Abstract
Background/Aim: The complex interactions between circulating platelets and tumor cells play important roles in tumor metastasis. Tumor cells can activate platelets by releasing mediators such as adenosine diphosphate (ADP). Treatments with anticoagulants have been shown to attenuate tumor metastasis. However, the role of ADP receptor P2Y12 in tumor cell metastasis has not been fully clarified.
Materials and Methods: In this study, highly metastatic B16-BL6 mouse melanoma cells were injected into the tail vein of mice as a model of hematogenic tumor metastasis to investigate the effects of P2Y12 antagonist clopidogrel on tumor metastasis.
Results: A high dose (25 mg/kg) of clopidogrel weakly but significantly inhibited lung metastasis and increased both the time to hemostasis and blood loss in the tail tip-excision mouse model.
Conclusion: Although it is necessary to consider increased bleeding as a side-effect, clopidogrel may be an effective antimetastatic drug.
Introduction
Almost one and a half centuries ago, a close relationship between tumor and thrombosis was reported, often referred to as Trousseau’s syndrome (1). Circulating platelets are traditionally viewed as major cellular components in hemostasis and thrombosis, but a growing body of evidence shows that the complex interactions between circulating platelets and tumor cells play important roles in critical steps during tumor metastasis, including epithelial–mesenchymal-like transitions, tumor cell migration and invasion. For instance, platelets promote invasiveness of prostate cancer cells via upregulation of matrix metalloproteinase-2 production (2-4).
Platelet membrane receptors are essential for platelet attachment, aggregation, and activation, the conclusive steps in platelet-mediated hemostasis. Important platelet membrane receptors include integrin αIIbβ3, which are indispensable for complete platelet attachment and aggregation (5). In addition, blood coagulation factor IIa (thrombin) induces activation of platelets via protease-activated receptor-1 and -4. Both these receptors are expressed on platelet membranes (6). Adenosine diphosphate (ADP) is contained in platelet-dense granules and is considered an inducer of secondary platelet aggregation. The main ADP receptors, P2Y1 and P2Y12, are both involved in platelet aggregation. P2Y12 plays a prominent role in secretion from dense granules, activation of fibrinogen receptor, and thrombus formation (7). In fact, only P2Y12 receptor antagonists are useful clinically.
Human cancer cells can activate platelets through direct contact, or by releasing mediators such as ADP, thrombin, thromboxane A2, or tumor-associated cysteine proteinases (8, 9). It was reported that the P2Y1 receptor-selective agonist 2-methylthioadenosine-5-diphosphate caused a decrease in A357 melanoma cell growth in vitro. On the other hand, the P2Y2 receptor agonist uridine triphosphate caused an increase in A357 melanoma cell growth in vitro (10). However, it remains unclear whether P2Y12 receptor antagonist also plays a role in tumor cell metastasis.
Clopidogrel is a P2Y12 receptor antagonist, widely used to treat coronary artery, peripheral vascular, and cerebrovascular diseases in clinical practice. In this study, we used B16-BL6 mouse melanoma cells as a metastasis model to investigate the safety of targeting P2Y12 and elucidate the effects of clopidogrel on tumor metastasis.
Materials and Methods
Materials. Fetal bovine serum was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium with L-glutamine was obtained from Invitrogen Co. (Grand Island, NY, USA). Dulbecco’s phosphate-buffered saline without calcium and magnesium [DPBS(−)] was from purchased Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). EDTA trypsin solution (EDTA, 0.02%; trypsin, 0.1%) and penicillin/streptomycin solution (penicillin, 50,000 U/ml; streptomycin, 50 mg/ml) were obtained from Cosmo Bio Co., Ltd. (Tokyo, Japan).
