33.    Effects of an N4-aryl substituted thiosemicarbazone on the metastasis of triple                    negative 4T1 mouse mammary cancer cells in vitro and in vivo

Triple negative breast cancer (TNBC) is defined by a lack of expression of estrogen (ER) and progesterone (PgR) receptors as well as HER2/neu. This pathology represents about 15% of all types of breast cancer and, among all subtypes, TNBC is associated with a worse prognosis and a higher incidence of visceral metastasis. The lack of targeted therapies and the poor prognosis of TNBC patients have fostered a major effort to develop new alternative treatments. Thiosemicarbazones are synthetic compounds that exhibit several pharmacological activities. Previously, we found that T2, an N4-aryl substituted thiosemicarbazone, had cytotoxic activity on 4T1 mouse mammary tumor cells. Now, we have investigated the action of T2 on metastasic properties of 4T1 cells. Cancer metastasis consists of a complex cascade of events, which ultimately allow for tumor cell escape and seeding of ectopic environments. For breast cancer cells to manifest their malignant potential, they must develop the ability to break through and dissolve extracellular matrix (ECM). One important class of ECM-degrading enzymes are the matrix metalloproteinases (MMPs). Consequently, we analyzed MMP9 activity of 4T1 cells after T2 treatment and we found that it was down-regulated (T2 2.5 􀀀M= 81.7 ± 0.1%; T2 5 􀀀M= 71.3 ± 0.1% and T2 10 􀀀M=60.0 ± 0.1 % respect to DMSO treated control cells, p˂0.05). Motility is another property of cancer cells that is required for migration from the primary site to a secondary organ to occur. Therefore, we quantified cell migration and we observed that treatment of 4T1 cells with T2 led to a dose dependent decrease in wound- healing cell migration (T2 2.5 􀀀M= 62 ± 2%; T2 5 􀀀M= 26 ± 4% and T2 10 􀀀M=20 ± 2% respect to control cells, p˂0.05). Cancer stem cells (CSC) are thought to be responsible for all of tumor characteristics including metastasis. Thereafter, we evaluated T2 action on mammospheres (enriched with CSC) from 4T1 cells and we noted that T2 significantly reduced mammospheres number (T2 5 􀀀M= 48.6± 0.1% and T2 10 􀀀M=51.8 ± 0.1 % respect to control cells, p˂0.05). Metastasis is a complex process that requires the regulation of both metastasis-promoting and metastasis suppressor genes. In consequence, we analyzed by western blot the modulation of NDRG-1 (N-myc downregulated gene 1), a metastasis suppressor gene, and we observed that T2
increased its expression in a dose-dependent manner. Finally, we evaluated T2 modulation on 4T1 experimental metastasis. The macroscopic appearance of the lungs from mice inoculated with control and treated cells clearly showed that treatment with T2 5 and 10 􀀀M reduced the number and the size of 4T1 colonies in the lungs of Balb/c mice. In conclusion, our results show that T2 inhibits metastasis of 4T1 cells, and this inhibition could be due in part to an up-regulation of NDRG-1 expression and to a downregulation of cell migration capacity and a reduction in MMP-9 activity and in CSC sub-population.