma Cells in Culture
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Table 3. Effect of -Tocopheryl Succinate (-TS), Retinoic Acid (RA) and Polar Carotenoids (PC) in Combination with Various Concentrations of Mevastatin on the Growth of Immortalized Dopamine Neurons (N27) in Culture
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DISCUSSION
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This study shows that a 50% growth inhibition in NB cells required 25.6 µM of mevastatin, whereas it needed only 5.12 µM of mevastatin for immortalized DA neurons, a difference in sensitivity of about fivefold. The anti-tumor activity of mevastatin is similar to that reported with other statins with a closed-ring structure (lovastatin and simastatin) [3–19]. This study shows that pravastatin with an open-ring structure at similar concentrations was ineffective in reducing the growth of NB cells in culture. This is consistent with a previous study in which pravastatin did not inhibit the growth of rat pheochromocytoma cells, PC-12, in culture [14]. These studies suggest that statins with a closed-ring structure exhibit anti-cancer activity, whereas statins with an open-ring structure are ineffective. This is further supported by the fact that the incidence of all types of cancer was reduced (observed 14 cases vs. expected 21 cases) among patients with hypercholesterolemia who were taking lovastatin, a statin with a closed-ring structure [28]. The above study is a small preliminary analysis of toxicity among people consuming lovastatin and must be confirmed by a well-designed epidemiologic study.
This study shows that RA, -TS and PC inhibited the growth of NB cells in culture in a dose-dependent manner. RA and PC produced similar effects on dopamine neurons, but -TS was ineffective. Polar carotenoid was most effective, whereas -TS was relatively less effective in reducing the growth of these cells. These results are consistent with those reported earlier [20–25]. The actions of these micronutrients on growth inhibition involves mechanisms that are not related to their known antioxidation effect [24]. To study the effect of mevastatin in combination with -TS, RA and PC, a dose of mevastatin that reduced the growth of NB cells and DA neurons by about 50% was selected. This study shows that mevastatin in combination with retinoic acid, -TS or polar carotenoids at high doses was more effective in reducing the growth of NB cells and DA neurons than the individual agents. The extent of the effect of the combined treatment–additive, synergistic or protective–depends upon the type of cells, dose of mevastatin and dose of micronutrient. It is interesting to note that the effect of mevastatin on NB cells was enhanced, even by concentrations of -TS which by themselves did not affect the growth of these cells. It is also interesting to note that low doses of mevastatin reduce the levels of PC-induced growth inhibition in DA neurons. These studies suggest that the combination of statins having a closed-ring structure with high doses of -TS, RA and PC may result in a need for lower doses of these cholesterol-lowering drugs for cancer prevention, thereby reducing the risk of long-term side-effects of statins. The mechanisms of action of -TS, RA and PC in enhancing the effect of mevastatin on tumor cells and immortalized cells unknown; however, these micronutrients produce effects on tumor cells that are not related to their antioxidation action.
At present, statins with both a closed-ring and an open-ring structure are used to manage cardiac disease among a large number of people in the U.S. Many of them are also taking some form of micronutrient supplements. This group of patients provides a unique opportunity to perform an epidemiologic study to establish whether an intake of statin alone or in combination with certain micronutrients is associated with the reduction in cancer incidence.
High-dose micronutrients are also used as an adjunct to standard cancer therapy [20,25, Pathak et al. unpublished observation]. High-dose lovastatin has exhibited anti-cancer activity in phase I trial for the treatment of human malignant astrocytoma [19]. This statin enhances the growth-inhibitory effect of a chemotherapeutic agent, NN'-Bis-(2-chloroethyl)-N-nitrosourea (BCNU), on human glioma cells in culture in a synergistic manner [12]. These studies suggest that statins having a closed-ring structure in combination with high-dose micronutrients could be used as an adjunct to standard cancer therapy. However, additional laboratory studies are needed to define the interaction between statins, micronutrients and standard cancer therapeutic agents in regulating growth of normal and tumor cells before they can be used in the treatment of cancer.
