[Gann, 75, 284-291; March, 1984]

EFFECTS OF ANTIPLATELET AGENTS ON PULMONARY METASTASES

Hiroyasu BANDO,Takashi YAMASHITAand Eiro TSUBURA ThirdDepartment of InternalMedicine, The Universityof Tokushima,School of Medicine, Kuramoto-cho3-chome,T okushima770

The role of platelets in cancer metastasis was studied by investigating the effects of the antiplatelet agents ticlopidine, diltiazem, dipyridamole and trapidil on arti ficialand spontaneous pulmonary metastases in mice. These agents were tested at their optimal inhibitory doses on adenosine diphosphate-induced platelet aggrega tion;namely, 100mg/kg for ticlopidine, 2mg/kg for diltiazem, 180mg/kg for trapidil and 60mg/kg for dipyridamole. At these doses, trapidil caused moderate inhibition of thrombin-induced platelet aggregation in mice, but the other agents had only slight effects. Artificial pulmonary metastasis was produced by inoculation of Lewis lung carcinoma (LLC) or B16 melanoma (B16) cells into C57BL/6 mice . For induction of spontaneous pulmonary metastases, these tumor cells were implanted subcutane ouslyinto the footpads of mice. The resulting primary tumors of LLC and B16 were removed 9-10 and 17 days later, respectively. Artificial pulmonary metastases were inhibited significantly by all the antiplatelet agents tested . Spontaneous pulmonary metastases were markedly reduced only when these agents were given after removal of the primary tumor. The role of platelets is discussed with respect to thrombus formation in the lodgement of tumor cells and the participation of platelet -derived growth factor in the growth of metastatic foci.

Key words: Blood-borne metastasis-Antiplatelet agents-B16 melanoma - Lewis lung carcinoma-Adenosine diphosphate-induced platelet aggregation

The outcome of hematogenous metastasis To clarify the role of platelets in cancer

depends on the interaction of tumor cells metastasis, we studied the effects of various

with various host factors in the metastatic antiplatelet agents (platelet aggregation in

process. The blood coagulation-fibrinolysis hibitors)with different modes of action (i .e., system is an important host factor , in which ticlopidine, diltiazem, dipyridamole and platelets are essential for blood coagulation. trapidil) on artificial and spontaneous pul Our previous studies on the inhibition of monarymetastases in mice . blood-borne metastases by sulfated poly

saccharides23) and the defibrinogenating MATERIALS AND METHODS agent ancrod10) showed that intravascular Animals Male C57BL/6 mice aged 6-8 weeks, coagulation in the target organ is an impor weighing 20-25g, were purchased from Shizuoka tantmechanism at an early stage of hemato Animal Farm Co. (Shizuoka). genousmetastasis. Tumors LLC* and B16, which both arose spontaneously in C57BL/6 mice, have been main Abbreviations used: LLC , Lewis lung carcinoma;PD tainedby serial biweekly subcutaneous passage in GF, platelet-derived growth factor; B16 , B16 the same strain of mice. Tumor fragments were melanoma; ADP, adenosine diphosphate; PRP , stirred in RPMI 1640 medium (Microbiological platelet-rich plasma; PPP, platelet-poor plasma; Associates, Md.) containing 0.2% trypsin (1:250, CPA, cyclophosphamide. Difco Lab., Detroit, Mich.) at 37•‹ for 30 min

284 Gann METASTASIS AND ANTIPLATELET AGENTS

(LLC) or 5min (B16). For intravenous inocula (B16) after tumor inoculation, and the lungs were tion,LLC tumor was treated with a solution of fixed in 10% formalin solution. Then the number DNase, collagenase I and anti-trypsin II-S (all of metastatic foci on the pulmonary surface was counted. purchased from Sigma Chemical Co., St. Louis) at 37•‹ for 45min. The isolated tumor cells were b) Spontaneous pulmonary metastasis: LLC

washed twice with RPMI 1640 medium contain cells (1•~106/0.05ml) and B16 cells (5•~105/0.05 ing10% fetal calf serum (FCS, Gibco, Grand ml) were inoculated into the footpads of C57BL/6 Island, New York), resuspended and counted in mice. The tumor-bearing foot was amputated on

a hemocytometer. B16 and LLC cells have been day 9 or 17 after tumor inoculation. The effects demonstrated to have strong and moderate ac of antiplatelet agents on different metastatic pro tivities,respectively, for aggregation of platelets.22) cesseswere examined, in mice bearing LLC, by