Cells. A mouse melanoma cell line, B16-F0, was established from a naturally occurring melanoma in the skin (ear) of a C57BL/6J mouse. The B16-F1 cell line was established from pulmonary-metastatic colonies produced by the administration of B16-F0 cells into a tail vein in a syngeneic C57BL/6J mouse. The B16-F10 cell line was then repeatedly selected by 10 successive passages of lung metastatic lesions (11). The B16-F10 cells were then administered into the urinary bladders of male C57BL/6J mice via the vas deferens, and the bladders were then ligated, excised, and maintained on semi-solid agar. Cells which had migrated through the wall of the bladder were recovered from the agar, cultured aseptically, and repassaged. This process was repeated six times, and the resulting cell line was named B16-BL6 (12) and generously provided by Professor Futoshi Okada of Tottori University (Yonago, Japan). According to our previous study, the metastatic potential was B16-F0<B16-F1<B16<F10=B16-BL6 (13). In the present experiments, we used B16-BL6 cells as a highly metastatic mouse melanoma cell line. Cells passaged less than 45 times were used in all experiments and cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and a 0.1% penicillin/streptomycin solution in 5% CO2 at 37°C.
Animals. Seven-week-old female C57BL/6N mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan). The mice were maintained in an air-conditioned room (23±2°C and 60±10% humidity) under a 12-hour light/dark cycle (lights on 7:00 a.m.). Food and water were given ad libitum during the experiment. All procedures followed the Guidelines for the Care and Use of Laboratory Animals at Mukogawa Women’s University (approval number: 2014PP-yakuri1-15).
Assay of experimental metastasis of tumor cells. This metastatic model was established by Fidler’s group (11, 12). Sub-confluent B16-BL6 cells were harvested with EDTA trypsin solution and resuspended to an appropriate density in DPBS(−). Cells (1×105/0.2 ml) were injected into the tail vein of syngeneic C57BL/6N mice, but 6 hours before the injection, and every day after injection, seven mice per group were treated with clopidogrel (0, 5, or 25 mg/kg diluted in 0.9% NaCl, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) by oral gavage. Control mice were administered vehicle only. These concentrations are somewhat higher than found in clinical use (1.5 mg/kg). Two weeks after tumor cell injection, mice were anesthetized with pentobarbital and sacrificed. Lungs were excised and fixed in a formaldehyde neutral buffer solution. Tumor nodules were enumerated with the aid of a magnifying glass.
Animal model of tail tip excision. The protocol was adapted from previous work (14). Briefly, seven mice per group were anesthetized with pentobarbital and, the lower segment of the tail was wiped clean with 70% ethanol and allowed to air dry. The tail tip excision point was marked at 1 mm from the distal end. Sterile razor blades were used to sever the tail tip in one quick slicing motion. The tail tip was held 1.5 cm above the surface of a gauze pad. Filter paper disks (5 mm in diameter) made from Whatman #4 filter paper were held up to the bleeding tail tip but not allowed to touch the tail and the blood was collected at 15-second intervals, until the disks were no longer impregnated with blood. Blood-impregnated disks were dropped into plastic tubes containing 2 ml of 10% NaOH at 1-minute intervals for 10 consecutive minutes or until hemostasis was achieved. Hemoglobin was determined and blood loss estimated by the enumeration of all collection tubes from each mouse.
Statistical analyses. Data are expressed as the mean±standard error of the mean of 6-7 animals. Statistics were calculated using SPSS version 29 (IBM, Armonk, NY, USA) and analyzed using Dunnett’s T3 test. Differences were considered significant at p<0.05.
Results
Effect of oral clopidogrel administration on metastasis formation in lungs. Clopidogrel was orally administered to mice 6 hours before and every day after tumor cell injection into the lateral tail vain. Two weeks later, mice were euthanized, and the metastatic nodules in the lungs were counted. A high dose (25 mg/kg) of clopidogrel weakly but significantly inhibited lung metastasis (Figure 1).
Inhibition of B16-BL6 lung metastasis by clopidogrel treatment. B16-BL6 melanoma cells (1×105) were injected into the lateral tail vein on day 0. The mice were orally administered normal saline or 5 or 25 mg/kg of clopidogrel, 6 hours before and every day after tumor cell injection for 2 weeks (n=7 mice per group). (A) Representative lungs of mice in the normal, 0 mg/kg (control), 5 mg/kg, and 25 mg/kg clopidogrel-treated groups. (B) Tumor nodules formed on the surfaces of lung tissues in mice from different groups of the B16-BL6 tumor model. Data indicate the mean±standard error of the mean of 6-7 mice. *Significantly different at p<0.05 compared with the control group using Dunnett’s T3 test.
