Neurotensin stimulates growth of colon cancer

Surgical Oncology 1992; 1: 127-l 34

Neurotensin stimulates growth of colon cancer K. YUSHlNAGA*,

B. M. EVERSt, M. lZUKURA$, Da PAREKH§, T. UCHIDA1I, C. M. TOWNSEND, JR AND J. C. THOMPSON

Department of Surgery and llOff.ce of Biostatisties, The University of Texas Medical Branch, Galveston, TX 77550, USA

Neurotensin

(NT),

stimulates

a peptide

the growth

study was to determine of a mouse ment

colon

kg-l) with

administered MC-26

kg-l)

or NT (600 t/g kg-‘). cantly stimulated

receiving

weight

and nucleic

play

an important

1992; 1: 127-134.

Keywords;

colon cancer,

in growth

(NT),

a

crine cells (N-cells) [I, 21. NT, which

physiological

is

system

of the distal small

is released

boweI

mucosa

[8-IO]

mucosa

in the gut in&d-

rats

*Visiting Scientist from the Second Department

of Surgery,

Tokyo Medical and Dental University, Tokyo, Japan. Surgical Association

Foundation

Fellowship Award and a UTMB Small Grant Award. the

§Visiting

Scientist

Johannesburg, the Michael

from

the

First Department

of Surgery,

131.

University

Miller

Foundation

and the Medical

127

the

and

given

NT

injections

demonstrated the

colleagues

[14],

from

NT stimulates

of

layer. our

an important and

tract;

conthe colon

adenocarcinomas

Recently,

laboratory,

growth

of

in both

Sumi have

of a human

(MIA Paca2) ,i7 vitro. Collectively, normal

colon

carcinogen

carcinogen-induced

incidence

muscle

and

an increase

of

gut

shown to pro-

with

the

intestinal

Research Council, South Africa.

on normal

NT has been

penetrating

both

effects

treated

and

pro-

[6, 71, small

Rats

tumours

of

NT stimulates

stomach

size

suggest

of small

stimulation

in the

and

cancer

of Witwatersrand,

and

[6], pancreas

trophic

and pancreas,

number

that

Osaka, Japan.

South Africa; supported by scholarships from

and Janie

Surgical

[I 11.

to these

comitantly

from

that

inhibition

[4]

151. Also,

carcinogenesis [12,

[3],

antrum

and colon

tn addition

fats, has

ton, Texas 77550, USA.

Scientist

cancers*

motility

of gastric

azoxymethane

*Visiting

area,

We conclude

vasodilation

secretion

Iiferation

Courtney M. Townsend, Jr, MD, Department

Osaka University Medical School,

group

(tumour

and LoVo in vr’vo. NT

colon

gastric

pancreatic

localized

bowel

and

of Surgery, The University of Texas Medical Branch, Galves-

TRecipient of an American

of MC-

and treated

the control

Lava.

of certain

signifi-

content

growth

cell lines MC-26

mote &respondence:

with

colon cancer,

regulation

and to endo-

by intraluminal

functions

nude

(control)

neurotensin.

tridecapeptide, nervous

saline

tumours

stimulated

ing mesenteric

to the central

numerous

MC-26

compared

boweI mainly

either

of NT (300 and 600 pg kg-‘)

decreased

INTRODUCTION Neurotensin

3, 16 athymic

to receive

NT (600 pg kg-‘) of the human

2, 60 mice

or NT (300 or 600 pg

and DNA, RNA and protein

for the colon cancer

role

Oncology

in viva. In experi-

or NT (150, 300 or 600 pg

(control)

rate of mice bearing

significantly

of this

affect the growth

(LoVo)

In experiment

randomized

area, weight

Similarly,

factor

ingestion,

cells (5 x 104) and then random-

saline

assessed.

were

the survival

acid contents)

NT acts as a trophic

The purpose

saline (control)

We found that administration

injections.

by fat

mucosa.

of NT would

MC-26

to receive

was then

mean tumour

is released

colon cancer

with

either

randomized

dose of NT was

saline

that

(s.c.) every 8 h for 21 days. In experiment

xenografts

In addition,

either

given

may

were

LoVo tumour

26 tumours.

