Vincristine and vinblastine lower plasma cholesterol concentrations in rhesus monkeys

482

Biochrmica et Bioph.vsica Acta, 752 (1983) 482-487 Elsevier

BBA 5 1450

VINCRISTINE AND VINBLASTINE RHESUS MONKEYS V. SAGAR

LOWER

SETH1 a, JON C. LEWIS b and RICHARD

PLASMA

W. ST.CLAIR

Departments of’ Medicine and ’ Pathology, Oncology and Atherosclerosis Forest University, Winston -Salem, NC 27103 (U.S.A.) (Received

Key words:

January

CHOLESTEROL

CONCENTRATIONS

IN

’ Research Centers, Bowman Gray School of Medicine of Wake

17th, 1983)

Vincristine;

Vinblastine;

Lipid metabolism;

Cholesterol; (Rhesus monkey)

3-4 days after a single clinical dose of vincristine or vinblastine in rhesus monkeys there was a marked decrease in plasma low-density lipoprotein cholesterol concentrations. There was also a concomittant increase in plasma triacylglycerol concentrations. Plasma lipid levels returned to normal concentrations within 7- 10 days after injection. Plasma high-density lipoprotein cholesterol concentrations were unaltered by the drugs. Electron micrographs of the hepatocytes from monkeys treated with vincristine or vinblastine showed an accumulation of glycogen particles and proliferation of smooth endoplasmic reticulum, which was accompanied by an increase in the number of lipoprotein-containing vesicles. These results indicate that vincristine and vinblastine alter plasma cholesterol and triacylglycerol concentrations in part by interfering with hepatic lipid and lipoprotein metabolism. These studies further suggest the possibility that other less cytotoxic alkaloids from Cutharanihus species with clinically useful hypocholesterolemic activity may be discovered.

Introduction The antitumor dimeric Cutharunthus alkaloids, vincristine and vinblastine, are used extensively as single agent or in combination with other anticancer drugs [ 1,2]. These alkaloids differ slightly in structure. The methyl group on the vindoline N atom in vinblastine is substituted by a formyl group in vincristine. These minor structural alterations bring forth profound differences in their clinical dosages, toxicities and antitumor spectrum [3,4]. The clinical intravenous bolus doses of vincristine and vinblastine are 1 and 5 mg/m’, respectively. At these doses there is a rapid disappearance of the alkaloids at 4-6 h post-injection

Abbreviations: HDL, density lipoprotein(s).

high-density

lipoprotein(s):

LDL,

000%2760/83/$03.00

0 1983 Elsevier Science Publishers

low-

B.V.

from blood with serum alkaloid levels in the range of 10-R-10-9 M in humans [5,6] and rhesus monkeys [7]. The Cuthurunthus alkaloids are known to bind to tubulin [8,9]. This association results in inhibition of microtubule synthesis [8], which in turn leads to metaphase arrest [ 10-121, and eventual cell necrosis [ 10,131. In cultured hepatoma cells a marked increase in the level of oxidized glutathione has been observed at alkaloid concentration of lo-’ M [14]. Reduction in the serum triacylglycerols and very-low-density lipoproteins has been reported in mice following intraperitoneal administration of vincristine (0.2 mg/kg body weight) [15]. Here we report the lowering of plasma cholesterol concentrations and electron microscopic examination of hepatocytes in rhesus monkeys given a single clinical dose of vincristine or vinblastine. A part of this work was reported previously at

483

the 13th International WA, U.S.A. [32].

