5-ALA Is a Potent Lactate Dehydrogenase Inhibitor but Not a Substrate: Implications for Cell Glycolysis and New Avenues in 5-ALA-Mediated Anticancer Action.

Abstract

Simple SummaryIn the present work, we found that 5-ALA, a natural precursor of heme, can hinder cell glycolysis, which is the main path of energy production for most cancer cells. More specifically, we found that 5-ALA can block an enzyme involved in glycolysis, called lactate dehydrogenase (LDH). We found that 5-ALA has a potency of LDH inhibition comparable to other established LDH inhibitors, such as oxamate or tartronic acid. Nevertheless, 5-ALA has a high accumulation rate in cancers and specifically in the incurable brain cancer glioblastoma multiforme (GBM), which is an important advantage. In fact, because of its high specificity to GBM, 5-ALA is used in the clinic to accurately guide the resection of the tumours, through the light emission of its photoactive product protoporphyrin IX (PpIX). PpIX is the penultimate step in the heme production. Importantly, we show here that continuous administration of 5-ALA killed GBM cells according to their dependence on glycolysis. We additionally found that 20% of externally administered 5-ALA is engaged in the inhibition of LDH, as when LDH was pre-loaded by another inhibitor, tartronic acid, then the cell production of PpIX from 5-ALA was increased by 20%. Since PpIX is an important drug for photodynamic therapy of cancer (excitation by light of PpIX produces oxygen by-products that can kill cancer cells), we additionally discovered that preloading LDH with its inhibitor tartronic acid before performing 5-ALA PDT increases the cancer cell death by 15%.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.