Antiplatelet Agents Ticlopidine (Daiichi Phar giving antiplatelet agents from day 5 to day 9 or maceuticalCo., Tokyo), diltiazem (Tanabe Phar from day 9 to day 14, and in mice bearing B16, maceuticalCo., Tokyo), dipyridamole (Japan by giving antiplatelet agents from day 9 to day Boehringer Ingelheim Co., Tokyo) and trapidil 17 or from day 17 to day 23. c) Combined effect of antiplatelet agents and (Mochida Pharmaceutical Co., Tokyo) were used as antiplatelet agents. These drugs were diluted anticancer agents: Inocula of 1•~106 LLC cells

with water to the desired concentration and ad were implanted into the footpad and the primary ministeredorally by stomach tube to mice in a tumor was removed 10 days later. Antiplatelet volume of 0.2ml. Ticlopidine and diltiazem were agents were given for 6 days after removal of the administered daily, whereas dipyridamole and tumor. A single dose of 50 mg/kg of CPA was in

trapidil were administered twice a day. jectedip on the day after removal of the implanted Ticlopidine, first described by Podesta et al.,20) tumor. is thought to enhance adenylate cyclase activity.) Mice were sacrificed on day 21 (LLC) or day

Diltiazern is a Ca antagonist enhancing adenylate 36 (B16) after tumor inoculation. Antimetastatic cyclase activity.21) Dipyridamole is an inhibitor activity was measured as described above. of cyclic-AMP phosphodiesterase, increasing pro

ductionof prostacyclin.18) Trapidil mainly inhibits RESULTS thromboxane synthetase activity and has the same

action as anti PDGF.19) Inhibitory Effect of Antiplatelet Agents Platelet Aggregating Agents ADP (Sigma on ADP-induced Platelet Aggregation Chemical Co.) and thrombin (Miles Labo., In Fig. 1 shows the inhibitory effect of anti diana)were used.

PRP PRP was obtained from citrated blood of plateletagents on ADP (400ƒÊM in 10mM mice. The number of platelets was adjusted to CaCl2)-induced PRP aggregation. Ticlopi approximately 2•~105/ƒÊl by adding PPP or saline. dineinhibited the aggregation of platelets PRP for platelet aggregation tests was prepared at a clinical dose of 12mg/kg, and had the 3hr after oral administration of ticlopidine or same inhibitory effect at doses of 60mg/kg, diltiazem, and 1hr after administration of dipy ridamoleor trapidil. PRP from mice treated with 120mg/kg and 360mg/kg. Diltiazem caused each agent for 2 days was also prepared 2, 5, 9 and only weak inhibition of platelet aggregation 12 days after the beginning of drug administration at any dose. Dipyridamale and trapidil in and the platelet activities were examined. hibitedaggregation only weakly at doses of Platelet Aggregation Platelet aggregation was measured turbidometrically with an Autoram-31 2mg/kg and 6mg/kg, respectively, but caused type aggregometer (Rika Denki Co., Tokyo). marked inhibition at doses of 60mg/kg and

Platelet aggregation was tested by adding 30ƒÊl 180mg/kg, respectively. Based on these re of a solution of aggregating agent, such as ADP sults,we used doses of 100mg/kg (ticlopidine), (400ƒÊM or 20ƒÊM in 10mM CaCl2) or thrombin 2mg/kg (diltiazem), 60mg/kg (dipyridamole) (20 NIH units/ml) to 0.27ml of PRP. Assay of Pulmonary Metastases and 180mg/kg (trapidil) for experiments a) Artificial pulmonary metastasis: LLC cells on metastases. (1•~106 in 0.2ml of RPMI 1640) or B16 cells (3•~ Inhibitory Effects of Antiplatelet Agents 105 in 0.2ml of RPMI 1640) were inoculated into on Thrombin-induced Platelet Aggrega C57BL/6 mice via the tail vein. Agents were given to mice orally for 2 days before tumor inoculation. tionAs shown in Fig. 2, trapidil (180mg/

The mice were killed 9 days (LLC) or 20 days kg) inhibited thrombin (20 NIH units/ml)-

75(3) 1984 285 H. BANDO, ET AL.

