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

on June 12, 2021. © 1958 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

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

on June 12, 2021. © 1958 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

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:

on June 12, 2021. © 1958 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

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

on June 12, 2021. © 1958 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

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

http://cancerres.aacrjournals.org/

384 CancerResearch Yushok

B Jugì

*3S*S2S82£CHO*

Yushok Cancer Chemotherapy Screening Data 385

ao "2U o "

o> o> o»

O O Ç —¿� 0, oo o M 41 00 00 M O»«• cnojooomcomo

t £ 1

OOC4iO OOoo

5H

B jss t- r-

OÃŒO OlOOOl?)

O uu-S 3O o e-

tDOiCnoO

«ss ini"\ r^o -

dSw « S

¿Z .

OO19>o-41OO

oomw v M

SIS

filis < < s8£ '3! IH O

- 6 S, SSSs i 18

iì ¡u *j.I I

OUND1Formulaa

gO

§•u u8M3uSo3uS

gS00Ssu

u8Su8CM3u8K99SusPiscomuIO1

8 8S1M «t>Sto

t-S0S

§SXT'^04s

sast~-t~(Ou

u uS*°

IA

oo0T**•S

S«IO

fO

386 CancerResearch Yushok

RELATIVERATEOFFRUCTOLYSISBASEDONFRUCTOSECONTROL

(15mM)aa100

litHour2ndHour3rdHour£

21ob-g¡8s"0

u"

-1U31

1J!Ills

ss000o*•«i

£St-*t-uoosSD

?B»-•à i2

u' ö•¿�"•gI »i•¿�y0a »"i! lS1

1Ε*mtojEn a00

CMIOcoIOIßs,11sUoNHoMUIBasic

fuchsinfC.le»a*TpHpHt-CO00s«£2ColemanO

gUo8tt-5P«O¿ssBrilliant

cresyl00Cl

Oïcoeos1ioo>oM^nO00IDHt~5tHSm0

0O02in|"c

Yushok Cancer Chemotherapy Screening Data 387

o to

z^s8£ s

inO

N IO

!zjjo.

"3SIsiw

w0)1Edr*ii

111i

Ico

s~Ü8

sz5

53ssoco•f ~*U0gü1I

1

1104

CO§

01uiINOnZu8SQuinao

rtne(HCl)^iO3

NatLAnllineC-I08oft-Rhodamine

B(C.IM»?O3

Natl.AnillnegO

nn8

uSIOi-Rhodamine

GÃ-C.ISo11cooMKU

A8)

UfrIO

r-Rhodamlne

6G(C.t0sEiftIpHJUKiKFLAVONOn11M

¿11

ir•^1£IOODlhydroquercetin0oCÃŒM1)Ã-üIO0mB)OD

UHesperldinS§u3iMS0SIOuMorinosRBMcC

(BRF)S•IOUPhloretin—Sf80CJüSco

üK04PhlorizinMOiPu8StouPhloroglucinolcooH.M.ChemicaleS0•IOQuere

etin^oH.M.Chemicale

KandKLabs.S

S§s£=S

SouQuercitrin

RhamnetinIO

toooi

ej£ 2®3il£ÜOBo

oS

*00Rutin

PYRroiNES3-Acetylpyridlnec^

coO0CiHhta3gODmC4

U4E6-Nitrophenylmei

nicotlnlcacidoÃ-iET

gH

fe09sgCOaus_Å“!O4195

inse0pyridine

on June 12, 2021. © 1958 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

388 CancerResearch Yushok

a jbssIoË -P " " § £S£1Äf*gSi"PS

Wrt«I

8 1fc PaO

U Ç * CM •¿�* -»omu

g 11âss«

°~Üli**

2 sss>JM«3S

Jigoaj

P co co go oCMQH '"' ea »^ m cimgèo"3°2O

M*8

3g0" C SSoo>H

S 9 « A - o oco>5 à Bfi«2«W°

~.&Q

|t>o>oot-MtfiOCOO>O> 0 COOONfi^^C^M^NUSjJ

*°^4 m .— —¿�'29

gè

lMI».

KHb.

di DìU U(x u.

itaul»

¡is "CI

i »SIli - 53 I

o-SzgaoO«18M02GE•uB9S•921—BoO«SXIOBIOu8MZ.MBu>OB9

MOM2&oS

o4DB

u

O

SCM

M

SBNO

O009

3 PIîû f-i

S Xt- oȟ U

s so Ss gg ss »« oo 5

o00S00U

2

Uco

3N—¿�

U

iXu

1

iÌ-.

S-(6-Nitthluronl

lsull(

SS fi.J&l ^14- mo

(3- per

IS

_. 8s s

S la £s t

I s,1 iS I

as

S 3§ § § S

on June 12, 2021. © 1958 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Yushok CancerChemotherapyScreening Data 389

ssIRE*SP§tg8§"EU

-1S •¿�^

1958;18:379-389. Cancer Res W. D. Yushok Carcinoma Cells by Chemical AgentsInhibition of Glucolysis and Fructolysis of Krebs 2 Ascites

Updated version

http://cancerres.aacrjournals.org/content/18/8_Part_2/379.citation

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

.pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/18/8_Part_2/379.citationTo request permission to re-use all or part of this article, use this link

on June 12, 2021. © 1958 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/content/18/8_Part_2/379.citationhttp://cancerres.aacrjournals.org/cgi/alertsmailto:pubs@aacr.orghttp://cancerres.aacrjournals.org/content/18/8_Part_2/379.citationhttp://cancerres.aacrjournals.org/