Effect of indomethacin on the growth of colon cancer cells in syngeneic rats

Int. J. Immunopharmac., Vol. 6 No. 4, pp. 329-334, 1984. Printed in Great Britain.

0192-0561/84 $3.00+ .00 © 1984 International Society for Immunopharmaeolngy

EFFECT OF INDOMETHACIN ON THE GROWTH OF COLON CANCER CELLS IN SYNGENEIC RATS NILS O. OLSSON, ANNE CAIGNARD, MONIQUE S. MARTIN and FRAN(~OISMARTIN Research Group on Human and Experimental Digestive Cancers, I.N.S.E.R.M.U. 252, Laboratoire d'Immunologie, Facult6 de M6decine, 21033 - Dijon, France (Received 3 June 1983 and in final form 18 November 1983)

Abstract - - A cell culture line has been established from a colonic tumor induced in a rat by dimethylhydrazine. Indomethacin had no effect on the growth of tumors induced in syngeneic rats by s.c. injection of this cell line. Neither indomethacin, nor prostaglandin E2 modify the growth of this established cell line in vitro or alter its destruction by endotoxin-activated macrophages. Indomethacin has been reported to suppress the growth of colonic tumors induced in rats by various carcinogenic drugs. The difference between these results obtained by others on chemically-induced colon cancer and the data we have obtained with a cell line derived from a similar cancer suggests that cancer cells in established culture may loose their sensitivityto prostaglandins, prostaglandin inhibitors or prostaglandin-dependent effector host cells.

Human and experimental colon cancers are poorly sensitive to chemotherapy and immunotherapy. However, two different groups have reported that indomethacin, an inhibitor of prostaglandin synthesis, strongly inhibited the growth of colonic tumors induced in rats by a variety of carcinogenic drugs: dimethylhydrazine (Pollard & Luckert, 1980; 1981a), methylazoxymethanol acetate (Kudo, Narisawa & Abo, 1980), dimethylnitrosamine-o-acetate (Pollard & Luckert, 1981b), methylnitrosourea (Narisawa, Sato, Tani, Kudo, Takahashi & Goto, 1981; Narisawa, Sato, Sano & Takahashi, 1982). In all these experiments, indomethacin was given after the end of administration of carcinogenic drugs, so that it probably did not interfere with the metabolism of the carcinogen. The mechanism of the inhibition of cancer growth by indomethacin is still unknown. It could directly act on cancer cell division or it could modulate cancer cell destruction by macrophages or other host effector cells (Ceuppens & Goodwin, 1981). In an effort to elucidate the mechanism, we have studied the effects of indomethacin on an established line of dimethylhydrazine-induced rat colon cancer cells (Martin, Knobel, Martin & Bordes, 1975) both in vitro and after inoculation of the syngeneic animal.

Reprints requests should be addressed to N. O. Olsson. 329

EXPERIMENTAL PROCEDURES

Materials Indomethacin (M.W. = 357.8) and prostaglandin E2 (PGE2) purchased from Sigma Chemicals (Saint Louis, MO, U.S.A.) were first dissolved in absolute ethanol. For the in vivo experiments, indomethacin was further diluted in tap water. For the in vitro experiments, indomethacin and PGE2 were further diluted in the culture medium. The same concentration of ethanol (0.1%0) was present in all cultures. Endotoxins extracted from E. Coli 0128B12 according to Westphal were obtained from Difco (Detroit, Michigan, U.S.A.). A n i m a l s and tumor cells All the animals used in this investigation were syngeneic BD IX rats bred in our laboratory. A permanent cancer cell line D H D K-12 was established from a transplantable colon carcinoma induced by 1.2 dimethylhydrazine in a BD IX rat (Martin et al., 1975). D H D K-12 cells were cultivated in Ham F 10 medium supplemented with 4% fetal bovine serum and 8% newborn calf serum (Microbiological Associates, Inc. Walkersville, MD, U.S.A.). Cells at the 66th passage were used in this work.

