chemotherapy to treatment did not produce a significant benefit in head and neck cancer. A new indirubin derivative, 50 nitro indirubinoxime, was designed and 2-ME2 clinical trial produced to boost its pharmacologic effectiveness. Previous studies have reported that 50 NIO exhibits higher anti-tumor action than indirubin or other derivatives in many different human cancer cells. 50 NIO inhibited the proliferation of cancer cell via G2/M cell cycle arrest and induced apoptosis through the activation of mitochondrial dependent caspase 3 and 7 in oral cancer cells. 50 NIO inhibited the proliferation of human salivary gland adenocarcinoma cells by arresting them at the G1 phase of the cell-cycle and by inhibiting Notch 1 and Notch 3 signaling. Additionally, 50 NIO inhibited a few kinases such as Plk1, Cdk1, and Cdk4/6, an essential regulator of cellular Gene expression functions that include cell cycle and cell proliferation. Recently, we’ve noted that 50 NIO inhibits the inflammatory reaction in TNF alpha stimulated human umbilical vein endothelial cells. In cDNA microarray, 50 NIO suppressed the expression of many proteins, which are related to migration, invasion and angiogenesis. The complete effect of 50 NIO remains uncertain on cancer invasion and migration, although it is very obvious that 50 NIO may prevent the development of numerous cancers by inducing apoptosis. We confirmed that 50 NIO suppressed the invasion and migrationability ofheadand neck cancer cells throughblocking Integrin b1/FAK/Akt signaling pathway in vitro for the very first time. We also discovered that 50 NIO considerably reduced angiogenesis in vivo chorioallantoic Cilengitide ic50 membrane assay model. Our results might support the future development of this compound as a potential therapy for metastatic ability and excessive angiogenesis in head and neck cancer. 2. Supplies and 2. 1. Cell culture Human head and neck cancer cell lines KB and FaDu were maintained in MEM media. SGT salivary gland adenocarcinoma cells were cultured in DMEMwith 10 % FBS, 100 units/ml penicillin, and 100 lg/ml streptomycin. 2. 2. Mobile proliferation assay Cells were cultured in 24 well plates at a density of 3 105 cells/well. One day later, the cells were treated with indirubin by-product for 24 h. Cell viability was based on performing the 3 2,5 diphenyl 2H tetrazolium bromide cell proliferation assay. The optical density value of the dissolved solute was then measured utilizing a Microplate Autoreader in a wavelength of 570 nm. The are reported as the mean SD of three split up studies. 2. 3. Cell colony formation assay The inhibition of the colony formation of head and neck cancer cells following therapy with 50 NIO was measured by soft agar assay as previously described. Briefly, 8 103 cells/ml were exposed or not exposed to different concentration of 50 NIO in 1 ml of 0. Three or four basal medium Eagle agar containing ten percent FBS, 2 mM L glutamine, and 25 lg/ml gentamicin.