Effect of clopidogrel on hemostasis of B16-BL6-bearing mice. The administration of clopidogrel for 2 weeks dose-dependently increased blood loss in the tail tip-excision model. The low (5 mg/kg) and high (25 mg/kg) doses of clopidogrel increased the total blood loss approximately 10- and 16-fold, respectively, compared with the control group (Figure 2). The time to hemostasis in both clopidogrel groups (5 and 25 mg/kg) was not within 10 minutes post-injury (data not shown). Although increased blood loss was observed, there was no effect on the body weight of clopidogrel-treated mice.
Effect of clopidogrel on hemostasis of B16-BL6-bearing mice. B16-BL6 cells (1×105) were injected into the lateral tail vein on day 0. The mice were orally administered normal saline (control) or 5 or 25 mg/kg of clopidogrel, 6 hours before and every day after tumor cell injection for 2 weeks. The tail tips of the mice were excised at 1 mm from the distal end. Blood loss is shown for each group. Data indicate the mean±standard error of the mean of 6-7 mice. *Significantly different at p<0.05 compared with the control group using Dunnett’s T3 test.
Discussion
In the present study, we demonstrated that clopidogrel, an antagonist of ADP receptor P2Y12, significantly inhibited the hematogenic metastasis of mouse melanoma B16-BL6 cells but also increased the time-to-hemostasis and blood loss in B16-BL6 tumor-bearing mice. However, the antimetastatic effect of clopidogrel in the present model of hematogenic metastasis appears to be weak because the metastasis rate in the high-dose (25 mg/kg) clopidogrel group was 24.3% lower compared with the control group. Considering thrombocytosis in patients with cancer and the anticoagulant effect of clopidogrel, other hematogenic metastasis models may be useful. For example, ADP and tumor cells injected into mice simultaneously to accelerate tumor cell metastasis (15).
Gareau et al. demonstrated that ticagrelor, another P2Y12 receptor antagonist, inhibited platelet–tumor cell interactions and metastasis in human and murine breast cancer cells. Furthermore, ticagrelor but not clopidogrel resulted in reduced metastasis and improved survival using an orthotopic 4T1 breast cancer mouse model (16). However, Palacios-Acedo et al. reported that clopidogrel resulted in better survival rates with smaller primary tumors and less metastasis than aspirin, a cyclooxygenase inhibitor, and clopidogrel was more effective than aspirin at dissolving spontaneous endogenous thrombi in an orthotopic mouse model of pancreatic cancer (17). Most recently, Jantsch et al. examined the effect of clopidogrel bisulfate on B16-F10 mouse melanoma cells and tumor development in a murine model of melanoma showing that clopidogrel bisulfate reduced cell viability and proliferation in B16-F10 cells in vitro and reduced tumor nodule counts in vivo (18).
In this study, we injected mouse melanoma B16-BL6 cells into the tail vein of mice, whereas Jantsch et al. administered mouse melanoma B16-F10 cells to mice intraperitoneally. In other words, we evaluated the antimetastatic action of clopidogrel, and Jantsch et al. investigated the antitumor action of clopidogrel bisulfate. Furthermore, Palacios-Acedo et al., Jantsch et al., and we orally administered clopidogrel to mice because clopidogrel is a prodrug that is metabolized to the active form by CYP2C19, a liver enzyme.
Considering that platelets protect tumors from natural killer cells and TNFα cytotoxicity (19, 20), it may be necessary to investigate the effect of clopidogrel on natural killer cell activity in B16-BL6-bearing mice.
In conclusion, although it is necessary to take into account increased bleeding as an adverse effect, clopidogrel may be a convincing candidate as an active antimetastatic drug.
Footnotes
Authors’ Contributions
Conceptualization: KN. Investigation: MX. Data curation: NY. Formal analysis: NY. Supervision: NY and KN. Writing-original draft: MX. Writing-review and editing: KN.
Funding
This study was supported by a Special Grant from the Smoking Research Foundation for Biomedical Research of Japan, and by MEXT’s Subsidy Program for Human Resource Development for Science and Technology, Initiative for Realizing Diversity in the Research Environment (Type of Women’s Leadership Development), Program for Developing the Next Generation of Aspiring Women Leaders at a Women’s University.
Conflicts of Interest
The Authors declare no conflicts of interest associated with this manuscript.
- Received January 16, 2025.
- Revision received February 8, 2025.
- Accepted February 11, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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