Nside the cell, glutamine is committed to glutaminolysis by the enzyme glutaminase (GLS), which converts glutamine to glutamate. The only way this metabolism could be reversed is through the action of glutamine synthetase (GLUL), which converts glutamate back into glutamine (7). As a result, in the absence of appreciable glutamine synthetase activity, glutamate can then be converted to -ketoglutarate where it enters the TCA cycle. The oncogene MYC is actually a recognized regulator of your initial actions of glutaminolysis, throughout which MYC up-regulates mitochondrial glutaminase as well as glutamine transporters, promoting influx with the amino acid and its subsequent metabolism (8). In prostate cancer, MYC can function as a transformative aspect. In the mouse prostate, Myc overexpression promotes prostatic intraepithelial neoplasia (PIN) followed by invasive adenocarcinoma within a dosedependent manner (9). Interestingly, current work has demonstrated that AR signaling can enhance glutamine metabolism in prostate cancer cells (10). On top of that, AR has been demonstrated to modulate MYC expression inside a context-dependent manner (11-13). Given MYC’s previously described part in glutamine metabolism, we hypothesized that androgens promoted prostate cancer cell growth in portion via augmenting MYC-mediated glutamine metabolism.Author Manuscript Author Manuscript Author Manuscript Author ManuscriptMol Cancer Res. Author manuscript; available in PMC 2018 August 01.White et al.PageMaterials and MethodsCell culture, plasmids, and reagents LNCaP and VCaP human prostate cancer cell lines had been obtained from ATCC (Manassas, VA) and maintained and tested for androgen responsiveness just before experiments as previously described (14). PTEN-P8 and PTEN-CaP8 have been obtained from ATCC and maintained in Dulbecco’s Modified Eagle’s Medium supplemented with eight fetal bovine serum (FBS), 25 g/ml bovine pituitary extract, 5 g/ml human recombinant insulin and six ng/ml human recombinant epidermal growth factor (15). PrEC-LHS, PrEC-LHSR and PrEC-LHMK human prostate cancer cells had been kindly provided by Dr. William Hahn (Dana-Farber Cancer Institute, Boston, MA, USA) and previously described (16). Cell lines were validated biannually by genotyping and mycoplasma-free confirmation by way of the use of a PCR-based assay. For all experiments, cells were first plated in phenol red-free medium containing charcoal-stripped FBS (CS-FBS) for 72 hours to lessen endogenous hormone signaling. Cells have been then switched for the remainder on the assay to a customized experimental medium (Sigma, St. Louis, MO) that lacked serum, non-essential amino acids, sodium pyruvate, further glucose and HEPES buffer. This experimental medium was supplemented with 2 mM L-glutamine unless otherwise noted (ex.5-Bromo-3-chloro-1,2,4-thiadiazole Chemscene Fig.Formula of 4,4′-Dibromo-2,2′-bipyridine 1A).PMID:24282960 Steady cell lines were developed employing a pINDUCER10 construct that enabled the expression of a brief hairpin RNA targeting MYC inside the presence of doxycycline (Supplementary Fig. 4A). Further lentiviral vectors have previously been described (17). Stealth siRNAs had been from Life Technologies (Grand Island, NY). The sequences for all shRNAs and siRNAs used within this study are listed in Supplementary Table 1. Antibodies recognizing MYC (cat#: 5605), SLC1A4 (cat#: 8442), SLC1A5 (cat#: 8057), phospho-S6 (cat#: 4856), total S6 (cat#: 2317) and cleaved poly (ADP-ribose) polymerase (PARP) (cat#: 5625) have been obtained from Cell Signaling (Beverly, MA). The antibody recognizing glutaminase (cat#: ab156876) was from Abcam.