and a human

subcutaneously

tumours

gut

and colonic

administration

inoculated

for 28 days, and survival

mice with

with

(MC-26)

groups

distal bowel

chronic

mice were

ized to four treatment

the

small

whether

cancer

1, male Balb/c

from

of normal

and found

pancreatic these data

role for NT in the growth

neoplastic

however,

tissue

of

of the gastro-

the in viva effect

of NT,

128

K. Yoshinaga et al.

without the simultaneous carcinogen,

administration

on the growth

xeno-

The

purpose

of this

administration

either a mouse

5 x IO4 MC-26

1, 56 mice were

study

was

to

determine

of NT affects the growth

colon cancer

(MC-26)

of

or a human

8 h for

21 days

tumour inoculation. perpendicular

AND

calipers

METHODS

colon

cancer

Research Laboratory maintained

in

chemically

cell line, CT26, (Worcester,

RPMI-1640

(FCS)

(Hyclone

LoVo is a human from

the American

culture

cancer

described tained

medium

Logan,

UT).

cell line obtained

Culture

ville, MD). Characterization

Mason

10% fetal calf

Laboratories,

Type

induced

MA) [I 51. MC-26 is

tissue

colon

in our

from

(Gibco, Grand Island, NY) containing serum

Collection

(Rock-

of this cell line has been

previously in detail [16, 171. LoVo is main-

in

Dulbecco’s

modified

Eagle

medium

(DMEM) (Gibco) and 10% FCS. atmosphere

of

air and 5% CO, at 37°C. Cells are routinely

passed by removing the medium cell monolayer

with 0.25%

diaminetetraacetic

acid

routinely monitored no mycoplasma

and overlaying the

trypsin:

(EDTA).

0.1%

Cell

for mycoplasma

ethylene-

cultures

are

contamination;

Experimental

experiments

twice as the

of these two diameters

from

the surrounding

stored

Sprague-Dawley,

used. The

mice were

environment

with

performed.

Indianapolis,

housed

controlled

chow

(Formulab

cells

cultures

were

by a I-min

RPM1 1640 without containing

harvested treatment

EDTA, centrifuged serum.

in an (22”(Z),

cycle. Mice were fed Chow

#5008,

Mills, Inc., St. Louis, MO) and tap water

and 0.1%

In

IN) were

(5 per cage) temperature

humidity and a 12 h light-dark standard

from with

Purina

ad libitum.

subconfluent 0.25%

trypsin

and resuspendend Single-cell

in

suspensions

50,000 MC-26 cells (0.2 ml) were injected

subcutaneously each mouse.

tissue,

and expressed frozen

at -20°C

until assayed

for DNA,

content.

In experiment

2, 60

protein

inoculated

with

5 x IO4 MC-26

(s.c.) into the interscapular

in

were dissected

weighed,

and

RNA and mice

were

cells as described

above. The mice were randomized

into three groups

receiving either saline or NT (300 and 600 pg kg-‘). An

additional

20

mice,

without

tumours,

were

assigned to a fourth group and treated with NT (600 lug kg-‘). the

Treatments

were continued

manner

as in experiment

same

stopped.

The

mice

daily to determine c, 20-25

g, 3-4

Petersburg, gen-free

were

for 28 days in 1 and

observed

then

several

times

mortality rates. 3, male athymic nude mice (Balb/ weeks

FI) were

of age;

housed

conditions

in

a

Life Science,

under specific

St.

patho-

temperature-controlled

isolation unit with 12-h light-dark

cycles in accord-

ance with the National Research Council’s Guide for

Rodent

Chow

no.

ad libitum. Initially, to establish were

vernier

weekly. The tumour area was then calculated

5010;

Purina, St. Louis, MO) and sterile water,

one and two, male Balb/c mice (21-24

g; Harlan

MC-26

using

Tokyo,

Research [18]. The mice were fed a standard

design experiments

of

and tumour

the two greatest

diameters

Corp.,

S.C.

the day

the Care and Use of the Nude Mouse in Biomedical

growth has been detected.

separate

either

Japan)

(Autoclavable

Three

tumour

For experiment

All cells are grown in a humidified 95%

from

mm’. At sacrifice (day 21), tumours cell line was developed

laboratory from a transplantable, mouse

starting

Mice were weighed

(Mitutoyo

product

Cell lines The MC-26 tumour

with

into four

saline (control) or NT (150, 300 and 600 ,ug kg-‘) every

growth was assessed by measuring

colon cancer (LoVo) in vivo.

