Figure 2 Alcohol induces cell SN-38 manufacturer invasion by suppressing Nm23 expression. T47D cells were treated with 0.5% v/v alcohol and the expression of known metastasis suppressor genes was determined by qRT-PCR. Nm23 mRNA expression levels significantly decreased following treatment. KAI1, RRM1, and BRMS1 expression were not affected by alcohol and expression of KISS1 and Mkk4 were increased by alcohol. (*p < 0.05,

as compared to the control cells with no alcohol treatment). To determine whether the effects of alcohol on the invasive ability of T47D cells can be blocked via Nm23, we transfected T47D cells with the pcDNA3-Nm23-H1 vector (kindly eFT-508 solubility dmso provided by Dr. Patricia Steeg at the National Cancer Institute, Bethesda, MD, USA) to overexpress Nm23. As expected, Nm23 overexpression resulted in a significant decrease in T47D cell invasion (Figure 3A, p < 0.05) while treatment of T47D control cells (transfected with an empty vector) with 0.5% v/v alcohol significantly increased cell invasive ability (Figure 3A, p < 0.05). (Note: Results from check details Figure 1A and 3A indicate

that 0.5% v/v ethanol increased cell invasion by 600% and 50%, respectively. This difference may be attributed to the addition of G418 (Gibco, St Louis, MO, USA) in the media used for the invasion assay shown in Figure 3A. As an inhibitor of protein synthesis, addition of G418 may have led to a decline in cell proliferation over the 24 hour invasion period.) However, 0.5% v/v alcohol was unable to increase the invasive ability of T47D cells overexpressing Nm23 AZD9291 concentration (Figure 3A, p > 0.05), suggesting that Nm23 expression is critical in alcohol-induced T47D breast cancer cell invasion. Nm23 protein levels are shown in Figure 3B. Figure 3 Overexpression of Nm23 suppressed cell invasion. The invasion assay was used to determine the invasive ability of T47D cells treated with 0.5% v/v ethanol and overexpressing Nm23, independently and in

combination. (A) Alcohol treatment increased the invasiveness of the T47D cells transfected with the empty vector; however, alcohol did not increase invasion in the T47D cells transfected with Nm23. (B) Western blot shows Nm23 expression levels following ethanol treatment, Nm23 overexpression, and the combination of ethanol and Nm23 overexpression. Quantification by ImageJ software indicates relative Nm23 expression. (*p < 0.05, as compared to the control cells transfected with empty vector). Down-regulation of Nm23 increases ITGA5 expression to promote breast cancer cell invasion To examine the downstream targets of Nm23 involved in alcohol induced cell invasion, we determined the effects of Nm23 overexpression and 0.5% v/v ethanol treatment on 84 genes associated with extracellular matrix regulation and adhesion molecules in the following groups of breast cancer cells: 1) T47D controls cells (empty vector), 2) T47D cells treated with 0.