Utilizing camelid-derived single domain antibodies to enhance immunotherapy of ovarian cancer

Abstract

growth, and cancer cells are well known to be “glutamine-addicted.” Glutamine is converted by glutaminase to glutamate, and glutamate is converted by glutamate dehydrogenase (GDH) to α-ketoglutarate, which enters the tricarboxylic acid cycle for the production of adenosine triphosphate (ATP). In addition, glutamine generates the antioxidant glutathione, which removes reactive oxygen species (ROS) and regulates critical cell signaling pathways. Our objective was to explore the effects of glutamine on ovarian cancer (OC) cell growth and metabolism. Methods: We used the OC cell lines HEY, SKOV3, and IGROV1 and assessed dell proliferation by MTT assay. Cell cycle progression and apoptosis were evaluated by Cellometer. Western immunoblotting was performed to evaluate the effects of glutamine on downstream targets of the mTOR and MAPK pathways and proteins related to cellular stress. Glucose uptake, GDH activity, lactate, ATP, and ROS production were all assessed by enzyme-linked immunosorbent assay. The effect of mTOR pathway inhibition on glutaminestimulated cell proliferation was assessed by 1) treatment with rapamycin, an mTOR inhibitor, and 2) knockdown of S6 by siRNA transfection. Results: Glutamine stimulated cell proliferation in a dose-dependent manner in the three OC cell lines. In addition, glutamine increased GDH activity, glucose uptake, and lactate and ATP production and resulted in increased phosphorylation of S6 and p42/44. As expected, glutamine starvation had the opposite effect on the OC cell lines, leading to inhibition of cell proliferation, induction of apoptosis and G1 cell cycle arrest, reduced glucose uptake, and decreased lactate and ATP production. Glutamine depletion also induced ROS production and increased expression of the cellular stress proteins PERK, PARP, Bip, and calnexin. Inhibition of S6 by siRNA or rapamycin resulted in inhibition of glutamine-induced cell proliferation as well as a decrease in GDH activity. Conclusions: Glutamine potently increased cell proliferation, glucose uptake, and ATP production whereas glutamine depletion had the opposite effects on OC cells. Glutamine-induced cell proliferation appeared dependent on the mTOR pathway. These studies suggest that targeting glutamine may be a promising therapeutic strategy for ovarian cancer.