Shindo Masahiro, Maeda Masatomo, Myat Ko, Mane Mayuresh M, Cohen Ivan J, Vemuri Kiranmayi, Albeg Avi S, Serganova Inna, Blasberg Ronald
Department of Neurology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box 52, New York, NY 10065, USA.
Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
Cancers (Basel). 2022 May 6;14(9):2303. doi: 10.3390/cancers14092303.
Three murine glioma cell lines (GL261, CT2A, and ALTS1C1) were modified to downregulate the expression of the murine 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 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.
使用短发夹RNA（shRNA）对三种小鼠胶质瘤细胞系（GL261、CT2A和ALTS1C1）进行改造，以下调小鼠基因的表达，并与shRNA乱序对照（NC）细胞系进行比较。在这六种细胞系中观察到乳酸脱氢酶A（LDH-A）和乳酸脱氢酶B（LDH-B）的mRNA、蛋白质和酶活性表达差异，以及它们的乳酸脱氢酶同工酶谱，证实了基因敲低（KD）成功。LDH-A基因敲低导致细胞代谢变化，糖酵解减少（GlycoPER），基础呼吸速率增加（线粒体氧消耗率，mitoOCR）。与CT2A和ALTS1C1细胞相比，GL261细胞的ATP产生能力更有限。对mRNA表达数据的分析表明：（i）GL261 LDH-A基因敲低细胞可能具有将乳酸代谢进入三羧酸循环的能力提高；（ii）GL261 LDH-A基因敲低细胞可以上调脂质代谢/脂肪酸氧化途径，而其他胶质瘤细胞系没有这种能力。这两个观察结果表明，GL261 LDH-A基因敲低细胞可以开发/激活替代代谢途径，以在营养受限的环境中提高存活率，并且特定的营养限制对肿瘤细胞代谢和增殖有不同的影响。将LDH-A基因敲低的表型效应与对照（NC）细胞和肿瘤中的效应进行比较。LDH-A基因敲低延长了GL261细胞在培养中的倍增时间，并阻止了免疫健全的C57BL/6小鼠皮下侧腹肿瘤的形成，而GL261 NC肿瘤在C57BL/6小鼠中的生长延迟延长。在裸鼠中，LDH-A基因敲低和NC GL261肿瘤均快速生长（比C57BL/6小鼠中的GL261 NC肿瘤生长更快），表明完整免疫系统对GL261肿瘤生长的影响。尽管CT2A和ALTS1C1细胞及肿瘤的LDH同工酶谱有微小变化，但在体外未观察到NC和KD细胞增殖或C57BL/6小鼠肿瘤生长（倍增时间）之间的差异。这些结果表明，GL261胶质瘤细胞（但不是CT2A和ALTS1C1细胞）预先编程具有激活具有更高三羧酸循环活性的不同代谢途径的能力，并且这种能力通过LDH-A的消耗而增强。我们观察到LDH-A消耗和免疫系统的联合影响对皮下定位的GL261肿瘤的生长有显著影响。
