Hypoglycemia Enhances Epithelial‐Mesenchymal Transition and Invasiveness, and Restrains the Warburg Phenotype, in Hypoxic HeLa Cell Cultures and Microspheroids

Abstract

The accelerated growth of solid tumors leads to episodes of both hypoxia and hypoglycemia (HH) affecting their intermediary metabolism, signal transduction, and transcriptional activity. A previous study showed that normoxia (20% O ) plus 24 h hypoglycemia (2.5 mM glucose) increased glycolytic flux whereas oxidative phosphorylation (OxPhos) was unchanged versus normoglycemia in HeLa cells. However, the simultaneous effect of HH on energy metabolism has not been yet examined. Therefore, the effect of hypoxia (0.1-1% O ) plus hypoglycemia on the energy metabolism of HeLa cells was analyzed by evaluating protein content and activity, along with fluxes of both glycolysis and OxPhos. Under hypoxia, in which cell growth ceased and OxPhos enzyme activities, ΔΨm and flux were depressed, hypoglycemia did not stimulate glycolytic flux despite increasing H-RAS, p-AMPK, GLUT1, GLUT3, and HKI levels, and further decreasing mitochondrial enzyme content. The impaired mitochondrial function in HH cells correlated with mitophagy activation. The depressed OxPhos and unchanged glycolysis pattern was also observed in quiescent cells from mature multicellular tumor spheroids, suggesting that these inner cell layers are similarly subjected to HH. The principal ATP supplier was glycolysis for HH 2D monolayer and 3D quiescent spheroid cells. Accordingly, the glycolytic inhibitors iodoacetate and gossypol were more effective than mitochondrial inhibitors in decreasing HH-cancer cell viability. Under HH, stem cell-, angiogenic-, and EMT-biomarkers, as well as glycoprotein-P content and invasiveness, were also enhanced. These observations indicate that HH cancer cells develop an attenuated Warburg and pronounced EMT- and invasive-phenotype. J. Cell. Physiol. 232: 1346-1359, 2017. © 2016 Wiley Periodicals, Inc.

Modeling tumor cell adaptations to hypoxia in multicellular tumor spheroids

“…It was recently shown that detachment of tumor cells from the ECM disrupts glucose metabolism and induces increased ROS species [ 100 ]. In agreement with this data, the formation and growth of MCTS is dependent on sufficient glucose availability and activation of antioxidant pathways [ 49 , 100 , 101 ]. To facilitate growth in MCTS and within hypoxic microenvironments, tumor cells activate signaling through HIF proteins [ 57 ].…”

Celecoxib inhibits mitochondrial O2 consumption, promoting ROS dependent death of murine and human metastatic cancer cells via the apoptotic signalling pathway

“…Many cells die from these stressful conditions, but the survivors, undergoing dramatic shifts in redox status with mitochondrial and nuclear damage from ensuing oxidative stress, promote further malignancy and metastasis [6,9]. This selection pressure, promoted by the combined stressful conditions of hypoxia plus hypoglycaemia [10] involves dysfunctional mitochondrial ATP synthesis (i.e., oxidative phosphorylation; OxPhos), causing enhanced release of ROS as a by-product of an impaired respiratory chain, which then causes mutations, particularly in the adjacent susceptible mitochondrial DNA, and eventually enhances metastasis [11][12][13][14]. As a consequence, cytosolic Ca 2+ levels become elevated which helps trigger epithelialmesenchymal transition, accelerated growth and invasiveness together with the emergence of more highly metastatic cancer stem cells [15].…”

Repurposing drugs as pro‐oxidant redox modifiers to eliminate cancer stem cells and improve the treatment of advanced stage cancers

“…33,77,78 The tumor microenvironment during rapacious growth phases will concomitantly cause intermittent periods of hypoxia, [79][80][81] acidosis, 82,83 and nutrient deprivation resulting in hypoglycemia, all factors which consequently induce a major reprogramming of cancer cell metabolism. 46,47,[84][85][86][87][88][89] These microenvironmental stress related changes in cancer cell metabolism are frequently associated with greater ROS production. 90 1.4 | Switch in redox control from de novo GSH synthesis to NADPH from the PPP in advanced cancer cells One major mechanism for enhancing ROS levels in cancer cells occurs via a feedback amplification loop involving ROS-mediated increase in cytosolic NADPH production.…”

“…ROS production systems become elevated in more advanced stage cancer cells to promote their growth and survival . The tumor microenvironment during rapacious growth phases will concomitantly cause intermittent periods of hypoxia, acidosis, and nutrient deprivation resulting in hypoglycemia, all factors which consequently induce a major reprogramming of cancer cell metabolism . These microenvironmental stress related changes in cancer cell metabolism are frequently associated with greater ROS production …”