Cancer

Congress,

Seattle,

Materials and Methods Rhesus monkeys. The four male rhesus monkeys (Mucaca mulatta) used for this study, No. 128 (11.3 kg body weight, 12.5 years old), No. 199 (13.3 kg, 12.5 years), No. 492 (11.7 kg, 15.6 years) and No. 722 (10.6 kg, 7 years) were kindly donated by Dr. Thomas B. Clarkson of the Department of Comparative Medicine of the Bowman Gray School of Medicine. The animals were kept in individual metabolic cages in a windowless room, artificially lighted from 6 a.m. to 5 p.m., and maintained at 72-78°F with 12 air changes/h. Complete medical profiles were maintained on the animals both prior to and during the study, and before entry all the animals were checked for normal liver and kidney functions. During this study they were fed either a low-fat, cholesterol-free diet of Purina Monkey Chow 25 (Ralston Purina Co., St. Louis, MO) containing 393 kcal/lOO g with 24% calories as protein, 10% calories as fat and 66% calories as carbohydrate, or a wheat flour/solid milk-based semi-purified diet containing 449 kcal/lOO g with 2 1% calories as protein, 41% calories as fat, 38% calories as carbohydrate and 0.70 mg cholesterol/kcal [ 161. These monkeys were initially fed the cholesterol-containing diet for 9 months prior to administration of the alkaloids, and then were switched to the cholesterol-free diet for 2 months before repeating the alkaloid administration. There was no weight loss or loss of appetite after the alkaloid administration. It took 50-60 days for the monkeys to reach stable levels of plasma cholesterol and triafter switching them to the acylglycerols, cholesterol-free diet. Alkaloid administration and determination. Prior to drug administration the monkeys were immobilized by intramuscular injection of ketamine hydrochloride (5- 10 mg/kg) (Bristol Laboratories, Syracuse, NY). Monkeys Nos. 128 and 199 were injected in the saphenous vein with vincristine sulfate (Eli Lilly & Co., Indianapolis, IN, 0.05 mg/kg body weight). Monkeys Nos. 492 and 722 were given a similar intravenous bolus injection of vinblastine sulfate (Eli Lilly & Co., 0.2 mg/kg).

Blood samples were withdrawn daily at 9 a.m. before and after the alkaloid injection, and plasma cholesterol, triacylglycerol and HDL-cholesterol levels were determined on overnight-fasted animals. The alkaloid content in the blood serum samples was determined by a sequential saturation radioimmunoassay, as previously described [5-71. Lipid determination. Total plasma cholesterol and triacylglycerol concentrations were determined using the Auto-Analyzer II methodology [ 171. HDL-cholesterol concentrations were determined by the heparin and manganese precipitation method [18]. These determinations were carried out in complete compliance with the Cooperative Lipid Standardization Program of the U.S. Department of Health and Human Services. Electron microscopy of hepatocytes. Liver biopsy samples were obtained by Dr. N. Lehner of the Department of Comparative Medicine by surgical procedures from the monkeys which were on Purina Monkey Chow and had been fasted overnight, both before and 72 h after injection of the alkaloids. The liver biopsy samples were fixed by immersion for 1 h in 0.1 M phosphate-buffered (pH 7.2-7.4) glutaraldehyde (2.5%). Subsequent to secondary fixation in 1% buffered OsO,, the samples were embedded in epoxy resin, sectioned with a diamond knife and double-stained with lead citrate and uranyl acetate for viewing in a Philip EM-400 electron microscope. Results and Discussion High-density lipoprotein-cholesterol concentrations in plasma were unaffected by the drug treatment, with the mean values showing no consistent trend either to increase or decrease, and the dayto-day variation being no more than that seen in multiple measurements on the same animals made over the 3 month period immediately following the drug trial (average S.D. of f7 mg HDL cholesterol/d1 while consuming the low-fat, cholesterol-free diet). In contrast, total plasma cholesterol concentrations consistently decreased in all animals for several days following administration of the drugs. This differs from the normal variation in plasma cholesterol concentrations within individual animals, in which changes occur randomly around the mean [ 191. The extent of

484

decrease varied in each animal, but ranged from 10 to 31% by day 3 with a single injection of vincristine sulfate or vinblastine sulfate (Tables I and II). This occurred in animals consuming either the cholesterol-containing or cholesterol-free diets. Since there was little influence on HDL-cholesterol concentrations, and since rhesus monkeys have very little of the triacylglycerol-rich very-low-density lipoproteins [20], the bulk of the decrease in total plasma cholesterol concentration must have been due to either a decrease in low-density lipoproteins (LDL) for the animals fed the cholesterol-free diet, or LDL plus cholesteryl ester-rich P-very low density lipoproteins in the cholesterol-fed animals [2 11. Triacylglycerol concentrations in non-human primates are generally much less than in human beings, and they either decrease or do not change with hypercholesterolemia [21,22]. There was, however, a small but consistent increase in plasma triacylglycerol con-

TABLE

I

TIME COURSE OF TOTAL PLASMA MALE RHESUS MONKEYS AFTER mg,‘kg

CHOLESTEROL, HDL CHOLESTEROL AND TRIACYLGLYCEROL A SINGLE INTRAVENOUS BOLUS INJECTION OF VINCRISTINE

LEVELS IN THE SULFATE (0.05

BODY WEIGHT)

H, high-fat,

cholesterol-rich

Experiment No.

