Post on 30-Jan-2021
INHIBITION OF GLUCOLYSIS AND FRUCTOLYSIS OF
KREBS 2 ASCITES CARCINOMA CELLS BY CHEMICAL AGENTS
W. D. YushokBiochemical Research Foundation, Newark, Delaware
A systematic search has been made for inhibitors of anaerobic glu-
colysis and fructolysis of Krebs 2 ascites carcinoma cells. The investigation
was at first limited to sugar analogs, which had been previously tested by
Woodward et al., (1) for effects on yeast fermentation, a biochemical pathway
similar to tumor glycolysis.
The program was eventually expanded to include the following types
of chemicals: (a) Chemotherapeutic agents which had been reported to inhibit
tumor growth in animals; (b) certain compounds which have a structural re
semblance to inhibitors of tumor growth; (c) several model compounds whose
effects on various biochemical systems are known and which have been tested
in order to help explain some of the unique responses of the intact cell under
in vitro conditions; (d) a few compounds which fitted into the general screening
program in progress here at the Biochemical Research Foundation. Fructose
was used as a substrate in addition to glucose in order to help elucidate the
mode of action of the inhibitors.
An inhibitory effect on glycolysis of the intact cancer cell does not
necessarily represent a direct action of these compounds on the glycolytic sys
tem. The effect on glycolysis may be indirect; the primary action may be on
an as yet unknown interrelated system or on the structural integrity of the
tumor cell. The modes of action of the inhibitory compounds reported here
are being investigated.
The long range objective of this program is not the suppression of
379
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380 Cancer Research Yushok
tumor growth by inhibition of glycolysis per se. Inhibition of glycolysis may
in addition influence (a) the intracellular concentration of intermediary metabo
lites of interacting metabolic pathways, (b) the supply of energy, and (c) the
shift in the equilibrium from anabolism to catabolism. A decrease in the con
centration of an intermediary metabolite may potentiate the action of different
types of inhibitors. This is based on the fact that the degree of inhibition by a
competitive antimetabolite is dependent on the metabolite concentration. Also,
the action of a noncompetitive enzyme inactivator may be affected by the capa
bility of a substrate to protect its enzyme. Therefore, a specific inhibitor of
glycolysis may be expected to potentiate the action of other unrelated inhibitors.
An eventual goal is the investigation of inhibitors of glycolysis in combination
with those of other pathways in the hope of finding a synergistic combination
which proves to be effective in controlling tumor growth.
Tumor-bearing ascitic fluid was obtained from female white Swiss
mice six to eight days following inoculation. The tumor cells were routinely
prepared by repeated centrifugations at low speed for short periods each fol
lowed by resuspension in Krebs-Henseleit bicarbonate buffer of pH7.4 accord
ing to a previously reported procedure (2).
Each of the test compounds and substrates was dissolved in Krebs-
Henseleit buffer. Compounds which were not completely soluble in Krebs-
Henseleit buffer at the given concentration are indicated by an asterisk (*).
Some of the relatively insoluble compounds were dissolved in dilute ethanol or
ethylene glycol. The quantity of ethanol or ethylene glycol used had little or
no effect on the substrate control. Acid compounds were neutralized with
sodium hydroxide before dilution with buffer.
Each substrate was added to the cells in the main chamber of the
Warburg vessel to give a final concentration of 15 millimolar (0.27%) in the
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Yushok Cancer ChemotherapyScreening Data 381
total fluid volume of three ml. At this level, both glucose and fructose were
glycolyzed at a maximal rate; the mean value was approximately 50 mi cr outer s
CC^/hr./mg. dry weight. Because the over-all range of rates of different cell
populations was 40 to 56, substrate controls were run with each experiment.
The results with each test compound were calculated relative to the substrate
control as 100.
The effects of 107 compounds on glucolysis and fructolysis were de
termined in the manometric system gassedwith 5% carbon dioxide and 95%pre-
purified nitrogen. Measurements were made throughout a 3-hour period of in
cubation at 37.5°C. The relative rate in the presence of test compound was
based on the total acid production during each hour.
At the end of these manometric measurements, trypan blue was
added, and the cells were examined microscopically for viability and for
changes in gross morphological characteristics. The results will be presented
in a subsequent publication.
ACKNOWLEDGEMENTS
The able technical assistance of Miss Lois Mallalieu and Mrs.
Elizabeth Nadler is gratefully acknowledged.
Compounds which were obtained through commercial channels are
designated by the name of the company. Compounds synthesized in this lab
oratory are indicated by the initials of the worker followed by the initials BRF.
We are indebted to the following companies and individuals for supplying gen
erous samples of some of the compounds:
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382 CancerResearch Yushok
N. K. Richtmyer (NIH)
Calco
du Pont
Lederle
Merck
Parke, Davis
Pfizer
Upjohn
Wellcome
Dr. Nelson K. RichtmyerLaboratory of ChemistryNational Institute of Arthritis and
Metabolic DiseasesNational Institutes of HealthBethesda 14, Maryland
Dr. J. J. DentónCalco Chemical DivisionAmerican Cyanamid CompanyBound Brook, New Jersey
Dr. H. S. HoltExperimental StationE. I. du Pont de Nemours and
Company, Inc.Wilmington, Delaware
Dr. J. M. RuegseggerLederle Laboratories DivisionAmerican Cyanamid CompanyPearl River, New York
Dr. F. J. WolfProcess Research and DevelopmentMerck and Company, Inc.Rahway, New Jersey
Dr. L. A. SweetParke, Davis and CompanyDetroit, Michigan
Mr. F. A. HochsteinChas. Pfizer and CompanyBrooklyn, New York
Drs. J. S. Evans, H. G. PeteringResearch LaboratoriesThe Upjohn CompanyKalamazoo, Michigan
Dr. George HitchingsWellcome Research LaboratoriesTuckahoe, New York
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Yushok Cancer ChemotherapyScreening Data 383
REFERENCES
1. Woodward, G. E., Cramer, F. B., and Hudson, M. T. Carbohydrate
Analogs as Antagonists of Glucose in Carbohydrate Metabolism in Yeast.
J. Franklin Inst., 256:577-587, 1953.
2. Yushok, W. D. Anaerobic Glucolysis of the Krebs Ascites Carcinoma on
the Basis of Cell Number. J. Franklin Inst., 259:359-361, 1955.
on June 12, 2021. © 1958 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
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1958;18:379-389. Cancer Res W. D. Yushok Carcinoma Cells by Chemical AgentsInhibition of Glucolysis and Fructolysis of Krebs 2 Ascites
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