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ACKNOWLEDGMENTS
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This study was supported by Shafroth Memorial Foundation and by NIH grants AG18285 and NS 38647.
Received February 6, 2001.
Accepted May 9, 2001.
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REFERENCES
|
|---|
- Harman JG, Gilman AG, Limbird LE (eds):
"Goodman & Gilman’s The Pharmacological Basis of Therapeutics," 9th Ed. New York: McGraw Hill, p
886,
1996.
- Rao S, Porter DC, Chen X, Herliczek T, Lowe M, Keyomarsi K: Lovastatin-mediated G1-arrest is through inhibition of the proteosome, independent of hydroxymethyl glutaryl-CoA reductase.
Proc Natl Acad Sci USA
96:
7797–7802,
1999.[Abstract/Free Full Text]
- Wejde J, Blegen H, Larsson O: Requirement for mevalonate in control of proliferation of human breast cancer cells.
Anticancer Research
12:
317–324,
1992.[Medline]
- Larsson O, Blegen H: Inhibitory effect of mevalonate on the EGF mitogenic signaling pathway in human breast cancer cells in culture.
Cancer Biochem Biophys
14:
193–200,
1994.[Medline]
- Larsson O: Cell cycle-specific growth inhibition of human breast cancer cells induced by metabolic inhibitors.
Glycobiology
3:
475–479,
1993.[Abstract/Free Full Text]
- Bonapace IM, Addeo R, Altucci L, Cicatiello L, Bifulco M, Laezza C, Salzano S, Sica V, Bresciani F, Weisz A: 17 beta-estradiol overcomes a G1 block induced by HMG-CoA reductase inhibitors and fosters cell cycle progression without inducing ERK-1 and -2 MAP kinases activation.
Oncogene
12:
753–763,
1996.[Medline]
- Addeo R, Altucci L, Battista T, Bonapace IM, Cancemi M, Cictiello L, Germano D, Pacilio C, Salzano S, Bresciani F, Weisz A: Stimulation of human breast cancer MCF-7 cells with estrogen prevents cell cycle arrest by HMG-CoA reductase inhibitors.
Biochem Biophys Res Comm
220:
864–870,
1996.[Medline]
- Rao S, Lowe M, Herliczek TW, Keyomarsi K: Lovastatin mediated G1-arrest in normal and tumor breast cells is through inhibition of CDK2 activity and redistribution of p21 and p27, independent of p53.
Oncogene
17:
2393–2402,
1998.[Medline]
- Lee SJ, Ha MJ, Lee J, Nguyen P, Choi YH, Pirnia F, Kang WK, Wang XF, Kim SJ, Trepel JB: Inhibition of the 3-hydroxyl-3-methylglutaryl-coenzyme A reductase pathway induces p53-independent transcriptional regulation of p21 (WAF1/CIP1) in human prostate carcinoma cells.
J Biol Chem
273:
10618–10623,
1998.[Abstract/Free Full Text]
- Jones KD, Couldwell WT, Hinton DR, Su Y, He S, Anker L, Law RE: Lovastatin induces growth inhibition and apoptosis in human malignant glioma cells.
Biochem Biophys Res Comm
205:
1681–1687,
1994.[Medline]
- Prasanna P, Thibault A, Liu L, Samid D: Lipid metabolism as a target for brain cancer therapy: synergistic activity of lovastatin and sodium phenylacetate against human glioma cells.
J Neurochem
66:
710–716,
1996.[Medline]
- Soma MR, Pagliarini P, Butti G, Paoletti R, Paoletti P, Fumagalli R: Simastatin, an inhibitor of cholesterol biosynthesis shows a synergistic effect with N,N'-Bis(2-chloroethyl)-N-nitrosourea and ß-interferon on human glioma cells.
Cancer Res
52:
4343–4345,
1992.
- Nordenberg J, Goldwasser I, Zoref-Shani E, Beery E, Sidi Y: Inhibition of B16 melanoma cell proliferation and alterations in p21 ras expression induced by interceptors of signal transduction pathways.
