Suppressing GFAT activity resulted in downregulation of BiP that activated inositol-requiring enzyme 1 (IRE1), a sensor protein of unfolded protein response, and exacerbated cisplatin-induced cell apoptosis. means in cell-based assays, and paired t test was used for mRNA expression results of lung cancer/normal tissue samples. All tests were two-tailed. P 0.05 was considered statistically significant. Results Overexpression of Zofenopril calcium GFAT in lung cancer cell lines and tissues GFAT has two isozymes, GFAT1[11] and GFAT2[12], encoded by different genes (GFPT1 and GFPT2, respectively; for simplicity, in this work both genes and proteins were referred to as GFAT1 and GFAT2 and as GFAT collectively). Human GFAT1 and GFAT2 have 75.6% homology in their protein sequences, presumably catalyze identical reactions without reported difference in catalytic activity, but have distinct distribution in normal tissues[12] and likely differential responses to stimuli[13C15]. We first examined the expression Zofenopril calcium of GFAT in various lung cancer cell lines. Compared with that of HBECs, all cancer cells lines had higher expression of GFAT mRNA, and correspondingly, GFAT protein levels and protein O-GlcNAcylation (Figure 1A and 1B), indicative of increased GFAT activity. To validate the findings in cell lines, we interrogated GFAT mRNA expression in lung cancer tissues, and found that average GFAT mRNA level was increased compared with that Zofenopril calcium of the corresponding normal tissues (Figure 1C). When examined individually, the majority of lung cancers (9/12 in adenocarcinomas and 11/12 in squamous cell carcinomas) had over two-fold increase of at least one isozyme (not shown). Open in a separate window Figure 1. Increased expression of GFAT in lung cancer cell lines and tissues. (A) Expression of GFAT mRNA in HBECs and lung cancer cell lines. Total RNA was extracted from cell lines; cDNA was synthesized by reverse transcription and used for PCR with specific primers for GFAT1, GFAT2, and -actin as loading control. Products were run in agarose gel with EB. (B) GFAT protein and O-GlcNAcylation levels in HBECs and lung cancer cell lines as examined with Western blot in total cell lysates. -Actin was probed as a loading control. (C) GFAT mRNA expression in human lung cancer tissues examined with TaqMan assay. GFAT expression in 12 adenocarcinomas, 12 squamous cell carcinomas, and their corresponding distant normal tissues was normalized to respective -actin, and MEKK13 then cancer over normal expression was calculated. * P 0.01; # P 0.05, in paired comparison with normal tissues as 1. Inhibition of GFAT is synergistic or additive to cisplatin cytotoxicity in lung cancer cells Having confirmed that GFAT was overexpressed in lung cancer cells, we used DON, a glutamine analog and an irreversible GFAT inhibitor[13,16C18], to investigate the potential of targeting the HBP pathway. DON displayed its effect on GFAT by decreasing protein O-GlcNAcylation in a dose-dependent Zofenopril calcium manner in A549 cells (Figure 2A). DON also inhibited lung cancer cell proliferation in a dose-dependent manner. Notably, cancer cells were more sensitive to DON treatment than HBECs, indicating that cancer cells are more dependent on HBP activity for proliferation (Figure 2B). We then tested DON in combination with cisplatin in three NSCLC cell lines with various concentrations. DON demonstrated mostly an additive effect (CI=1) in inhibiting cancer cell growth in A549 cells (Table 1), but synergistic effects (CI 1) in Calu-3 and H2009 cells (Table 2). Therefore, DON was able to enhance the efficacy of cisplatin in all the.