Monkey No.

I

128

1

2

2

centrations as a result of drug treatment. This increase showed a similar time course to the decrease in plasma cholesterol. By 7- 10 days post-injection plasma cholesterol and triacylglycerol concentrations had returned to predrug levels. Although the mechanism(s) of the plasma cholesterol-lowering effects of vincristine and vinblastine cannot be determined from this study, there are at least two possible mechanisms that are suggested from the literature. Vinblastine has been shown to inhibit the rate-limiting enzyme in cholesterol synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase, in C-6 glial cell in culture [23], and to inhibit the incorporation of radioactively labeled acetate into lipids by cultured ascites cells [24,25]. This does not appear to be the result of a general toxic effect of the alkaloid since inhibition of cholesterol synthesis was greater than the inhibition of synthesis of nonlipid components such as RNA, DNA or protein [25]. Consequently,

diet; L, low-fat,

cholesterol-free

Triacylglycerol

HDLcholesterol

(mg/dl)

% decrease in total plasma cholesterol

0 1 2 3 4

802 148 702 666 647

_ 6.7 12.5 16.9 19.3

25 24 28 24 26

8 10 II 42 21

H

0 1 2 3 4

253 227 210 190 188

_ 10.3 17.0 24.9 25.7

82 72 92 72 70

12 17 13 14 24

L

0 1 2 3 6

176 177 139 122 153

_ 21.0 30.7 13.1

72 84 68 68 72

17 14 47 66 36

0 1 2 3 6

126 125 114 96 119

_ _ 9.5 23.8 5.5

69 78 72 66 63

17 15 19 35 21

Day after injection

Total plasma

H

199

128

199

diet.

Diet

L

cholesterol

(mg/dl)

(mg/dl)

485

TABLE

II

KINETICS OF TOTAL PLASMA CHOLESTEROL, HDL CHOLESTEROL MALE RHESUS MONKEYS AFTER A SINGLE INTRAVENOUS BOLUS mg/kg BODY WEIGHT) H, high-fat,

cholesterol-rich

diet; L, low-fat,

Experiment

Monkey No.

Diet

492

H

No. 1

722

H

Total plasma cholesterol

0

606 580 596 541 591

0

2

492

122

L

L

(mg/dl)

2 3 4

529 521 466 426 471

0 1 2 3 6

184 182 159 133 156

0 1 2 3 6

82 73 67 106 106

I

2

diet.

Day after injection

1 2 3 4

1

cholesterol-free

AND TRIACYLGLYCEROL LEVELS IN THE INJECTION OF VINBLASTINE SULFATE (0.2

even though an inhibition of endogenous cholesterol synthesis is one possible mechanism for the lowering of plasma cholesterol concentrations with these drugs, it would not appear to be a likely possibility, since a decrease in plasma cholesterol concentrations also occurred in the cholesterol-fed animals under conditions in which endogenous cholesterol synthesis and, in particular, liver cholesterol synthesis has been shown to be markedly suppressed [26]. Perhaps the most likely mechanism for the plasma cholesterol lowering is by interference with secretion of lipoproteins from the liver, secondary to the disruption of microtubules by the alkaloids. Microtubules are thought to play a role in the translocation of lipoproteins from their point of final synthesis, via Golgi-derived secretory vesicles, to the plasma membrane of liver parenchymal cells [27]. As a result, inhibition of this mechanism would be expected to result in the retention of lipoprotein particles within the

% decrease in total plasma cholesterol

_ 4.3 1.6 9.7 2.5

_ 1.5 11.9 19.5 10.9

_ 1.1 13.6 27.7 15.2

_ 11.0 18.3 _

HDLcholesterol

Triacylglycerol (mg/dl)

