Aromatic Bis[aminomethylidenebis(phosphonic)] Acids Prevent Ovariectomy-Induced Bone Loss and Suppress Osteoclastogenesis in Mice

Osteoporosis is a skeletal disease associated with excessive bone turnover. Among the compounds with antiresorptive activity, nitrogen-containing bisphosphonates play the most important role in antiosteoporotic treatment. In previous studies, we obtained two aminomethylidenebisphosphonates—benzene-1,4-bis[aminomethylidene(bisphosphonic)] (WG12399C) acid and naphthalene-1,5-bis[aminomethylidene(bisphosphonic)] (WG12592A) acid—which showed a significant antiproliferative activity toward J774E macrophages, a model of osteoclast precursors. The aim of these studies was to evaluate the antiresorptive activity of these aminobisphosphonates in ovariectomized (OVX) Balb/c mice. The influence of WG12399C and WG12592A administration on bone microstructure and bone strength was studied. Intravenous injections of WG12399C and WG12592A bisphosphonates remarkably prevented OVX-induced bone loss; for example, they sustained bone mineral density at control levels and restored other bone parameters such as trabecular separation. This was accompanied by a remarkable reduction in the number of TRAP-positive cells in bone tissue. However, a significant improvement in the quality of bone structure did not correlate with a parallel increase in bone strength. In ex vivo studies, WG12399C and WG12592A remarkably bisphosphonates reduced osteoclastogenesis and partially inhibited the resorptive activity of mature osteoclasts. Our results show interesting biological activity of two aminobisphosphonates, which may be of interest in the context of antiresorptive therapy

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

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

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

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

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

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