330

NILS O. OLSSON, ANNE CAIGNARD, MONIQUE S. MARTIN and FRANCOIS MARTIN

Effect o f indomethacin on D H D K-12 cell growth in vivo Two groups of 14 BD IX rats, matched for age and sex, were challenged by a s.c. injection of l0 s DHD K-12 cells in the anterior thoracic wall. Immediately after tumor challenge and until the end of the experiment, one group received indomethacin at a concentration of 20 mg 1-1 in their drinking water. The other group was not treated. Diameters of s.c. tumors were measured weekly with callipers in 2 perpendicular dimensions and the tumor volume was estimated by the product of half the length by the square of the width (Simpson, Sanford & Holmquist, 1974). Three months after challenge, all the animals were killed, the s.c. tumors were excised and weighed and the animals examined for metastases and indomethacin-induced gastric or intestinal lesions. Tumors, metastases and digestive lesions were systematically processed for histology. Effect o f indomethacin and prostaglandin on D H D K-12 cell growth in vitro A microassay which has been previously reported (Olsson, Leclerc, Jeannin & Martin, 1982) was used to study this effect. A thousand cancer cells were seeded in the wells of a Falcon 3040 F Microtest II tissue culture plate in 200/al of Ham F 10 medium containing 10% fetal bovine serum and various concentrations of indomethacin or prostaglandin E2. The plate was incubated for 72 h at 37°C, fixed by formalin, and stained by methylene blue. The absorbance, proportional to the cancer cell number, was read on a scanning photometer after elution by 0.1 M HC1. For each concentration of indomethacin or PGE2, 8 microculture wells were used.

Effect o f indomethacin on macrophage-mediated cytotox&ity on D H D 1(-12 cells Peritoneal exudate cells were obtained by washing out the peritoneal cavity of BD IX rats with 10 ml cold Ham F 10 medium supplemented with heparin (0.05 mg ml-1). The macrophage content of the recovered fluid was evaluated by counting the cells adhering to a hemocytometer after 30 min incubation at 37°C. One hundred microliters of peritoneal cell suspension containing l0 s macrophages were transferred into the wells of Falcon 3040 F Microtest II tissue culture plates After 1 h incubation at 37°C, the wells were washed with warm Ham F 10 medium to remove the nonadherent ceils. Each well received 200/al of Ham F 10 medium, supplemented with 10% endotoxin-free fetal bovine serum, to which could be added various concentrations of endotoxin, indomethacin or PGE2.

After preincubation of the macrophages in this medium for various times, the medium was aspirated and replaced by the same amount of the same medium containing 10" DHD K-12 cells. The plates were incubated at 37°C for 72 h and the tumor cell density was determined by the methylene blue assay as described above. We had previously determined that residual macrophages do not significantly contribute to the methylene blue absorbance (Olsson et al., 1982). For each assay the culture conditions were identical in the eight wells of the same column. The absorbance obtained with mixed cultures of macrophages and cancer cells were compared with the absorbances of the adjacent row containing only cancer cells with the same concentrations of endotoxin, indomethacin or prostaglandin. Macrophage-mediated cytotoxicity was calculated in percentage by the formula P = 100 (5~b-), where a is the mean absorbance of the wells containing cancer cells alone and b the mean absorbance of the wells containing macrophages and cancer cells. The statistical signification of the calculated cytotoxicities was determined by the Student's t-test for paired samples. The Mann and Whitney's U test was used to compare two cytotoxicities.

RESULTS

Indomethacin, used in the in vivo experiments, was well tolerated by the rats since there was no difference in the body weight between control and treated groups. However in 2 treated animals, an intestinal ulcer was found, one in the cecum, the other one in the ileum. Tumors grew after challenge in all the animals. There was no significant difference in the tumor growth curves (Fig. 1), nor in the tumor weight at the time

cm 3 12-

........indomethacin

8642-

/ // ,, /

control

loJ

/ 2

4

6

8

~0

~2

~4

wk

Fig. 1. Effect of oral administration of iudomethacin on tumor growth. The tumor volume was estimated from two perpendicular dimensions measured each week after the injection of l0s cancer cells in the thoracic wall.