(mg/dl) 50 47 60 50 56

18 20 28 31 34

21 19 24 23 24

3 2 6 16 13

84 96 83 72 92

23 18 42 148 31

43 39 36 46 59

12 19 51 43 17

hepatocytes. This possibility was examined by electron microscopy of the biopsied liver hepatocytes before and 72 h after treatment with the clinical doses of vinblastine and vincristine (Fig. IA-F). The hepatocytes, prior to the alkaloid treatment, were characterized by a prominent centrally located nucleus surrounded by moderately electron opaque cytoplasm containing numerous mitochondria, lysosomes, peroxisomes and elements of both rough and smooth endoplasmic reticulum (SER) (Fig. 1A and B). This normal hepatocyte ultrastructure was in dramatic contrast to that observed with either of the two alkaloids. As is illustrated in Fig. 1C and D, vinblastine treatment resulted in a generally foamy cytoplasmic appearance due primarily to an increase in the amount and dilation of smooth endoplasmic reticulum. This smooth endoplasmic reticulum alteration was accompanied by an increase in the glycogen content and by a decrease in the

486

amount of rough endoplasmic reticulum (Fig. 1E). The nucleus, mitochondria and lysosomes appeared unaffected; however, an increase in the number of electron-dense vacuoles containing 250-500 I\ diameter nascent lipoprotein particles. was evident. The changes in the hepatocytes of vincristine-treated animals were comparable to

those observed in the vinblastine-treated monkey. (see Fig. IE and F). Furthermore, there was no detectable necrosis of the hepatocytes by either alkaloid. Other investigators [28-301, by using a much higher concentration (50- lOO-fold) of vinblastine or vincristine in rodents have shown clustering of lipoprotein-containing vesicles and in-

Fig. I. Transmission electron micrographs of hepatocytes from rhesus monkeys before (A,B) and 3 days after treatment with either vinblastine (C. D and E) or vincristine (F). Magnifications: A, 4930 x ; B, 10330 x : C, I360 x : D. 4930 x : E. 10330 X : F. IO.330 X N. nucleus; M, mitochondria; L, lysosome: SER. smooth endoplasmic reticulum; P, peroxisome: G. glycogen; LPP. lipoprotein partlclc.

487

hibition of secretion of very-low-density lipoprotein and albumin by hepatocytes. Our results are consistent with these observations. Although inhibition of lipoprotein secretion from the liver could explain the lowering in plasma cholesterol concentrations, it is more difficult to see how a similar mechanism could also result in the small increase in triacylglycerol concentration. Since the major low-density-lipoprotein species to be secreted from the liver is thought to be a triacylglycerol-rich very-low-density lipoprotein [31] one might also expect triacylglycerol levels to decrease as well. Consequently, there may be multiple effects of the alkaloids on lipoprotein metabolism, with the mechanism for the lowering of plasma cholesterol levels being different from that responsible for the increase in triacylglycerols. In conclusion, vincristine and vinblastine at clinical doses in rhesus monkeys lower plasma cholesterol levels, presumably by interfering with lipid and lipoprotein metabolism. These results suggest the possibility that other alkaloids from Catharanthus species, but with less cytotoxic properties and more pronounced hypocholesterolemic activities may be discovered. Acknowledgements We thank Drs. Robert L. Capizzi, Charles L. Spurr and Thomas B. Clarkson for support and encouragement. We gratefully acknowledge the help of Dr. Noel Lehner in performing liver biopsies. We also thank Dr. Larry Rude1 for helpful discussion. This work was supported by USPHS grants CA 12197 and HL 14164, and a grant from the National Dairy Council. References Livingston, R.B. and Carter, SK. (1970) Single Agents in Cancer Chemotherapy, pp 279-297, IFI/Plenum, New York See-Lasley, K. and Ignoffo, R.J. (1981) Manual of Oncology Therapeutics, pp. l-170, The C.V. Mosby Company, St. Louis Creasey, W.A. (1975) in Antineoplastic and Immunosuppressive Agents (Sartorelli, A.C. and Johns, D.G., eds.), pp. 670-694, Springer Verlag, New York Gerzon, J. (1981) in Development of Anticancer Drugs Based on Natural Products Prototype (Cassady, J.M. and Douros. J.D., eds.), pp. 271-317, Academic Press, New York Sethi, V.S., Jackson, D.V., White, D.R., Richards, F., Stuart, J.J., Muss, H.B., Cooper, M.R. and Spurr, C.L. (1981) Cancer Res. 41, 3551-3555

