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5A), suggesting that the decrease in 67LR expression occurs at the level of protein stability. To test this, we examined the involvement of the ubiquitin�Cproteasome proteolytic system in the decrease of 67LR under 5% O2. HeLa cells were transfected with His-tagged 67LR, immunoprecipitated with anti-His antibody and analyzed by Western blot using anti-ubiquitin antibody. AZ191 Ubiquitinated 67LR was observed in cells cultured in 5% O2 (Fig. 5B), and treatment with the proteasome inhibitor MG132, protected 67LR from degradation induced by exposure to 5% O2 (Fig. 5C). Moreover, in cells treated with MG132, EGCG induced MRLC dephosphorylation at Thr18/Ser19, even at 5% O2 (Fig. 5D). These observations indicate that exposure of cells to 5% O2 accelerates 67LR degradation, thus abrogating the anti-cancer activity of EGCG. Tumor hypoxia, caused by an imbalance in O2 supply and demand, occurs at a distance of 100�C200?��m from blood vessels [20]. Previous studies showed that at least 25% of cancer cells are exposed to hypoxic conditions (pO2?LGK-974 nmr EGCG resistance in solid tumor cells exposed to hypoxic conditions. Exposure of solid tumor cells to low O2 levels led to the degradation of the EGCG-sensing molecule, 67LR, resulting in the abolishment of EGCG anti-proliferative activity. EGCG is unstable, and generates oxidative products such as quinones, which form a covalent interaction with a nucleophilic cysteine residue in a peptide or protein [22]. It is known that SOD and catalase in the culture medium stabilized EGCG and increased its half-life to longer than 24?h [17]. Therefore, we examined the inhibitory effects Apoptosis Compound Library in vivo of EGCG on cancer cell proliferation with addition of SOD and catalase to the cell culture medium. To examine whether or not O2 condition is involved in the oxidation of EGCG, we evaluated the stability of EGCG under different O2 conditions (21% or 5% O2). LC/MS analysis revealed that O2 condition did not affect on the amount of intact form of EGCG (data not shown). Additionally, in the cells treated by proteasome inhibitor, EGCG induced anti-melanoma activity even at 5% O2 (Fig. 5D). These results suggested that the EGCG resistance under low O2 condition is not due to the generation of oxidative products. Our data indicate that 67LR expression is highly susceptible to O2 partial pressure. 67LR plays a critical role in tumor cell attachment, metastasis and invasion [23].