Fig. 1. Inhibition of ADP-induced platelet aggregation by antiplatelet agents

Control (• ). Ticlopidine (•› 12, •¢ 60, •£ 120, •¡ 360mg/kg), diltiazem (•› 2, •¢ 20, •£ 60mg/kg), dipyri damole(•› 2, •¢ 10, •£ 20, •¡ 60mg/kg) and trapidil (•› 6, •¢ 30, •£ 60, •¡ 180mg/kg) were given orally to mice. induced PRP aggregation moderately, while Effects of Antiplatelet Agents on Arti ticlopidine (100mg/kg), diltiazem (2mg/kg) ficialPulmonary Metastases All anti and dipyridamole (60mg/kg) inhibited this plateletagents tested inhibited the develop aggregation only slightly. mentof artificial pulmonary metastases of Change in Platelet Function in Mice both LLC and B16. Metastases of LLC were Treated with Antiplatelet Agents Next inhibited markedly in mice treated with we studied the time course of change in diltiazem or dipyriamole, while metastases platelet aggregation after treatment with of B16 were significantly inhibited by ticlo antiplatelet agents (Fig. 3). The platelet- pidine,diltiazem and trapidil (Table I). aggregating activity decreased on the day Effects of Antiplatelet Agents on Spon after treatment, but returned to normal 3 taneousPulmonary Metastases As days after treatment. No rebound phenome shown in Table II, no significant inhibitory nonor delayed inhibitory effect on platelet effects on spontaneous pulmonary metastases aggregation was observed. were observed in mice treated with anti-

286 Gann METASTASIS AND ANTIPLATELET AGENTS

Fig. 2. Inhibitory effects of antiplatelet agents on thrombin-induced platelet aggregation

Control (• ). Ticlopidine (•£ 100mg/kg), diltiazem (•› 2mg/kg), dipyridamole (•¡ 60mg/kg) and trapidil

(•¡ 180mg/kg) were given orally to mice. platelet agents before removal of the primary examined. The mean number of pulmonary tumor; diltiazem showed slight inhibition, metastatic nodules was 51.3•}5.6 in the con but ticlopidine, dipyridamole and trapidil trolgroup, and was reduced to 31.5•}6.8 tended to enhance pulmonary metastases. in mice treated with CPA alone. In mice In contrast, after removal of the primary treated with CPA plus ticlopidine, diltiazem tumor, spontaneous pulmonary metastases or dipyridamole, pulmonary metastases were of LLC and B16 were inhibited by all kinds significantly inhibited compared with those of antiplatelet agents used (Table II). in mice treated with CPA alone (Table III). Combined Effects of Antiplatelet Agents and Cyclophosphamide on Spontaneous DISCUSSION

Pulmonary Metastases The effects of Since Gasic et al.8) reported that treatment combined therapy with antiplatelet agents of platelets with neuraminidase reduced and CPA on pulmonary metastases were pulmonary metastasis of TA3 ascites tumor,

75(3) 1984 287 H. BANDO, ET AL.

Table I. Effects of Antiplatelet Agents on Arti. ficial Pulmonary Metastases

LLC cells (1•~106) or B16 cells (3•~105) were in oculatedinto the tail vein of mice. Antiplatelet agents

Fig. 3. Change in PRP aggregation activity after were given orally 2 days before tumor inoculation. The incidences of pulmonary metastases are shown in pa administration of antiplatelet agents rentheses(number of mice with pulmonary metastases/ Mice were treated with each antiplatelet agent for two number of mice tested). **P<0.02, ***P<0.01, days (n=4 in each point). compared with control. Aggregation ratio (%)

=maximal aggregation of treated PRP/maximal aggregation of control PRP•~100

•£ Ticlopidine, •¡ diltiazem, •› dipyridamole, •¢ trapidil.

Table II. Effects of Antiplatelet Agents on Spontaneous Pulmonary Metastases

LLC cells (1•~106) or B16 cells (5•~105) were implanted into the footpads of mice. The implanted tumor in each mouse was removed on day 9 or day 17 after tumor implantation. Antiplatelet agents were given as follows:

The incidences of pulmonary metastases are shown in parentheses (number of mice with pulmonary me tastases/number of mice tested). *P<0.05, **P<0.02, ****P<0.001, compared with control.