Indomethacin and Colon Cancer Cells

331

Table 1. Effect of indomethacin concentration on colon cancer cell line D H D K-12 cultivated in vitro lndomethacin

Absorbance*

S.D.t

Change

956 933 946 961 951 951 833

41 53 53 39 51 47 55

--207o - lO70 + 1°70 - 107o - 107o -- 13070

(M) 0 10-s 10-7 10-4 10-s 5.10 -s 10-4

D H D K-12 ceils (los) were cultivated for 3 days in medium supplemented with various concentrations of indomethacin. The terminal density was evaluated by the methylene blue photometric assay. Change was calculated as the difference in percentage between the absorbances of the treated and the untreated cells. Results are mean of two different experiments. * Mean of 8 wells. t S.D. = standard deviation.

Table 2. Effect of prostaglandin E2 concentration on colon cancer cell line D H D K-12 cultivated in vitro PGE2

Absorbance*

S.D.-I"

Change

817 856 837 835 814 811

31 39 24 25 23 36

-+ 5% + 2070 + 2% 0070 - l°7o

(M) 0 10-j° 10-9 10-8 10-7 10-~

D H D K-12 cells (104) were cultivated for 3 days in medium supplemented with various concentrations of PGE2 and the terminal density was evaluated by the methylene blue photometric assay. Change was calculated as the difference in percentage between the absorbance of the treated and the untreated cells. * Mean of 8 wells. "I" S.D. = standard deviation.

Table 3. Effect of indomethacin on the in vitro cytotoxicity of endotoxin-activated macrophages on D H D K-12 colon cancer cells

Preincubation time

(h)

Macrophage-mediated cytotoxic effect (°70) Without LPS With LPS or indomethacin at indomethacin concentrations of 0 10-rM 10-SM

0 4

- 5 - 12

24

- 17

47

44

66

20 -9

15 -6

14 -2

Macrophages, l 0 s per well, were incubated 0, 4 or 24 h at 37°C with 100 ng endotoxin (LPS) per ml and different concentrations o f indomethacin in 200/.d of complete medium. After washing with medium, lip D H D cancer cells were a d d e d in 200/A complete medium containing the same respective concentration of LPS and indomethacin. The plates were incubated for 72 h at 37°C. Results are given as percentages of cytotoxicity calculated from the mean of 8 wells in two different experiments. Negative figures mean an increase of the absorbance in the wells containing macrophages. Indomethacin concentration did not significantly modify macrophage-mediated cytotoxic effect.

NILS O, OLSSON,ANNECAIGNARD,MONIQUE S. MARTIN and FRANCOIS MARTIN

332

Table 4. Effect of prostaglandin E2 on the in vitro cytotoxicity of endotoxin-activated macrophages on DHD K-12 colon cancer cells Macrophage-mediated cytotoxic effect (%) Without LPS With LPS or PGE2 at PGE2 concentrations of 0 10-1°M 10-SM

Preincubation time (h) 0 4 24

- 45 - 47 - 36

51 30 4

48 25 12

10-6M

56 41 13

67" 48* 10

Macrophages, l0 s per well, were incubated 0, 4 or 24 h at 37°C with 100 ng endotoxin (LPS) per ml and different concentrations of PGE2 in 200 tal of complete medium. After washing with medium, 104 DHD K-12 cancer cells were added in 200/al complete medium containing the same respective concentrations of LPS and PGE2. The plates were incubated for 72 h at 37°C. Results are given as percentages of cytotoxicity calculated from the mean of 8 wells. Negative figures mean an increase of the absorbance in the wells containing macrophages. PGE2 did not significantly modify macrophage-mediated cytotoxic effect (P >0.05, Mann and Whitney's U test, except for (*): P <0.01). Table 5. Reduction of the incidence of carcinogen-induced intestinal tumors in rats treated by indomethacin Carcinogen* Indomethacint

No. Rats~.

o70 rats with tumor No tumors/rat

Reference

MAM-Ac

No i.r.

30 30

87 50

2.9 1.0

Kudo et al., 1980

MNU

No i.p.

30 29

67 31

1.1 0.45

Narisawa et al., 1981

MNU

No d.w.

27 52

67 15

1.04 0.22

Narisawa et al., 1982

DMH

No d.w.

