5% alcohol. Results are presented in Table 2, with only the most significantly affected genes shown. Interestingly, one gene observed to be affected by alcohol and Nm23 in the opposite Doramapimod cell line manner was fibronectin receptor subunit integrin alpha 5 (ITGA5). In cells overexpressing Nm23,
alcohol treatment was no longer able to increase ITGA5 expression (Table 2). Additionally, alcohol exposure increased the expression of ITGA5 nine-fold; however, this effect was eliminated by the overexpression of Nm23 (Figure 4A and Table 2), suggesting that Nm23 blocked the effects of alcohol. Thus, our data suggests that the effects of alcohol on ITGA5 are Nm23-dependent. Table 2 Effects of alcohol and Nm23 overexpression on extracellular matrix and adhesion proteins expression Gene Name 0.5% EtOH Nm23-H1 0.5% EtOH TH-302 nmr + Nm23-H1 VCAN 4.1125 3.1514 4.359 COL8A1 -18.2522 -18.6875 -8.9755 CTGF -4.3772 -5.712 -4.1296 CTNNA1 -15.455 Selleck Ilomastat -20.1681 -14.5808 CTNNB1 5.6569 5.5251 5.9134 CTNND1 -69.551 -18.9483 -26.4647 CTNND2 16.9123 12.9601 17.9262 ITGA1 -1.7777 -2.3168 -1.6771 ITGA2 -6.4531 -8.421 -6.0881 ITGA4 -5.3889 -7.0323 -5.0841 ITGA5 9.3827 -12.0754 -9.038 ITGA6 -1.1408 -1.4886 -1.0762 ITGA7 -8.1681 -7.5371 -5.4869 ITGAL -6.3643 -8.3051 -6.0043 ITGAV -2.042 -2.6647 -1.9265 ITGB1 -3.0314 -3.2355 -1.554
ITGB2 -2.3295 -3.0398 -2.1977 ITGB3 -5.2416 -4.8032 -3.8798 ITGB4 -1.021 1.8226 1.6066 ITGB5 -19.4271 -15.3908 -3.62 KAL1 1.454 1.1142 1.5411 LAMA1 1.1096 -1.1761 1.1761 MMP1 4.1487 -1.136 1.2176 MMP10 -12.5533 -11.3451 -5.191 MMP13 24.761 18.9746 26.2455 MMP16 4.1989 4.1583 5.6334 MMP2 3.249 1.7363 2.3685 NCAM1 -3.8106 -4.9726 -3.595 PECAM1 -13.4543 -17.5573 -12.6933 SELE 1.2483 -1.0454 1.3232 SELL 7.0128 5.374 7.4333 SELP -7.1107 -9.2792 -6.7085 SGCE 1.021 -1.2781 1.0822 SPG7 10.4107 6.0043 8.2477 CLEC3B -1.4641 -1.9106 -1.3813 TNC -3.9177 -5.1124 -3.6961 VCAM1 1.0281 1.325 1.0898 Figure
4 Nm23 down-regulates ITGA5 expression. Nm23 regulates cell invasion through ITGA5 expression. (A) ITGA5 mRNA levels were determined by qRT-PCR in T47D cells treated with 0.5% v/v ethanol and overexpressing Nm23, independently and in combination. Alcohol promotes ITGA5 mRNA expression approximately 17-DMAG (Alvespimycin) HCl nine-fold. This effect was blocked by the overexpression of Nm23. (B) Western blot shows Nm23 and ITGA5 protein level in T47D cells with ethanol treatment, Nm23 overexpression, and in combination. (C) Western blots show Nm23 and ITGA5 protein level in MCF-7 (left) and MDA-MB-231 (right) cells following various doses of ethanol treatment. (*p < 0.05, as compared to the control cells transfected with empty vector). To determine the relationship between Nm23 and ITGA5 in alcohol-treated T47D breast cancer cells, we knocked down each gene separately and in combination, using small interfering RNA (siRNA), and subsequently measured cell invasion.