288 Gann METASTASIS AND ANTIPLATELET AGENTS mentof tumor cells and subsequent growth Table III. Combined Effect of Antiplatelet of metastatic foci. The experimental model Agents and Cyclophosphamide on Pulmonary Metastases of Lewis of spontaneous metastases is useful for study Lung Carcinoma ingthe metastatic process from the release of tumor cells to the growth of metastatic foci. Reduction of artificial pulmonary metas tasesby antiplatelet agents is thought to be due to inhibition of thrombus formation during lodgement of tumor cells in the target organ. It has been reported that platelet aggregation inhibitors prevented a decrease of platelet count after tumor inoculation. 16,17)

LLC cells (1•~106) were implanted into the footpads We did not use trypsin in the preparation of of mice. The implanted tumor was removed 10 days LLC cells for artificial metastases, because after tumor implantation. Antiplatelet agents were it may decrease the interaction between given for 6 days after removal of the tumor. A single dose of 50mg/kg of cyclophosphamide (CPA) was platelets and tumor cells.7) In fact, when injected ip on the day after removal of the implanted LLC cells treated with trypsin were used in tumor. *P<0.05, ***P<0.01, ****P<0.001, com experiments on artificial pulmonary metas paredwith control. tases,no inhibitory effect of antiplatelet agents on pulmonary metastasis was ob there have been many papers on the rela served(data not shown). However, pulmo tionof platelet function to metastases.5,6) narymetastases were inhibited in our experi Platelets are considered to play an essential mentalsystem when antiplatelet agents were role in the formation of tumor emboli, which given up to 6 days after tumor inoculation are an initial trigger of tumor lodgement in (data not shown). the metastatic process. There are many re In spontaneous metastasis, reduction of portsof reduction of metastasis by antiplatelet pulmonary metastases by antiplatelet agents therapy.11,13) Kohga et al.15) reported that was observed only after removal of the pri ticlopidine inhibited the aggregation of marytumor. At the time of removal of the platelets induced by B16 and LLC tumor primary tumor, tumor cells had already cells and also inhibited artificial and spon become lodged in capillary beds and micro taneouspulmonary metastases. In particular, metastaticfoci in the lung were in a growth spontaneous pulmonary metastases were phase. It has been demonstrated that tumor significantly inhibited when this compound promotingfactors,14,24) such as PDGF, were was given for 2 days before and one day after released from platelets after their aggre removal of primary tumor. However, Hilgard gation.2)PDGF is a polypeptide mitogen et al.12)observed enhancement of pulmonary which has recently been purified to homo metastasis on treatment with platelet aggre geneityfrom clinically outdated human gationinhibitors, so the significance of plate blood platelets.1) PDGF has been shown to letsin cancer metastasis is still uncertain. stimulate the multiplication of rat epithelial To clarify the role of platelets in cancer mammary tumor cells4) and many cancer metastasis, in this work we studied the effect - derived cells lines.9) Inhibition of platelet of antiplatelet agents on spontaneous and aggregation may prevent release of PDGF, artificial pulmonary metastases of LLC or and result in reduction of pulmonary metas B16 in C57BL/6 mice. This experimental tasis.These results support the hypothesis model of artificial metastases is thought to that antiplatelet agents reduce pulmonary be advantageous for analysis of the lodge metastases by inhibiting the release of PDGF.

75(3) 1984 289 H. BANDO, ET AL.

