NIO inhibited the activation of Akt and FAK as shown by the decline in the phosphorylation of FAK and Akt. Cytotoxicity Checkpoint inhibitor effect of 50 NIO on cell growth and cell colony formation of head and neck cancer cells To look at the effect of 50 NIO on the growth of head and neck cancer cells, we performed MTT assay and colony formation assay. Head and neck cancer cells, FaDu, KB, and SGT, were exposed to increasing concentrations of 50 NIO for 24 h and cell viability was checked. Most of the cell lines showed a significant dose dependent decline in cell viability after more than 2. 5 lM 50 NIO treatment. The best strength of 50 NIO on cell viability was SGT cells. But, 1 lM 50 NIO was small cytotoxic to head and neck cancer cell lines. These demonstrated that healing with 50 NIO with doses higher than 2. 5 lM for 24 h led to concentration dependent loss in cell viability in three head and neck cancer cell lines, but doses below 1 lM did not cause cytotoxicity. Next, we used low concentration of fifty NIO to accomplish for subsequent studies. To try the lower dose efficiency of 50 NIO, cells were treated with 0. 1 or 1 lM 50 NIO for 10 days and Extispicy assayed by clonogenic formation. Treatment with 0. 1 lM 50 NIO had no significant influence on cell colony formation. However, colony formation was decreased approximately 50% in 1. 0 lM 50 NIO treated cells. 3. 2. 50 NIO inhibits migration and invasion of KB and FaDu cells in vitro To look at whether 50 NIO inhibits the cell invasion and migration, we performed in vitro Matrigel trans well chamber assays applying FaDu and KB cells. Once the FaDu and KB cells were cultured with 50 NIO, occupied cells were significantly inhibited in a concentration dependent manner. 50 NIO, at concentrations of 2. 5 lM, inhibited the cell invasion of KB and FaDu cells to 5000-rpm and 450-pound of get a handle on after 22 h treatment. Treatment with 1 lM of fifty NIO just inhibited 25% of cell invasion in KB cells.. But, migration assays showed that 1 lM 50 NIO somewhat inhibited migration activities by over 257 and 500-pages compared to the get a grip on in both cells, respectively. These indicated that 50 NIO substantially inhibited the migration and invasion of FaDu and KB cells. 3. 3. 50 NIO inhibits Integrin b1/FAK/Akt and ERK1/2/MMPs signaling Several studies have indicated that Integrin b1/FAK/Akt and ERK1/2/ MMPs signaling pathway play an important part on tumor invasion and migration. To elucidate the mechanism by which 50 NIO causes the inhibition of migration and invasion in head and neck cancer cells, we monitored the phosphorylation and/or expression of Integrin b1, FAK, Akt, ERK1/2, and MMPs. The level of Integrin b1 was reduced by 50 NIO treatment in a concentration dependent manner, having a 50% lowering of 1 lM 50 NIO handled FaDu and KB cells. Inhibition of the Integrin b1 reaction by 50 NIO was also observed in SGT cells.