Conclusions We present data that shows hypoxia mediated increase in MMP1 expression and chondrosarcoma invasion is partially mediated by CXCR4 signaling. CXCR4 block ade can inhibit the effects of hypoxia on MMP1 expression and chondrosarcoma invasion in vitro, sug gesting that CXCR4 blockade could be a therapeutic tar inhibitor Vismodegib get to inhibit chondrosarcoma invasion and metastasis. The effectiveness of this strategy requires in vivo confirmation. Methods Tissue Articular cartilage, chondrosarcoma tissue, and cancel lous bone were obtained Inhibitors,Modulators,Libraries from surgical specimens, and either preserved in RNAlater Inhibitors,Modulators,Libraries Solution or snap frozen in liquid nitrogen for later use. There were 8 articular cartilage specimens and 16 chondrosarcoma. IRB approval was obtained.

Cell lines and cell culture Human chondrocytes isolated from normal adult articu lar cartilage and chondrosarcoma cell line JJ were cultured in complete medium with 10% FBS. All cells were cultured in a humidified incubator under Inhibitors,Modulators,Libraries 5% CO2 and either normoxia or hypoxia. JJ was derived from a human grade II chondrosarcoma. The drugs and inhibitors used were AMD3100, human recombinant SDF 1, MMP inhibitor O phenanthroline, MAP kinase inhibitors MEK1/2 inhibitor U0126, JNK inhibitor SP600125, p38 inhibitor SB203580 or DMSO, solvent for the inhibitors. Transfections Cells were transiently transfected with an expression construct for human Hif 1a in pcDNA3. 1 vector, or empty vector using Fugene HD in 6 or 12 well plates 24 h after seeding. Cells were then incubated for 48 h and harvested for the following experiments.

RNA interference Cells were transfected with Hif 1a siRNA, CXCR4 siRNA, ERK1/2 siRNA or control siRNA by HiPerFect transfec tion Inhibitors,Modulators,Libraries reagent. RNA and protein were obtained 72 h after transfection for qRT PCR and Western Blot analysis. Real time RT PCR RNA was isolated Inhibitors,Modulators,Libraries from cells with RNAqueous Kit or tissues with Trizol Reagent. After treatment with TURBO DNase, one microgram of RNA was reverse tran scribed with random hexamers to obtain first strand cDNA using iScript cDNA kit. The quantification of mRNA for Hif 1a, CXCR4, SDF 1, and MMP1 was performed by two step real time quantitative RT PCR. 18S was used as an internal control since it has been shown to be the optimal reference gene. Amplifica tion conditions were as follows 2 min preincubation at 50 C, 10 minutes at 95 C for enzyme activation, and 40 cycles at 95 C denaturation for 10 s, 55 C annealing for 30 s and 72 C extension for 30 s. The comparative threshold cycle method, i. e, 2 Ct method was used for the calculation of fold amplifica tion. Each experiment was evaluated with three PCR reactions and each experiment was repeated Nilotinib structure three times. Data are presented as mean value SD.