ZIA BC 011701 (ZIA) | |||
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Title | Metabolic characterization of FH- and SDH-deficient RCC cell lines | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Moscow, Jeffrey | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $103,268 | Project Dates | 00/00/0000 - 00/00/0000 |
Fiscal Year | 2016 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) |
Kidney Disease (100.0%) Urinary System (100.0%) |
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Research Type | |||
Systemic Therapies - Discovery and Development | |||
Abstract | |||
Since there was a difference in the OCR and glycolytic reserve between the deficient and restored cell lines, and evidence that restoration of enzyme activity altered metabolic signaling in both pairs of cells, we believed that these pairs of cell lines offered models to examine strategies that would exploit the Krebs cycle enzyme deficiencies of the SDH-deplete and FH-deplete cells. We first examined the fate of pyruvate in these cells, looking for evidence that disrupting the metabolism of pyruvate could provide a therapeutic window between the deplete and the replete cells. Pyruvate can take one of four metabolic routes: pyruvate can enter the Krebs cycle either through thiamine-dependent PDH, or through the anapleurotic pathway via pyruvate carboxylase, which is a biotin-dependent enzyme; pyruvate can take the last step in glycolysis through conversion to lactate by LDH; and it can be metabolized to alanine by ALT. We found: - No difference in UOK262 or UOK269 deficient or restored in sensitivity to thiaminase, suggesting that PDH did not play an important role distinguishing deplete from replete cells. - No effect of thiaminase on UOK262 and UOK269 restored cells on OCR, also indicating that transit into and through the Krebs cycle is independent of PDH. - No difference in UOK262 or UOK269 deplete or restored in sensitivity to dichloroacetate, an inhibitor of PDH kinase, which negatively regulates PDH, also indicating that PDH may not be a critical node in the metabolism of these cells. - We devised a procedure to deplete serum of biotin, and then compared the UOK 262 and UOK269 cells grown in biotin depleted serum to biotin repleted serum. The cells can grow up to 5 passages in depleted serum before they stop growing and appear to undergo apoptosis. Initial results indicate that biotin depletion did not sensitize UOK262 cell lines to thiaminase, but did sensitize UOK 269 SDH deficient cells but not SDH repleted cells to thiaminase, indicating that pyruvate carboxylase may play an important role in UOK269 SDH deficient cells than in the UOK262 FH-deficient cells. - There was no difference in the sensitivity of the pairs of cell lines to NHI-2, an inhibitor of LDH-A, which regulates one of the other fates of pyruvate, conversion into lactate. However, NHI-2 completely protected against the cytotoxicity of thiaminase in UOK262 FH repleted cells, but had not effect on the FH deficient cells. This suggests that cellular pyruvate is important in the repleted cells but not important in the FH-deficient cells when PDH is inhibited by thiaminase. - No difference in UOK262 or UOK266 deficient or restored cells in sensitivity to CB339, the glutaminase inhibitor; no difference in the dose response to CB839 whether glutamine is present in the medium or not; no difference in thiaminase growth inhibition in the presence of CB839; all suggesting that glutamine is not a critical nutrient. - No difference in UOK262 or UOK266 deficient or restored cells in sensitivity to lonidamine, a putative inhibitor of both hexokinase and the mitochondrial pyruvate carrier. Hexokinase catalyzes the first step in glycolysis. We also found no difference in thiaminase growth inhibition in the presence of lonidamine. - However, we found that restoration of FH activity of UOK262 cells, but not SDH activity of UOK269 cells, sensitizes the cells to 3-bromopyruvate, another inhibitor of hexokinase II. This suggests that restoration of FH activity actually increases flux through the glycolytic pathway. - No difference in UOK262 or UOK269 deficient or restored cells in sensitivity to AZD3965, an inhibitor of the monocarboxylate transporters that mediate transport of lactate and pyruvate - in fact AZD3965 was essentially inert in these cell lines, with no growth inhibition in concentrations up to 100 uM, suggesting that the fate of lactate and possibly pyruvate may not be relevant in these cells. - No difference in UOK262 or UOK266 deficient or restored cells in |