The effects of chemically modified tetracyclines on vascular endothelial growth factor production in models of breast cancer

Chemically Modified Tetracyclines (CMTs) are non-antibiotic, anti-proteolytic agents that inhibit invasiveness and metastasis in tumors. This study aimed to examine CMTs as inhibitors of the angiogenic response in breast cancer cells, associated monocytes and endothelial cells. CMT 308, a 9-amino, non-phototoxic derivative of CMT 300 (6-demethyl-6-deoxy-4-dedimethylaminotetracycline) reduced basal Vascular Endothelial Growth Factor (VEGF) secretion from MCF-7 and MDA-MB-453 cells that are models of early breast cancer and from MDA-MB-435s and MDA-MB-231 cells that are models of late breast cancer. Subcytotoxic levels of CMT 308 also specifically reduced basal VEGF secretion from MonoMac 6 cells used as a model of infiltrating monocytes in the tumor microenvironment. Time course studies showed that VEGF was secreted into the conditioned media of MCF-7 cells within 4 hours of plating and that CMT 308 inhibited VEGF secretion up to 8 hours. The duration of CMT 308's effectiveness was maximal up to 8 hours in the MCF-7 cells, with CMT 308 inhibiting new secretion of VEGF rather than pre-existing protein. However, with MonoMac 6 cells, CMT 308 treatment was effective up to 24 hours. Treatment with TGFbeta increased intracellular VEGF protein and secretion of VEGF, but not VEGF mRNA levels in breast cancer cells. CMT 308 decreased levels of TGFbeta-induced VEGF protein in cellular lysates of MDA-MB-435s cells. Neither CMT 300 nor CMT 308 affected VEGF mRNA levels in the breast cancer cells. Lysates of MonoMac 6 cells treated with CMT 300 showed a reduction in VEGF protein, while those treated with CMT 308 showed a small decrease in VEGF immuno-reactivity as determined by western blot. Additionally, CMT 308 treatment also decreased VEGF mRNA levels in MonoMac 6 cells. Both, CMT 300 and CMT 308 reduced tube formation, migration and invasion of endothelial cells through layers of Matrigel, a reconstituted basement membrane. These results indicate that CMT 308 inhibited basal and some pathways) of TGFbeta-mediated stimulation of VEGF secretion at a post-transcriptional step, leading to decreased secretion of VEGF in breast cancer and monocytic cells. This novel mode of inhibition of release of the pro-angiogenic cytokine as well as the angiogenic response of the endothelial cells strongly recommends CMT 308 as a potential therapeutic agent for breast cancer.