Grigalavicius Mantas, Ezzatpanah Somayeh, Papakyriakou Athanasios, Raabe Tine Therese Henriksen, Yannakopoulou Konstantina, Theodossiou Theodossis A
Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, 0379 Oslo, Norway.
Institute of Biosciences and Applications, National Center for Scientific Research "Demokritos", 15341 Aghia Paraskevi, Greece.
Cancers (Basel). 2022 Aug 18;14(16):4003. doi: 10.3390/cancers14164003.
In a course of metabolic experiments, we determined that the addition of δ-aminolevulinic acid (5-ALA) to a panel of glioblastoma multiforme (GBM) cells caused a steep reduction in their glycolytic activity. This reduction was accompanied by a decrease in adenosine triphosphate (ATP) production from glycolysis. These results suggested that 5-ALA is an inhibitor of glycolysis; due to the structural similarity of 5-ALA to the established lactate dehydrogenase (LDH) inhibitors oxamate (OXM) and tartronate (TART), we initially investigated LDH inhibition by 5-ALA in silico. The modelling revealed that 5-ALA could indeed be a competitive inhibitor of LDH but not a substrate. These theoretical findings were corroborated by enzymatic and cell lysate assays in which 5-ALA was found to confer a potent LDH inhibition comparable to that of OXM and TART. We subsequently evaluated the effect of 5-ALA-induced glycolysis inhibition on the viability of GBM cells with diverse metabolic phenotypes. In the Warburg-type cell lines Ln18 and U87, incubation with 5-ALA elicited profound and irreversible cell death (90-98%) at 10 mM after merely 24 h. In T98G, however, which exhibited both high respiratory and glycolytic rates, LD95 was achieved after 72 h of incubation with 20 mM 5-ALA. We additionally examined the production of the 5-ALA photosensitive metadrug protoporphyrin IX (PpIX), with and without prior LDH inhibition by TART. These studies revealed that ~20% of the 5-ALA taken up by the cells was engaged in LDH inhibition. We subsequently performed 5-ALA photodynamic therapy (PDT) on Ln18 GBM cells, again with and without prior LDH inhibition with TART, and found a PDT outcome enhancement of ~15% upon LDH pre-inhibition. We expect our findings to have a profound impact on contemporary oncology, particularly for the treatment of otherwise incurable brain cancers such as GBM, where the specific accumulation of 5-ALA is very high compared to the surrounding normal tissue.
在一系列代谢实验中，我们确定向一组多形性胶质母细胞瘤（GBM）细胞中添加δ-氨基乙酰丙酸（5-ALA）会导致其糖酵解活性急剧降低。这种降低伴随着糖酵解产生的三磷酸腺苷（ATP）减少。这些结果表明5-ALA是糖酵解的抑制剂；由于5-ALA与已确定的乳酸脱氢酶（LDH）抑制剂草氨酸盐（OXM）和丙醇酸盐（TART）结构相似，我们最初在计算机上研究了5-ALA对LDH的抑制作用。建模显示5-ALA确实可能是LDH的竞争性抑制剂，但不是底物。这些理论发现得到了酶促和细胞裂解物测定结果的证实，在这些测定中发现5-ALA具有与OXM和TART相当的有效LDH抑制作用。我们随后评估了5-ALA诱导的糖酵解抑制对具有不同代谢表型的GBM细胞活力的影响。在沃伯格型细胞系Ln18和U87中，仅在24小时后，用10 mM的5-ALA孵育就引发了严重且不可逆的细胞死亡（90 - 98%）。然而，在T98G细胞中，其呼吸率和糖酵解率都很高，用20 mM的5-ALA孵育72小时后达到了半数致死剂量95%（LD95）。我们还研究了在有和没有TART预先抑制LDH的情况下5-ALA光敏代谢前体原卟啉IX（PpIX）的产生。这些研究表明，细胞摄取的5-ALA中约20%参与了对LDH的抑制。我们随后对Ln18 GBM细胞进行了5-ALA光动力疗法（PDT），同样是在有和没有TART预先抑制LDH的情况下，发现预先抑制LDH后PDT效果提高了约15%。我们期望我们的发现对当代肿瘤学产生深远影响，特别是对于治疗如GBM这种难以治愈的脑癌，与周围正常组织相比，5-ALA在其中的特异性积累非常高。
