Moreover, CA IX possesses clinical po tential as a target for anticancer treatment, indeed, functional inhibition of CA IX has been proposed as an attractive option for therapeutic targeting of various kinase inhibitor Tipifarnib hypoxic tumors. Transcription of the gene encoding CA IX is primarily activated by the hypoxia inducible HIF 1 transcription factor that binds to the hypoxia re sponse element Inhibitors,Modulators,Libraries located next to the transcription initiation site. Phosphorylation of Thr443 of CA IX by protein kinase A in hypoxic cells is critical for its activation. Inhibitors,Modulators,Libraries Because kinetic and X ray crystallographic studies sug gest that carnosine is a potent activator of the carbonic anhydrase isoforms hCA I, II, and IV and the studies described above indicate that carnosine affects the HIF 1 signaling pathway, we initially examined whether CA IX is involved in the antitumor activity of carnosine.

We subse quently investigated whether carnosine exerts its effect on CA IX through modulation of transcription and transla tion Inhibitors,Modulators,Libraries levels of HIF 1 and CA IX and or through altering CA IX function. Methods Cell culture and spheroid preparation Madin Darby canine kidney, HeLa, HT 29, and SiHa cell lines were obtained from the American Type Culture Collection and cultured in Dulbeccos modified Eagles medium supplemented with 10% fetal calf serum and gentamicin at 37 C and 5% CO2 in humidified air. The cells were counted, seeded in 3 or 6 cm Petri dishes for 24 h, and treated with L carnosine under normoxic and hypoxic conditions. HeLa spheroids were generated by seeding cells in 96 well plates coated with Inhibitors,Modulators,Libraries 1% agarose.

After 4 days of incubation at 37 C and 5% CO2, the spheroids were photographed and treatment was initiated by addition of fresh medium with or without carnosine. In all experiments, at least 30 replicate wells were set up for the control and the carnosine treatment groups. Photographs were taken every 48 h. At the end of the experiment, extracellular pH was measured Inhibitors,Modulators,Libraries and the spheroids were subjected to flow cytometric analysis to determine cell viability. Measurement of extracellular pH using sensor dish reader The sensor dish reader monitors pH in real time in special plates using a non invasive technique that detects the luminescence lifetime of a sensor spot at the bottom of each well that is dependent on the pH of the surrounding sample. Cells were seeded into Imatinib Mesylate chemical structure wells and allowed to attach. Measure ment was started on the second day, when the cells reached 80% confluence. Cells were cultured in the pres ence or absence of carnosine under hypoxic or normoxic conditions as described above. The pH was measured by the SDR every 30 min.