29 30

100 50

3.40 0.76

Pollard and Luckert, 1980

DMH

No d.w.

10 9

90 22

1.30 0.22

Pollard and Luckert, 1981a

MAM-Ac

No d.w.

14 12

71 8

1.36 0.08

Pollard and Luckert, 1981a

DMN-O-Ac

No d.w.

18 12

78 8

1.44 0.08

Pollard and Luckert, 1981b

* Carcinogenic drugs; MAM-Ac = Methylazoxymethanol acetate; MNU = Methylnitrosourea; DMH = 1,2 Dimethylhydrazine; DMN-o-Ac = dimethylnitrosamine-o-acetate. "I" No = no indomethacin; i.r. = intrarectal indomethacin; i.p. = intraperitoneal indomethacin; d.w. = indomethacin in drinking water. Number of rats in each group. o f sacrifice between control (8.4 g, S.E. 1.7) and treated rats (10.0 g, S.E. 1.8). Metastases to lung or lymph nodes were f o u n d in 4 o f 14 control animals and 1 o f 14 treated rats ( P = 0.16). In the in vitro experiments, neither i n d o m e t h a c i n , at c o n c e n t r a t i o n s between 10-8 and 5 x 10-s M, nor PGE2, between 10-1° and 10-6 M significantly modified t u m o r cell g r o w t h (Tables 1 and 2). I n d o m e t h a c i n inhibited t u m o r cell growth at concentrations equal to or larger than 10-* M, but these concentrations were quite superior to the i n d o m e t h a c i n p l a s m a levels expected (Hucker, Zacchei, Cox, Brodie & Cantwell, 1966) f r o m the oral dose given in the in vivo assay. Resident peritoneal m a c r o p h a g e s were rendered cytotoxic to D H D K-12 ceils by endotoxin (100 ng m1-1) but this cytotoxic activity was lost if the m a c r o p h a g e s were incubated for 24 h b e f o r e the addition o f t u m o r

cells. I n d o m e t h a c i n did not significantly modify the cytotoxic activity o f peritoneal macrophages on D H D K-12 t u m o r cells (Table 3). The only variation o f m a c r o p h a g e - m e d i a t e d cytotoxicity obtained with P G E 2 was an increase when the m a c r o p h a g e s were preincubated 4 h or less with 10 -6 M PGE2. Both agents were unable to prevent or enhance the loss o f t u m o r i c i d a l activity by e n d o t o x i n - a c t i v a t e d m a c r o p h a g e s during their incubation before the addition o f t u m o r cells. DISCUSSION

I n d o m e t h a c i n has b e e n r e p o r t e d by several investigators to inhibit the growth o f chemicallyinduced rat colon t u m o r s (Table 5). In contrast, our experiments failed to reveal any effect of indomethacin t r e a t m e n t on the in vivo growth o f an established line