Combinations of antiplatelet agents and in the spread of malignant disease. Int. J. anticancer agents were found to cause a Cancer. 11, 704-718 (1973). 7) Gasic, G. J., Gasic, T. B. and Jimenez, S.A greater reduction in the number of metastatic . Effects of trypsin on the platelet-aggre. foci than anticancer agents alone. These re gating activity of mouse tumor cells. Thromb. sultssuggest the possibility of clinical appli Res., 10, 33-45 (1977). cationof combination therapy in the pre 8) Gasic, G. J., Gasic, T. B. and Stewart, C.C ventionof cancer metastases. . Antimetastatic effects associated with Measurement of the thickness of footpads platelet reduction. Proc. Natl. Acad. Sci. U.S.A., 61, 46-52 (1968). of tumor-bearing mice treated with anti 9) Hara, Y., Steiner, M. and Baldini, M. G. plateletagents before removal of the primary Platelets as a source of growth-promoting tumor revealed no influence of antiplatelet factor(s) for tumor cells. Cancer Res., 40,1212 -1216 (1980). agents on the growth of the primary tumor. 10) Higuchi, Y., Yamashita, T., Yamamoto, T.,K Thus, platelets probably play little role in agawa, K. and Tsubura, E. Effect of the growth of massive tumors. defibrinogenation on tumor growth and In this study we found that platelets are metastasis in mice. Ketsueki-to-Myakukan important not only for the lodgement of (Blood & Vessel), 11, 35-39 (1980) (in Jap anese). tumor cells but also for the growth of micro 11) Hilgard, P. The role of blood platelets in metastasesin the cancer metastatic process. experimental metastases. Br. J. Cancer,28, 429-435 (1973). ACKNOWLEDGMENTS 12) Hilgard, P., Heller, H. and Schmidt, C. G. This work was supported in part by grants from The influence of platelet aggregation inhib the Ministry of Education, Science and Culture. itorson metastasis formation in mice (3LL). Z. Krebsforsh., 86, 243-250 (1976). (ReceivedOct. 21, 1983/AcceptedJan. 26, 1984) 13) Karpatkin, S. and Pearlstein, E. Role of platelets in tumor cell metastases. Ann. Intern. Med., 95, 636-641 (1981). 14) Kepner, N. and Lipton, A. A mitogenic REFERENCES factor for transformed fibroblasts from hu manplatelets. CancerRes., 41, 430-432 (1981). 1) Antoniades, H. N., Scher, C. D. and Stiles, C. D. Purification of human platelet deri 15) Kohga, S., Kinjo, M. Tanaka, K., Ogawa, vedgrowth factor. Proc. Natl. Acad. Sci. H., Ishihara, M. and Tanaka, N. Effects U.S.A., 76, 1809-1813 (1979). of 5-(2-chlorobenzyl)-4,5,6,7-tetrahydro 2) Antoniades, H. N., Stathokos, D. and Scher thieno[3,2-C]pyridine hydrochloride (ticlo C. D. Isolation of a cationic polypeptide pidine),a platelet aggregation inhibitor, on from human serum that stimulates prolifer blood-borne metastasis. Cancer Res., 41, 4710-4714 (1981). ationof 3T3 cells. Proc. Natl. Acad.Sci. U.S.A., 72, 2635-2639 (1975). 16) Kolenich, J. J., Mansour, E. G. and Flynn, 3) Ashida, S. and Abiko, Y. Mode of action A. Haematological effects of aspirin. Lan of ticlopidine in inhibition of platelet aggre cet,ii, 714 (1972). gationin the rat. Thromb. Haemostasis, 41, 17) Lichtner, R. and Haarmann, W. Antime 436-449 (1979). tastaticaction of RX-RA 69, a new potent 4) Eastment, C. T. and Sibrasku, D. A. Plate PDE-inhibitor in the Lewis lung carcinoma letderived growth factor(s) for a hormone of the mouse. In "Interaction of Platelets -responsive rat mammary tumor cell line. J. and Tumor Cells," ed. G. A. Jamieson, pp. Cell. Physiol., 97, 17-28 (1978). 131-141 (1982). Alan R. Liss, Inc., New 5) Gasic, G. J. and Gasic, T. B. Removal of York. sialic acid from the cell coat in tumor cells 18) Mills, D. C. B. and Smith, J. B. The influ and vascular endothelium, and its effects on enceon platelet aggregation of drugs that metastasis. Proc. Natl. Acad. Sci. U.S.A., 48, affect the accumulation of adenosine 3'5'- 1172-1177 (1962). cyclic monophosphate in platelets. Biochem. 6) Gasic, G. J., Gasic, T. B., Galanti , N., John J., 121, 185-196 (1971). son,T. and Murphy, S. Platelet-tumor 19) Ohnishi, H., Yamaguchi, K., Shimada, S., cell interactions in mice. The role of platelets Suzuki, Y. and Kumagai, A. A new approach 290 Gann METASTASIS AND ANTIPLATELET AGENTS

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