MATERIALS

inoculated

cells and then randomized

groups of 14 mice each. Each group received

grafts has not been determined. whether

In experiment

of a known

of colon cancer

region of

LoVo cells

(1 X IO’)

mice. When tumours

were

became

in area, the mice were

both given

tumours,

inoculated

chow

Ralston dispersed

S.C. in nude

approximately

10 cm2

killed and tumours

minced

into 3-mm* pieces that were then implanted

bilater-

ally into the flanks of 16 nude mice. On day 10, after tumour

mice were randomly

allocated

to receive either saline or NT (600 pug kg-‘)

implantation,

S.C. every

8 h for 14 days. Mice were tumours observer. culated

were measured

weekly,

and

by the same

The surface area of the tumours was calas described

after tumour weighed,

weighed

twice weekly

above.

implantation),

and frozen

at

At sacrifice

tumours were -20°C

DNA, RNA and protein content.

(day

25

removed,

until assayed

for

Neurotensin Peptide

and colon cancer

preparation

that

A stock solution of NT (Bachem,

Inc., Torrance,

was prepared

the amount

by first dissolving

CA)

of NT

needed for the study in 1 ml of sterile water containing 0.1% (w : v) bovine serum albumin (BSA) (Calbiothem-Behring, required

La Jolla, CA) and then diluted to the

concentration

with saline containing

0.1%

BSA. Equal portions of this solution, sufficient for a single injection of all animals of a given group, were stored 0.1%

in glass vials at

- 20°C. Saline

BSA (control) was likewise

aliquots

and stored at -20°C.

absorption,

divided

into equal

In order to prolong

saline or NT was mixed

15% (w : v) hydrolysed

containing

(v: v) with

I:4

gelatin (Sigma Chemical

Co.,

St. Louis, MO) prior to administration.

DNA, RNA and protein Tissues were

thawed

tion

of

the

by means

procedure

modificawith

calf

of the orcinol procedure

with

yeast RNA as the standard [20]. Protein content was determined

by the method of Lowry and colleagues

[21] with BSA as the standard.

Statistical

body

a

palpable

tumour

was

weights

of control

mice

or mice

NT (data not shown).

with

On day 21, the mean tumour area of mice treated with NT (either 300 or 600 pg kg-‘) increased

compared

with

was significantly

tumours

of the

group (Fig. 1). There were no differences size between

NT 160 ,ug kg-’ kg-’

tumour weight

significantly

increased

by 5g%, DNA content

content by 62% and protein content 600 ,ug kg-’

increased

control

in tumour

and control. Similarly,

NT at 300 pg

MC-26

MC-26

by 54%, RNA by 45%.

tumour

NT at

weight

protein content by 53% (Fig. 2).

of mice with

MC-26

2, all 60 mice inoculated

In experiment

tumour

cells

developed

survival of tumour-bearing (control), 46,

NT 300 ,ug kg-’

30 and

29 days,

tumour-bearing mice with

tumours.

with MC-26 The

mice treated

median

with saline

or NT 600 ,ug kg-’

respectively.

All

were

NT-treated

mice died by day 43; 50% of control

MC-26

tumours

were

still alive (Fig. 3).

MC-26

analysis

150

as mean& SEM. For experi-

Results are expressed ment

1, the data of tumour

were

analysed

size and body weight

as a two-factor

factorial

(one factor was days after tumour

experiment

inoculation

and

1

the second factor was the dose of NT) with repeated measures.

Statistical

analysis of tumour weight

DNA, RNA and protein content was performed a

one-way

Duncan’s separation

classification

multiple-range [22].

analysis test was

For experiment

of

For experiment

Student’s

3, data

was considered

variance.

used for

mean data

Wilcoxon

test

analysed

by

were

t-test. In all instances,

and using

2, survival

were analysed by Gehan’s generalized [23].

a value of PcO.05

significant. Figure 1. MC-26 tumour area (mm’) in relation to time

RESULTS

from tumour inoculation treatments

Effect of NT on MC-26 tumour

tumours),

growth

tumours)

In experiment

1, all mice, except for one mouse in

the control group,

developed

by

66%, DNA content by 65%, RNA content by 67% and

The DNA

by the Burton [lg]

thymus DNA used as the standard. RNA content was measured

in mean treated

develop

tumours

and homogenized.

diphenylamine

not

from the study. There were no differences

Effect of NT on survival

determination

content was measured

did

excluded

129

tumours.

The mouse

comparing

of 150 pg kg-’ 300 ,ug kg-’

neurotensin

(single-hatched

(double-hatched

or 600 ,ug kg-’

bars; n = 14

(closed bars; n = 14 tumours)

with control group (open bars; n = 13 tumours) with saline. * = PcO.05

(NT)

bars; n = 14

compared

with control.

treated

K. Yoshinaga et al.

130

NT 600 fig kg-’ (tumour-free) 0

-bearing)

50

40 Days

60

Figure 3. Survival rates of MC-26 tumour-bearing mice treated with saline (control), NT 300 pg kg-’ or NT 600 ,ug kg-’ and tumour-free mice treated with NT 600 ,ug kg-’ (n = 20 mice/group).

