We not long ago showed that AZD1480 is often a potent, aggressive compact molecule inhibitor of JAK1/2 kinase, and that it is actually capable of inhibiting STAT3 phosphorylation and tumor growth within a STAT3 dependent method. Whilst tumor growth was inhibited right in vivo in each and every tumor model examined, in some tumor cell lines AZD1480 did not block tumor cell development in vitro at amounts that generated maximal inhibition of STAT3 phosphorylation. This suggests the likely essential effects of AZD1480 within the tumor microenvironment by inhibiting JAK/STAT signaling. A ZD1480 is at present in early clinical trials for sound and hematologic malignancies. Our present study shows that AZD1480 inhibits tumor angiogenesis and metastasis in aspect by affecting the tumor microenvironment. Materials and Methods Reagents AZD1480 was supplied by AstraZeneca and dissolved in DMSO for in vitro research. For in vivo experiments, AZD1480 was suspended in water supplemented with 0.
5% Hypromellose and 0. 1% Tween 80. All solvents are from Sigma. Mouse IL 6 was purchased from R&D Systems. Antibodies Sunitinib solubility against p STAT3, p JAK2, JAK2, cleaved caspase 3 and matrix metalloproteinase 9 were obtained from Cell Signaling Technology. Antibodies against STAT3 and VEGF were obtained from Santa Cruz Biotechnology. Cell lines Renca murine cell line was a gift from Dr. Alfred Chang. Human renal cell carcinoma cell line, 786 O, was generously provided by Dr. William G. Kaelin. The 4T1 mouse mammary tumor cell line and the Calu six lung carcinoma cell line were from ATCC. Mouse EC line derived from prostate and colon was kindly offered by S. Huang and J. Fidler. All the cell lines above were grown in DMEM or RPMI 1640 with 10% fetal bovine serum.
Human umbilical vein endothelial cells were obtained from Clonetics and cultured on collagen I coated plates in their complete medium. 786 O STAT3C and vector expressing control cell lines were generated and maintained as described previously. Animal models and drug Motesanib administration Female BALB/c and athymic nude mice were obtained from National Cancer Institute and Taconic Laboratories. Animal use procedures were approved by the institutional animal care and use committees of Beckman Research Institute at City of Hope and AstraZeneca. For subcutaneous tumor model, 2. 5106 Renca or 786 O cells suspended in 100 ul PBS were injected into the flank of BALB/c or nude mice, respectively. When average tumor volume reached approximately 100 150 mm3, AZD1480 or vehicle was administered by oral gavage either once a day at the dose of 50 mg/kg, or twice daily at 30 mg/kg, as indicated.
Tumor size was measured by caliper every other day. For experimental lung metastasis model, 0. 1106 Renca or 1106 786 O cells suspended in 500 ul PBS were injected via tail vein to BALB/c or nude mice, respectively.