Clopidogrel in a combined therapy with anticancer drugs—effect on tumor growth, metastasis, and treatment toxicity: Studies in animal models

<div><p>Clopidogrel, a thienopyridine derivative with antiplatelet activity, is widely prescribed for patients with cardiovascular diseases. In addition to antiplatelet activity, antiplatelet agents possess anticancer and antimetastatic properties. Contrary to this, results of some studies have suggested that the use of clopidogrel and other thienopyridines accelerates the progression of breast, colorectal, and prostate cancer. Therefore, in this study, we aimed to evaluate the efficacy of clopidogrel and various anticancer agents as a combined treatment using mouse models of breast, colorectal, and prostate cancer. Metastatic dissemination, selected parameters of platelet morphology and biochemistry, as well as angiogenesis were assessed. In addition, body weight, blood morphology, and biochemistry were evaluated to test toxicity of the studied compounds. According to the results, clopidogrel increased antitumor and/or antimetastatic activity of chemotherapeutics such as 5-fluorouracil, cyclophosphamide, and mitoxantrone, whereas it decreased the anticancer activity of doxorubicin, cisplatin, and tamoxifen. The mechanisms of such divergent activities may be based on the modulation of tumor vasculature <i>via</i> factors, such as transforming growth factor β1 released from platelets. Moreover, clopidogrel increased the toxicity of docetaxel and protected against mitoxantrone-induced toxicity, which may be due to the modulation of hepatic enzymes and protection of the vasculature, respectively. These results demonstrate that antiplatelet agents can be useful but also dangerous in anticancer treatment and therefore use of thienopyridines in patients undergoing chemotherapy should be carefully evaluated.</p></div

Summarized effects of the combination therapy with clopidogrel and anticancer drugs on tumor growth and metastasis, as well as platelets condition.

<p>Summarized effects of the combination therapy with clopidogrel and anticancer drugs on tumor growth and metastasis, as well as platelets condition.</p

Combination therapy with clopidogrel and 5-fluorouracil (5-FU) reduce the growth and metastasis of 4T1 tumors.

<p>(A) Tumor weight on day 26 and kinetics of tumor growth. (B) Number of lung metastatic foci. (C) Expression of E- and N-cadherin in tumor tissue (left graph), E:N-cadherin ratio (middle) and representative blots (right). (D) Platelets’ morphological parameters, including platelet count, platelet distribution width (PDW), mean platelet volume (MPV), and plateletcrit (PCT). (E) ELISA measurements of plasma proteins corresponding to platelets activity: von Willebrant factor (vWF), thromboxane b2 (TXB2), transforming growth factor beta 1 (TGF-β1), P-selectin, and prostacyclin metabolite (6-keto-PGF1α). All graphs show values for individual animals with median line; the exception is panel D: mean with standard error of mean (SEM) and kinetics of tumor growth: mean with standard deviation (SD) is presented. N = 10 mice per group; some tests were performed on tissue or plasma from selected animals from each group (at least 3 in western blot tests; data for individual blots presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188740#pone.0188740.s015" target="_blank">S1 Table</a>). All mice were euthanized on day 26. Statistical analysis: Kruskal–Wallis test for multiple comparisons; *<i>p</i><0.05. The values of selected morphological parameters of platelets in healthy BALB/c mice: platelet count: 245 ± 95 [×10³/μL]; MPV: 6.6 ± 0.3 [fL]; PDW: 47 ± 1 [fL]; PCT: 0.2 ± 0.06 [%]. The level of TGF-β1: 320 ± 449; P-selectin: 100 ± 10 in healthy BALB/c mice.</p

Platelet parameters in mice bearing 4T1 tumors treated with clopidogrel combined with doxorubicin (DOX), cisplatin (CDDP), and tamoxifen (TMX).

<p>Morphological parameters of platelets: (A) Platelet count. (B) Platelet distribution width (PDW). (C) Mean platelet volume (MPV). (D) Plateletcrit (PCT). (E) Concentration of transforming growth factor β1 (TGF-β1) in mice plasma. All graphs show values for individual animals with median line. N = 4–10 mice per group. Statistical analysis: Kruskal–Wallis test for multiple comparisons; *<i>p</i><0.05.</p

Combination therapy with clopidogrel and cyclophosphamide (CP) reduce the growth and metastasis of orthotopic 4T1 tumors.