Figure 2. Tumour weight (g) and protein, DNA and RNA content (mg) of MC-26 tumours in control group (open bars; n= 13), NT 150 fig kg-’ (single-hatched bars; n = 14), NT 300 ,ug kg-’ (double-hatched bars; n = 14), and NT 600 ,ug kg-’ (closed bars; n = 14) at sacrifice. * = P CO.05 compared with control.

There

were

rates kg-’

no

between

significant

mice

treated

or NT 600 pg kg-‘;

in NT-treated

differences with

either

however,

tumour-bearing

rate

mice was significantly control,

a group of mice, which did not have tumours, None

was

Figure 4. LoVo tumour area (mm2) in relation to time from tumour implantation comparing NT 600 pg kg-’ (singlehatched bars; n = 16 tumours) with control group (open bars; n = 16 tumours) treated with saline. * = P < 0.05 compared with control.

these

mice

died

during

the

treatment treatment areas

Effect of NT on LoVo tumour experiment

placed

25

22

19

Days

with the highest dose of NT (600 ,ug kg-‘). of

period.

In

15

NT 300 pg

the survival

lower than that of controls. As an additional treated

10

in survival

into

3, athymic

then randomized NT 600 ,ug kg-‘; after tumour

LoVo

growth

tumour

nude

the

mice.

These

were

mice were

to receive either saline (control) or treatment

implantation.

was started

control

tained

xenografts

on day 10

After only 5 days of NT

(day

were

15 of tumour

significantly group.

These

until sacrifice

implantation),

increased

tumour

compared

differences

were

(25 days after tumour

with main-

implanta-

tion) (Fig. 4). In addition,

LoVo tumour

weight

was increased

by

69%. DNA content

by 59%, RNA content

by 88% and

protein

content

by

with

tumours

of mice treated with saline (Fig. 5).

54%

compared

LoVo

131

Neurotensin and colon cancer

Figure 5. Tumour weight (g) and protein, DNA and RNA content (mg) of L&o tumours in control gruup (open bars; n = t6 tumours) and NT 600 pg kg-’

(single-hatched

bars; n = t 6

tumours) at sacrifice. * = P < 0.05 compared

with control.

Control

cDNA, we have identified certain human colon can-

D1SCUSS10N

cer cell lines that express In this study, we have demonstrated tion of NT stimulates

the growth

that administra-

of a murine colon

receptor on these

of mice with MC-26 tumours

which

NT stimulated

by accelerating

MC-26

tumour

tumour

growth

in a

manner, that is, at doses of 300 and

dose-related

colon

involves

cancers binding

via

a direct

One prublem,

increased tumour area, weight and nucleic acid con-

nature of the transduction

pathway

lar, the identity

tumours.

In addition,

NT stimulated

growth of human colon cancer xenografts

(LoVo) in

athymic nude mice.

be

by a direct,

Receptors play

an

growth

role

presence

action

hormones

in regulation

receptors

of NT.

appear

to

of tumour

intracellular GMP

growth

[I 51 and

compared

proglu-

increases

of pentagastrin

and which

antagonist,

by a prostaglandin inhibits

gastrin

of NT receptors

331. In addition,

The

high-affinity

NT

in a variety of malig-

the human

line, l-IT29 [28, 291, human [30, 311 and a murine

[27].

has not been determined

have been identified

cell lines including

E, analogue,

release

colon

cancer

small cell lung cancers

neuroblastoma,

NIE-115

[32,

using a recently cloned NT receptor

evidence

differentiation

content with

is greater

mechanism,

pathway

is involved

including both fibroblasts

messengers

of NT on the growth

Considerable

and

cGMP

and, in particu-

cells is by a direct

(cGMP)

is the

and lead to eventual

signal transduction

be elucidated. cyclic

is stimu-

by the gastrin receptor

If the effects

of the

action,

of the cellular second DNA synthesis

colon cancer

of MC-26

for either MC-26 or LoVo; however, nant

that stimulate

the growth

[26],

enprostil,

mechanisms

which possess gastrin receptors,

by administration

inhibited mide

receptor-mediated

for gastrointestinal

[24, 251. For example,

fumours, lated

that one of the mechanisms

growth of MC-26 and LoVo may

important

cellular

cell growth.

