LDH-A—Modulation and the Variability of LDH Isoenzyme Profiles in Murine Gliomas: A Link with Metabolic and Growth Responses

SIMPLE SUMMARY: Three different murine glioma cell lines were modified to downregulate the expression of the murine LDH-A gene using shRNA knock-down (KD), and compared to shRNA scrambled control (NC) cell lines. Successful LDH-A KD was confirmed by differences in the expression of LDH-A and LDH-B mRNA, protein and enzymatic activity, as well as their LDH isoenzyme profiles. mRNA expression data indicated that: (i) GL261 LDH-A KD cells may have an improved ability to metabolize lactate into the TCA cycle; and (ii) that GL261 LDH-A KD cells can upregulate lipid metabolism/fatty acid oxidation pathways, whereas the other glioma cell lines (CT2A, and ALTS1C1) do not have this capacity. The data suggest that GL261 LDH-A KD cells can develop/activate alternative metabolic pathways for enhanced survival in a nutrient-limited environment. Furthermore, LDH-A KD prolonged the doubling time of GL261 cells in culture and prevented the formation of subcutaneous flank tumors in immune-competent C57BL/6 mice. No differences between NC and KD cell proliferation (in vitro) or tumor growth in C57BL/6 mice (doubling time) were observed for CT2A and ALTS1C1 cells and tumors. Our results suggest that GL261 glioma cells (but not CT2A and ALTS1C1 cells) are pre-programmed with a capacity for activating different metabolic pathways with higher TCA cycle activity, and that this capacity is enhanced by LDH-A depletion, leading to a difference in growth and survival in an immune-competent environment. ABSTRACT: Three murine glioma cell lines (GL261, CT2A, and ALTS1C1) were modified to downregulate the expression of the murine LDH-A gene using shRNA, and compared to shRNA scrambled control (NC) cell lines. Differences in the expression of LDH-A and LDH-B mRNA, protein and enzymatic activity, as well as their LDH isoenzyme profiles, were observed in the six cell lines, and confirmed successful LDH-A KD. LDH-A KD (knock-down) resulted in metabolic changes in cells with a reduction in glycolysis (GlycoPER) and an increase in basal respiratory rate (mitoOCR). GL261 cells had a more limited ATP production capacity compared to CT2A and ALTS1C1 cells. An analysis of mRNA expression data indicated that: (i) GL261 LDH-A KD cells may have an improved ability to metabolize lactate into the TCA cycle; and (ii) that GL261 LDH-A KD cells can upregulate lipid metabolism/fatty acid oxidation pathways, whereas the other glioma cell lines do not have this capacity. These two observations suggest that GL261 LDH-A KD cells can develop/activate alternative metabolic pathways for enhanced survival in a nutrient-limited environment, and that specific nutrient limitations have a variable impact on tumor cell metabolism and proliferation. The phenotypic effects of LDH-A KD were compared to those in control (NC) cells and tumors. LDH-A KD prolonged the doubling time of GL261 cells in culture and prevented the formation of subcutaneous flank tumors in immune-competent C57BL/6 mice, whereas GL261 NC tumors had a prolonged growth delay in C57BL/6 mice. In nude mice, both LDH-A KD and NC GL261 tumors grew rapidly (more rapidly than GL261 NC tumors in C57BL/6 mice), demonstrating the impact of an intact immune system on GL261 tumor growth. No differences between NC and KD cell proliferation (in vitro) or tumor growth in C57BL/6 mice (doubling time) were observed for CT2A and ALTS1C1 cells and tumors, despite the small changes to their LDH isoenzyme profiles. These results suggest that GL261 glioma cells (but not CT2A and ALTS1C1 cells) are pre-programmed to have the capacity for activating different metabolic pathways with higher TCA cycle activity, and that this capacity is enhanced by LDH-A depletion. We observed that the combined impact of LDH-A depletion and the immune system had a significant impact on the growth of subcutaneous-located GL261 tumors.