<p>(A) Tumor weight on day 31 and kinetics of tumor growth. (B) Number of lung metastatic foci. (C) Tumor blood vessel permeability measured as the accumulation of IRDye 800CW PEG Contrast Agent 24 h after injection. (D) Example photographs of fluorescence measurements are presented. (E) Expression of E- and N-cadherin in tumor tissue (left graph), E:N-cadherin ratio (middle), and representative blots (right). (F) Platelet count. (G) ELISA measurements of plasma proteins connected with platelets activity: von Willebrant factor (vWF) and thromboxane B2 (TXB2). All graphs show values for individual animals with median line; the exception is panel (E): mean with standard error of mean (SEM) and kinetics of tumor growth: mean with standard deviation (SD) is presented. N = 10 mice per group; some tests were performed on tissue or plasma from selected animals from each group (at least 3 in western blot tests; data for individual blots presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188740#pone.0188740.s015" target="_blank">S1 Table</a>). All mice were euthanized on day 31. Statistical analysis: Kruskal–Wallis test for multiple comparisons; *<i>p</i><0.05.</p

Platelet morphological parameters in PC-3M-luc2 bearing mice treated with clopidogrel combined with DTX.

<p>(A) Platelet morphological parameters, including platelet count, platelet distribution width (PDW), mean platelet volume (MPV), and plateletcrit (PCT). (B) ELISA measurements of plasma proteins related to platelets activity: von Willebrant factor (vWF), thromboxane b2 (TXB2), transforming growth factor beta 1 (TGF-β1), P-selectin, and prostacyclin metabolite (6-keto-PGF1α). All graphs show values for individual animals with median line. Plasma from 2 to 9 mice per group was analyzed. Statistical analysis: Kruskal–Wallis test for multiple comparisons; *<i>p</i><0.05. The values of selected platelet parameters for healthy nude mice: platelet count: 525 ± 30 [×10³/μL]; MPV: 5.0 ± 0.3 [fL]; PDW: 31 ± 5 [fL]; PCT: 0.3 ± 0.03 [%].</p

Clopidogrel did not affect tumor growth and metastasis in the combination therapy with 5-FU of mice bearing MC38/EGFP subcutaneous tumors.

<p>(A) Tumor weight on day 53. (B) Kinetics of tumor growth. (C) Score for microvessel density (MVD) measured in tumor tissue sections stained with anti-CD31 antibody. (D) Microphotographs of tumor tissue stained with anti-CD31 antibody from control and clopidogrel treated mice. (E) Expression of E- and N-cadherin in tumor tissue (left graph), E:N-cadgherin ratio (middle) and representative blots (right). (F) Morphological parameters of platelets, including platelet count, platelet distribution width (PDW), mean platelet volume (MPV), and plateletcrit (PCT). (G) ELISA measurements of plasma proteins connected with platelets activity: von Willebrant factor (vWF) and thromboxane B2 (TXB2). All graphs show values for individual animals with median line; the exception is panel E: mean with standard error of mean (SEM) and B: mean with standard deviation (SD) is presented. All mice were euthanized on day 53. N = 6–9 mice per group; some tests were performed on tissue or plasma from selected animals from each group (at least 3 for western blot; data for individual blots presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188740#pone.0188740.s015" target="_blank">S1 Table</a>). Statistical analysis: Kruskal–Wallis test for multiple comparisons; *<i>p</i><0.05. The values of selected platelet parameters for healthy C57Bl/6 mice: platelet count: 571 ± 94 [×10³/μL]; MPV: 5.4 ± 0.2 [fL]; PDW: 41 ± 4 [fL]; PCT: 0.3 ± 0.04 [%].</p

Dosing and treatment schedules used in experiments.

<p>Dosing and treatment schedules used in experiments.</p

Lactate dehydrogenase (LDH) and creatine kinase (CK) levels in animals treated with clopidogrel combined with 5-fluorouracil (5-FU), cyclophosphamide (CP), cisplatin (CDDP), doxorubicin (DOX), and tamoxifen (TMX) in 4T1 tumor model.

<p>LDH values in mice treated with clopidogrel combined with: (A) 5-FU, (B) CP, (C) CDDP, (D) DOX, and (E) TMX. CK values for clopidogrel in the combination therapy with (F) DOX and (G) TMX. All graphs show values for individual animals with median line. Plasma from 4 to 7 mice per group was analyzed. Statistical analysis: Kruskal–Wallis test for multiple comparisons; *<i>p</i><0.05.</p

Clopidogrel improved tumor growth inhibition of 5-FU in mice transplanted intrasplenically with HT-29-luc2 human colon cancer.

<p>(A) Luminescence of intrasplenic tumors measured on days 20, 27, and 34. (B) Photographs of luminescence on day 27 of experiment. (C) Weight of spleens with primary tumors harvested on day 51. (D) Number of metastatic foci counted in the liver harvested on day 51. (E) Morphological parameters of platelets, including platelet count, platelet distribution width (PDW), mean platelet volume (MPV), and plateletcrit (PCT). (F) Plasma level of transforming growth factor β1 (TGF-β1). All graphs show values for individual animals with median line. All mice were euthanized on day 51. N = 7–10 mice per group (A–E); TGF-β1 was measured in plasma from five randomly selected mice (F). Statistical analysis: Kruskal–Wallis test for multiple comparisons; *<i>p</i><0.05.</p