The effects of 1,4-dimethylpyridine in metastatic prostate cancer in mice

BACKGROUND: We previously showed that 1-methylnicotinamide (1-MNA) and its analog 1,4-dimethylpyridine (1,4-DMP) could inhibit the formation of lung metastases and enhance the efficacy of cyclophosphamide-based chemotherapy in the model of spontaneously metastasizing 4T1 mouse mammary gland tumors. In the present study, we aimed to investigate whether the previously observed activity of pyridine compounds pertains also to the prevention and the treatment of metastatic prostate tumors, in a combined chemotherapy with docetaxel. METHODS: Cancer-preventing activity of 1,4-DMP was studied in the model of prostate tumors spontaneously arising in C57BL/6-Tg (TRAMP)8247Ng/J (TRAMP) mice. The efficacy of the combined chemotherapy, comprising simultaneous use of 1,4-DMP and docetaxel, was evaluated in the orthotopic mouse model of human PC-3M-luc2 prostate cancer. The toxicity of the applied treatment was also determined. RESULTS: The development of prostate tumors in TRAMP mice remained unaffected after administration of 1,4-DMP. Similarly, no effect of 1,4-DMP was found on the growth of orthotopically transplanted PC-3M-luc2 tumors. However, when 1,4-DMP was administered along with docetaxel, it enhanced the anticancer activity of the chemotherapy. As a result, in PC-3M-luc2-bearing mice statistically significant inhibition of the tumor growth and lower metastases incidence were observed. The decreased metastatic yield is probably related to the diminished platelet activity observed in mice treated with combined therapeutic regimen. Finally, the combined treatment exhibited lowered side effects accompanying docetaxel administration. CONCLUSIONS: Results presented herein confirm previously published data on the anticancer activity of pyridine compounds and demonstrate that 1,4-DMP may be beneficially implemented into chemotherapy utilizing various cytotoxic agents, directed against multiple metastatic tumor types. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12885-017-3161-4) contains supplementary material, which is available to authorized users

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

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

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

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

Unfavorable effect of clopidogrel on cisplatin (CDDP), doxorubicin (DOX), and tamoxifen (TMX) anticancer activity in 4T1 mammary gland cancer model.

<p>(A) Tumor size on the last day of experiment. (B) Number of lung metastatic foci. (C) Lung weight and (D) kinetics of tumor growth in mice treated with clopidogrel combined with DOX, CDDP, and TMX. All graphs show values for individual animals with median line. The exception is (D): mean with standard deviation (SD) is presented. N = 10–12 mice per group. All mice were euthanized: DOX and TMX experiment on 29, CDDP experiment on day 26. Statistical analysis: Kruskal–Wallis test for multiple comparisons, *<i>p</i><0.05.</p

Clopidogrel increases the toxicity of docetaxel (DTX) in mice bearing human PC-3M-luc2 prostate tumors.

<p>(A) Luminescence of prostatic tumors on day 15 (day of treatment initiation), 23, and 27. (B) Photographs of mice on day 27 of experiment. (C) Tumor weight of tumors harvested on day 46. (D) Primary tumor tissue stained with hematoxylin and eosin (H&E) showing visible mitotic spindles. (E) Survival curves. (F) Number of organs with diagnosed metastases. (G) H&E-stained lung (left), liver (middle), and lymph node (right) tissue with micrometastases. All graphs show values for individual animals with median line with the exception of (E) shows percent of survival in indicated days. All mice were euthanized on day 46; the number of animals euthanized before: 12. N = 9 mice per group. Statistical analysis: Kruskal–Wallis test for multiple comparisons, *<i>p</i><0.05. Survival analysis: Mantel–Cox test showed significant differences between survival curves (<i>p</i><0.0001).</p

Clopidogrel protects against the toxicity of MTX in mice bearing human PC-3M-luc2 prostate tumors.

<p>(A) Luminescence of prostatic tumors on day 14 (a day prior to the treatment initiation) and 29. (B) Photographs of mice on day 29 of experiment. (C) Tumor weight of tumors harvested on day 37. (D) Number of organs with diagnosed metastases. (E) Survival curves. (A)–(E) Data from mice treated with 3 mg/kg/dose of MTX. All mice were euthanized on day 37; the number of animals euthanized before: 10. N = 9 mice per group. (F) Tumor weight of tumors harvested on day 47. (G) Number of organs with diagnosed metastases. (H) Transforming growth factor β1 (TGF-β1) plasma level. (F)–(H) Data from mice treated with 1 mg/kg/dose of MTX. All mice were euthanized on day 47. N = 7–9 mice per group. All graphs show values for individual animals with median line; the exception: (E) shows percent of survival on indicated days. Statistical analysis: Kruskal–Wallis test for multiple comparisons, *<i>p</i><0.05. Survival analysis: Mantel–Cox test showed significant differences between survival curves (<i>p</i><0.0001).</p