Israel J Med Sci
32:
1153–1157,
1996.[Medline]
- Sato-Suzuki I, Murota S-I: Simastatin inhibits the division and induces neurite like outgrowth in PC-12 cells.
Neuroscience Letters
220:
21–24,
1996.[Medline]
- Maltese WA, Defendini R, Green RA, Sheridan KM, Donley DK: Suppression of murine neuroblastoma growth in vivo by mevinolin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
J Clin Invest
76:
1748–1754,
1985.
- Sebti SM, Tkalcevic GT, Jani JP: Lovastatin, a cholesterol inhibitor, inhibits the growth of human H-ras oncogene transformed cells in nude mice.
Cancer Commun
3:
141–147,
1991.[Medline]
- Jani JP, Tkalcevic GT, Sebti SM: Lovastatin inhibits in vivo tumor growth of H-ras and c-myc but not v-src oncogene-transformed cells.
Cell Pharmacol
1:
67–72,
1994.
- Jani JP, Specht S, Stemmler N, Blanock K, Singh SV, Gupta V, Katoh A: Metastasis of B16F10 mouse melanoma inhibited by lovastatin, an inhibitor of cholesterol biosynthesis.
Invasion Metastasis
13:
314–324,
1993.[Medline]
- Thibaul A, Cooper MR, Figg WD, Weinberger MS, Tompkins AC, Trapel J, Samid D, Myers CE: A phase I trial of hig-dose lovastatin shows activity against malignant astrocytoma.
Proc Am Assoc Cancer Res
35:
226,
1994.
- Prasad KN, Kumar A, Kochpillai V, Cole WC: High doses of multiple antioxidant vitamins: Essential ingredients in improving the efficacy of standard cancer therapy.
J Am Coll Nutr
18:
13–25,
1999.[Abstract/Free Full Text]
- Garewal H: Beta-carotene and antioxidant nutrients in oral cancer prevention. In Prasad KN, Santamaria L, Williams RM (eds):
"Nutrients in Cancer Prevention and Treatment." Totowa, NJ: Humana Press, pp
235–247,
1995.
- Meyskens Jr FL: Role of vitamin A and its derivatives in the treatment of human cancer. In Prasad KN, Santamaria L, Williams RM (eds):
"Nutrients in Cancer Prevention and Treatment." Totowa, NJ: Humana Press, pp
349–362,
1995.
- Schwartz JL: Molecular and biochemical control of tumor growth following treatment with carotenoids or tocopherols. In Prasad KN, Santamaria L, Williams RM (eds):
"Nutrients in Cancer Prevention and Treatment." Totowa, NJ: Humana Press, pp
287–316,
1995.
- Kline K, Yu W, Zhao B: Vitamin E succinate: Mechanisms of action as tumor growth inhibitor. In Prasad KN, Santamaria L, Williams RM (eds):
"Nutrients in Cancer Prevention and Treatment," Totowa, NJ: Humana Press, pp
39–55,
1995.
- Conklin KA: Dietary antioxidants during cancer chemotherapy: Impact on chemotherapeutic effectiveness and development of side effects.
Nutr Cancer
37:
1–18,
2000.[Medline]
- Prasad KN, Mandal B, Waymire JC, Lees GJ, Vernadakis A, Weiner N: Basal level of neurotransmitter synthesizing enzymes and effect of cyclic AMP agents on the morphological differentiation of isolated neuroblastoma clones.
Nat New Biol
241:
117–119,
1973.[Medline]
- Prasad KN, Carvalho E, Kentroti S, Edwards-Prasad J, Freed CR, Vernadakis A: Establishment and characterization of immortalized clonal cell lines from fetal rat mesencephalic tissue.
In Vitro Cell Dev Biol
30:
596–603,
1994.
- Stein EA, Lazkarzewski P, Steiner P: The Lovastatin Study Group I–IV: Lovastatin 5-year safety and efficacy study.
Arch Intern Med
153:
1079–1085,
1993.[Abstract/Free Full Text]
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ABSTRACT
INTRODUCTION
