We not long ago showed that AZD1480 is really a potent, aggressive tiny molecule inhibitor of JAK1/2 kinase, and that it really is capable of inhibiting STAT3 phosphorylation and tumor development inside a STAT3 dependent method. Despite the fact that tumor development was inhibited right in vivo in every tumor model examined, in some tumor cell lines AZD1480 did not block tumor cell growth in vitro at amounts that produced maximal inhibition of STAT3 phosphorylation. This suggests the prospective significant effects of AZD1480 over the tumor microenvironment by inhibiting JAK/STAT signaling. A ZD1480 is currently in early clinical trials for sound and hematologic malignancies. Our existing study shows that AZD1480 inhibits tumor angiogenesis and metastasis in portion by affecting the tumor microenvironment. Materials and Techniques Reagents AZD1480 was supplied by AstraZeneca and dissolved in DMSO for in vitro studies. 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 six was obtained from R&D Systems. Antibodies Perifosine Akt inhibitor 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 presented by Dr. William G. Kaelin. The 4T1 mouse mammary tumor cell line and the Calu 6 lung carcinoma cell line were from ATCC. Mouse EC line derived from prostate and colon was kindly supplied 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 DCC-2036 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.