6 Sethi, VS. and Kimball, J.C. (1981) Cancer Chemother. Pharmacol. 6, I 11- I 15 7 Sethi, V.S. and Surratt, P. (1983) in Proceedings of the First International Conference on Modulation of Cancer by Vitamins(Meyskens, F.L. and Prasad, K.N., eds.), S. Karger, Basel, in the press B. and Wolff, J. 8 Bhattacharyya, Sci. U.S.A. 73, 2375-2378 K. and 9 Wilson, L., Anderson, Motility (Goldman, R., Polland, eds.), pp. 105 1- 1064, Cold Spring Spring Harbor H. and Mauro, F. 10 Madoc-Jones,

(1976) Proc. Natl.

Acad.

Chin, D. (1976) in Cell T. and Rosenbaum, J.. Harbor Laboratory, Cold (1968) J. Cell Physiol.

72,

185-196 11 Krishan, A. and Frei, E. (1975) Cancer Res. 35, 487-501 12 Olmsted, J.B. and Borisy, G.G. (1973) Annu. Rev. Biochem. 42, 507-540 R.S. (1980) Cell Tiss. Kinet. 13, 327-335 13 Camplejohn, 14 Beck, W.T. (1980) Biochem. Pharmacol. 29, 2333-2337 L. and Somfai-Relle, S. (1979) 15 Kremmer, T., Holczinger, Biochem. Pharmacol. 28, 227-230 J.A. and Bullock, B.C. (1981) J. 16 Rudel, L.L., Reynolds, Lipid Res. 22, 278-286 17 Rush, R., Leon, L. and Turrel, J. (1971) in Advances in Automated Analysis Vol. I, pp. 503-507, Thurman Associates, Miami Technicon International Congress 18 Lipid Research Clinic Program (1975) Manual of Laboratory Operations, Vol. 1, Lipid and Lipoprotein Analysis, pp. I-81, NHLI, NIH, Bethesda G.R., Heaster, V., Wagner, 19 St. Clair, R.W., Henderson, W.D., Bond, M.D. and McMahon, M.R. (:980) Atherosclerosis 37, 103-121 20 Rudel, L.L., Greene, D.G. and Shah, R. (1977) J. Lipid Res. 18, 734-744 21 Rudel, L.L., Shah, R. and Greene, D.G. (1979) J. Lipid Res. 20, 55-65 22 Clarkson, T.B., Lehner, N.D.M., Wagner, W.D., St. Clair, R.W., Bond, M.G. and Bullock B.E. (1979) Exptl. Mol. Pathol. 30, 360-385 23 Volpe, J.J. (1979) J. Biol. Chem. 254, 2568-2571 24 Creasey, W.A. (1969) Biochem. Pharmacol. 18, 227-232 25 Creasey, W.A. (1981) Br. J. Cancer 44, 921-924 26 Gould, R.G., Taylor, C.B., Hagerman, J.S., Warner, I. and Campbell, D.J. (1953) J. BioI. Chem. 201, 519-528 27 Stein, 0. and Stein, Y. (1976) in Membrane Alterations as Basis of Liver Injury (Popper, H., Bianchi, L. and Reutter, W., eds.), pp. 257-266, University Park Press, Baltimore 28 LeMarchand, Y., Singh, A., Assimacopoulos. Jeannet, F., Orci, L., Rouiller, C. and Jeanrenand, B. (1973) J. Biol. Chem. 248, 6862-6870 29 Marzella, L. and Glaumann, H. (1980) Lab. Invest. 42, 18-27 30 Sandberg, P.O. and Glaumann, H. (1982) Exp. Mol. Path. 36,242-261 31 Schaefer, E.J., Eisenberg, S. and Levy, R.I. (1978) J. Lipid Res. 19, 667-687 32 Sethi, V.S., Lewis, J.C. and St. Clair, R.W. (1982) in Proceedings of the 13th International Cancer Congress, Seattle, WA, Abst. No. 724