We have postulated for the NT-induced

membrane

central to an understanding

tent of MC-26

NT significantly

mechanism

to its specific

hormone

but not 150 pg kg-‘,

These

receptor. of

600 pg kg-‘,

for the NT

results].

findings suggest that NT may exert its trophic effect

cancer, MC-26, li7 v&o and reduces the survival rate growth.

the mRNA

[B. M. Evers, unpublished

suggests that control

of various

cell

and lymphocytes

the surrounding

cGMP concentration

the

remains tu

in the

in human

of

of

types

[34, 351.

colon cancers

mucosa

[36].

NT

in the murine neuro-

blastoma

cell line, Nl E-l 15 [32, 331. Other studies,

however,

have shown that NT does not affect levels

of either CAMP or cGMP in the human colon cancer, HT 29 1291, which suggests that stimulation CAMP or cGMP may nut be involved the signals

that trigger

the growth

of either

in transmitting

of colon cancers

by NT. Another mediate

possible

the trophic

second effects

messenger

pathway

of NT on certain

to cell

132

K. Yoshinaga

lines is via turnover This pathway yield

PIP, which

lipase

is then

C to form

receptors

of polyphosphoinositide

involves the phosphorylation hydrolysed

diacylglycerol

in HT29

This

activation

inositol

mobilization appears

of

an

and

in turn,

triggers

a

cellular

of

eventually

of

early

cytoplasmic

of NT induces

a rapid

cancers

[42],

increase

growth

of various

other

have demonstrated

that glucagon can stimulate

pro-

liferation

colon cancer cells in vitro. The

ism

secondary

to

the

release. Administration

of

manuscript. work

was

35608)

and

be an indirect mechan-

stimulation

of

of glucagon,

I. Polak JM, Sullivan

glucagon

however,

does

[4, 441, but the

release

of

of MC-26 [15] and

other colon cancers [45], is not altered

by NT [46],

so that the possibility that the trophic effect of NT is mediated

through

gastrin release

In addition, to our knowledge, stimulate growth

appears

neurotensin

the release or production factors

(e.g.

epidermal

unlikely. does not

of other known

growth

factor

by

2. Helmstaedter

regulation

in the small

that

of patients

responsive

with

manner

of specific

analogous

successfully

cancers

to NT may have important

cations for the development (by means

in the treatment

receptor

employed

regimens

antagonists) strategies

in the treatment

with breast and prostate cancer.

are

future impli-

of therapeutic

to current

that

that

in a are

of patients

SR, et al. Specific

locali-

intestine

immunocytochemistry.

CH, Feurle

GE, Forssmann cells mammals.

1977; 53: 35-41.

SL, Barrowman

ity and blood flow. 4. Andersson

JA,

Kvietys

on intestinal

PR, Granger capillary

Am J Physiol1984;

S, Rosell S, Hjelmquist of gastric

DN.

permeabil-

247: G161-6.

U, Chang

and intestinal

by (gln4) neurotensin.

D, Folkers

motor

activity

in

Acta Physiol Scan 1977;

100: 231-5. 5. Baca I, Feurle Lehnert

T.

creatic

GE, Schawab

Effect

secretion

plasia

of

antrum

the

of

A, Mittmann

neurotensin

in dogs.

6. Feurle GE, Muller

on

Digestion and

W, pan-

1982; 23: 174-83.

B, Rix E. Neurotensin

pancreas

U, Knauf exocrine

growth

induces

hyper-

of

gastric

the

in rats. Gut 1987; 28: 19-23.

7. Wood

8. Wood

colon cancers. Iden-

colon

(PDT 220).

of man and various

Effect of neurotensin

NT plays

of MC-26 and LoVo

clinical applications

of patients with NT-responsive tification

the

PO1 DK

of neurotensin-immunoreactive

intestine

Histochemistry 3. Harper

Effect

colon cancers, and that this trophic effect of NT may have important

Society

and

V, Taugner

WG. Localization

JG,

Hoang

HD,

of neurotensin

tion and growth

our study has shown

a role in the growth

from

to the N cell in human

radioimmunoassay

or

insulin-like growth factor). In conclusion,

grants DK 15241,

Nature 1977; 270: 183-4.

dogs

growth

Cancer

SN, Bloom

of neurotensin

in viva [24, 251. Also, NT inhibits gastric motility and

gastrin, which stimulates

by (5R37

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