Light-induced photoactivation of hypericin affects the energy metabolism of human glioma cells by inhibiting hexokinase bound to mitochondria

Glucose-dependent energy required for glioma metabolism depends on hexokina se, which is mainly bound to mitochondria, A decrease in intracellular pH l eads to a release of hexokinase-binding, which in turn decreases glucose ph osphorylation, ATP content, and cell proliferation. Thus, intracellular pH might be a target for therapy of gliomas, and a search for agents able to m odulate intracellular pH was initiated. Hypericin, a natural photosensitize r, displays numerous biological activities when exposed to light. Its mecha nism and site of action at the cellular level remain unclear, but it probab ly acts by a type II oxygen-dependent photosensitization mechanism producin g singlet oxygen. Hypericin is also able to induce a photogenerated intrace llular pH drop, which could constitute an alternative mechanism of hyperici n action. In human glioma cells treated for 1 h with 2.5 mu g/ml hypericin, light exposure induced a fall in intracellular pH. In these conditions, mi tochondria-bound hexokinase was inhibited in a light- and dose-dependent ma nner, associated with a decreased ATF content, a decrease of mitochondrial transmembrane potential, and a depletion of intracellular glutathione, Hexo kinase protein was effectively released from mitochondria, as measured by a n ELISA using a specific anti-hexokinase antibody. In addition to decreased glutathione, a response to oxidative stress was confirmed by the concomita nt increase in mRNA expression of gamma-glutamyl cysteine synthetase, which catalyzes the rate-limiting step in overall glutathione biosynthesis, and is subject to feedback regulation by glutathione. Hypericin also induced a dose- and light-dependent inhibition of [H-3]thymidine uptake and induced a poptosis, as demonstrated by annexin V-FITC binding and cell morphology. Th is study confirmed the mitochondria as a primary target of photodynamic act ion. The multifaceted action of hypericin involves the alteration of mitoch ondria-bound hexokinase, initiating a cascade of events that converge to al ter the energy metabolism of glioma cells and their survival. In view of th e complex mechanism of action of hypericin, further exploration is warrante d in a perspective of its clinical application as a potential phototoxic ag ent in the treatment of glioma tumors.