Antiproliferative Effect Of Carboxyamidotriazole Against Human Breast Cancer Cells And Underlying Mechanisms

Background.Carboxyamidotriazole (CAI) is an inhibitor of transmembrane calcium influx and intracellular calcium-requiring signal transduction pathway. CAI inhibits the proliferation and invasive characteristics of several tumor cell lines in vitro, including prostate, glioblastoma, bladder, hepatoma, leukemia and breast. CAI also demonstrates antiangiogenic activity in the chick chorioallantotic membrane assay and rat aortic ring assay, as well as the activity of inhibiting the proliferation of human umbilical vein endothelial cells in vitro. Recent studies have indicated that CAI can inhibit proliferation ofcultured cancer cells by inducing apoptosis.Although the antiproliferative, antiangiogenic, anti-invasive and apoptosis inductive effects of CAI have been well-documented, the cell cycle regulating activity of CAI itself in cancer cells has not been systematically assessed. And little is known about the molecular mechanism of the anticancer effect of CAI. The purpose of our present study is to evaluate the antiproliferative activity of CAI against MCF-7 human breast cancer cells and 293T cells, explore the mechanisms and signaling pathways of CAI-induced cell cycle arrest and apoptosis in human breast cancer cells and provide basic evidence for on-going clinical trial and the applicability of CAI as a potential preventive and/or therapeutic agent against human breast cancer.Methods.Survival/proliferation of cell lines treated with CAI was determined by trypan blue dye exclusion assay or MTT assay. The morphological changes of MCF-7 cells were observed and photographed by a phase-contrast microscope. [³H]-thymidine incorporation assay was performed to assess DNA synthesis of cells. Cell cycle distribution was determined by flow cytometric analysis of DNA content of nuclei of cells following staining with propidium iodide. Apoptosis induction by CAI in MCF-7 cells was assessed by (i) flow cytometry analysis of sub-G₀-G₁ DNA content and (ii) quantification of phosphatidylserine exposure using annexin V-FITC kit. Alterations in mitochondrial membrane potential (△ψ_m) were analyzed by flow cytometry using the △ψ_m-sensitive dye rhodamine123.