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Glutamine depletion alters choline metabolism and reduces survival of pancreatic cancer cells
Noriko Mori1, Balaji Krishnamachary1, Yelena Mironchik1, and Zaver M. Bhujwalla1,2,3
1The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University, Baltimore, MD, United States, 2The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, Baltimore, MD, United States, 3Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
1The Russell H. Morgan Department of Radiology and Radiological Science, The Johns Hopkins University, Baltimore, MD, United States, 2The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, Baltimore, MD, United States, 3Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, United States
Synopsis
Pancreatic ductal adenocarcinoma (PDAC) has poor prognosis due to a combination of late-stage diagnosis and limited response to radiation and chemotherapy. Effective treatments for PDAC are urgently needed. Mutant Kras has been reported as a major cause of glutamine (Gln) addiction in PDAC. Here we investigated the effects of Gln depletion on PDAC cell survival and metabolism. Our data identified Gln as critical for PDAC cell viability. Gln depletion significantly altered choline metabolism in the cell lines investigated.
Introduction
The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) arises from a combination of late-stage diagnosis and limited response to chemotherapy and radiotherapy. Effective treatments for PDAC are urgently needed. Mutant Kras that occurs in almost 90% of PDAC is a major cause of glutamine (Gln) addiction in these cells1. Elevated levels of phosphocholine and total choline have been previously reported in human pancreatic cancer xenografts2. Here we investigated the effects of glutamine depletion on the survival and metabolism of human PDAC cells.Methods
Human PDAC Panc 1, Pa04C, Pa09C, and Pa20C cells were cultured in DMEM high glucose (4.5 g/L) with 10% FBS. To evaluate the effect of Gln depletion on cell growth, cells were incubated in DMEM with or without Gln for 72h and the cell viability was determined using an automated cell counter or a cell counting kit (CCK-8, Dojindo Molecular Technologies). To evaluate the effects of Gln depletion on metabolism, dual-phase cell extraction was performed and water-soluble as well as lipid-soluble extracts were obtained from approximately 20 million cells. Fully relaxed proton spectra from the aqueous phase were obtained on a Bruker Avance 500 spectrometer. Signal integrals of the aqueous phase were determined and normalized to cell number, and compared to the standard (TSP). Three independent experiments with or without Gln depletion were performed for the cell viability and metabolism studies.Results and Discussion:
Cell viability, assayed with cell counting (Figure 1), was significantly reduced with Gln depletion in Panc 1 (~23%), Pa04C (~35%) and Pa09C (~51%) cells. The viability of Pa20C cells also decreased to 54% although this was not statistically significant. CCK-8 assay results showed similar changes in cell viabilities. We investigated changes in metabolism induced by Gln depletion in Panc 1 and Pa04C cells. Representative spectra are presented in Figure 2a and summarized in Figure 2b. Gln depletion resulted in significantly higher free choline (~ 5 fold), glycerophosphocholine (GPC) (~10-20 fold), and total choline (choline + phosphocholine (PC) + GPC) levels (~2-4 fold) after Gln depletion (Figure 2b). PC levels did not change consistently with Gln depletion. PC increased in Panc 1 cells with Gln depletion, but not in Pa04C cells. Our data confirmed the critical importance of Gln for maintaining PDAC cell viability. The changes in choline metabolism may reflect the effects on cell viability, or the adaptive response of cells to Gln depletion. We are currently investigating molecular changes in choline and glutamine pathways induced by glutamine depletion. Our data highlight the importance of targeting glutamine metabolism in the treatment of PDAC.Acknowledgements
Supported by NIH R35 CA209960 and R01 CA193365.References
1. Son J, Lyssiotis CA, Ying H, et al. Glutamine supports pancreatic cancer growth through a Kras-regulated metabolic pathway. Nature. 2013;496 (7443):101–105.
2. Penet MF, Shah T, Bharti S, et al. Metabolic imaging of pancreatic ductal adenocarcinoma detects altered choline metabolism. Clin Cancer Res. 2015 21(2):386-95.
Figures
Figure 1: Panc 1, Pa04C, Pa09C and Pa20C cell numbers with or without Gln depletion. ~105 cells were seeded in 6 well plates in triplicates. Cells were cultured in Gln +/- medium for 72h. Cell numbers were quantified with an automated cell counter at the end of the time point. Values represent mean ± SEM, ** P ≤ 0.01, * P ≤ 0.05 (n=3).
Figure 2: a. Choline metabolite region of representative 1H MR spectra obtained from the aqueous phase of Panc 1 and Pa04C cells. GPC; glycerophosphocholine, PC; phosphocholine, Cho: choline Cells were collected after culturing in Gln +/- medium for 72h. b. Choline metabolite levels in arbitrary unit (A.U.) obtained from 1H MR spectra of the aqueous phase of Panc 1 and Pa04C cells. Values represent mean ± SEM, ** P ≤ 0.01, * P ≤ 0.05 (n=3).