Indomethacin and Colon Cancer Cells of cancer cells obtained from a chemically-induced rat colon carcinoma. This discrepancy could be attributed to the difference in experimental conditions. The dosage or the route of administration of indomethacin is not the cause of the discrepancy, since the drug was given in drinking water at the same concentration which efficiently suppressed growth of colonic (Narisawa et al., 1982; Pollard & Luckert, 1980; 1981a; 1981b) as well as other (Fulton & Levy, 1980; Lynch, Castes, Astoin & Salomon, 1978; Lynch & Salomon, 1979) tumors. The kind of tumor studied in our experiments is probably not an important point, since indomethacin has been reported to inhibit the in vivo growth of numerous chemically-induced colonic tumors as well as various other experimental tumors. The latter include chemically-induced sarcomas (Fulton & Levy, 1980; Lynch et aL, 1978; Lynch & Salomon, 1979; Plescia, Smith & Grinwich, 1975), virus-induced sarcomas (Strausser & Humes, 1975), the ¥oshida hepatoma (Trevisani, Ferretti, Capuzzo & Tomasi, 1980), the P815 mast-cell tumor and the Lewis lung carcinoma (Hial, Horakova, Shaff & Beaven, 1976). Finally, the mode of tumor initiation in the animals might be responsible for our negative results. Most of the experiments where indomethacin inhibited tumor growth were performed with primary chemically-induced tumors (Kudo et al., 1980; Narisawa et aL, 1981, 1982; Pollard & Luckert, 1980; 1981a; 1981b), primary virus-induced tumors (Strausser & Humes, 1975), serially transplantable tumor grafts (Fulton & Levy, 1980; Lynch et al., 1978; Lynch & Salomon, 1979), transplantable ascite tumors (Hial et aL, 1976; Trevisani et aL, 1980) or tumors induced by inoculation of cells freshly prepared from excised tumors (Plescia et al., 1975). In contrast, very few results have been reported with solid tumors developed after inoculation of cancer cells from an established line. In one such model, Favalli, Garaci, Etheredge, Santoro & Jaffe (1980) even obtained enhancement of the tumor growth in indomethacintreated mice inoculated with cells from the B16 melanoma line. The inhibition of tumor growth by indomethacin in vivo may be due to several mechanisms. First, indomethacin could exert a direct effect on cancer cell growth. High concentrations of this drug (4 × 10-4 M) actually reduced the growth of rat hepatoma cells used early after establishment of the culture (passages 4 to 10) with arrest of the cell cycle at the G1 phase (Bayer, Kruth, Vaughan & Beaven, 1979). On the other hand 10-8 M indomethacin has been reported to stimulate in vitro the growth of B16 melanoma cells (Santoro, Philpott & Jaffe, 1976) as well as three other long-time established cell cultures, Hela, Hep-2 and

333

L929 (Thomas, Philpott & Jaffe, 1974). In our in vitro experiments, also using a long-time established cancer cell line, DHD K-12, indomethacin was cytotoxic at concentrations higher than 10-4 M but had no effect at lower, pharmacological concentrations. In a second hypothetical mechanism, indomethacin, which is a well-known inhibitor of prostaglandin synthesis, could act by preventing the stimulation of cancer cells growth by prostaglandins. However, experimental results are in disagreement with this hypothesis, since PGEI slowed the in vitro proliferation of B16 melanoma cells (Santoro, 1976) as well as Hela, Hep-2 and L 929 cells (Thomas et al., 1974). In our experimental system, PGE2, as well as PGEI (results not shown), were also unable to modify cancer ceils growth. Indomethacin may also act through various modifications of the host immune response to cancer (Ceuppens & Goodwin, 1981). Special attention has been paid to its influence on tumor cell destruction by activated macrophages through prostaglandin synthesis inhibition. Using LPS-activated resident mouse macrophages, Taffet & Russel (1981) showed that 10-6 M indomethacin prevented the shut-off of their cytolytic activity on P815 mastocytoma cells in 24 h incubation experiments. Nevertheless in different experimental models, indomethacin had no effect on macrophage-mediated cytotoxicity on cancer cells (Drysdale & Shin, 1981; Meltzer & Wahl, 1978; Shaw, Russel, Printz & Skidgel, 1979; Schultz, Pavlidis, Stylos & Chirigos, 1978). In our experiments no enhancing effect of indomethacin on macrophagemediated cytotoxicity on the DHD K-12 cancer cells could be detected, though we used an experimental pattern similar to Taffet & Russel's (1981). These discrepancies between Taffet & Russel's results and results from other investigators, including those reported here, can be attributed to differences in the experimental models, particularly: the origin and mode of activation of the macrophages, the target cells, the animal species, the incubation schedules and the cytotoxicity assay. As an example, Drysdale & Shin (1981) showed that macrophages activated by inflammatory peritoneal cells or antibody-coated tumor cells were insensitive to indomethacin, whereas this drug blocked the cytotoxicity of LPS- or lymphokine-activated macrophages assayed under the same experimental pattern. "Our experiments are the first to investigate simultaneously the effect of indomethacin on the in vivo and the in vitro growth of the same cancer cells and on the cytotoxicity of activated macrophages on these cells. The disagreement of our negative results with part of the literature is possibly due to the use of long time established ceil line.

334

N1LS O. OLSSON, ANNE CAIGNARD, MONIQUES. MARTIN and FRAN(,2OISMARTIN REFERENCES

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