The molecular mode of action of lonidamine, a therapeutic agent employed in cancer chemotherapy, has been elusive. Here we provide evidence that lonidamine (LND) acts on mitochondria to induce apoptosis. LND provokes a disruption of the mitochondrial transmembrane potential which precedes signs of nuclear apoptosis and cytolysis. The mitochondrial and cytocidal eects of LND are not prevented by inhibitors of caspases or of mRNA or protein synthesis. However, they are prevented by transfection-enforced overexpression of Bcl-2, an oncoprotein which inhibits apoptosis by stabilizing the mitochondrial membrane barrier function. Accordingly, the cell death-inducing eect of LND is ampli®ed by simultaneous addition of PK11195, an isoquinoline ligand of the peripheral benzodiazepine receptor which antagonizes the cytoprotective eect of Bcl-2. When added to isolated nuclei, LND fails to provoke DNA degradation unless mitochondria are added simultaneously. In isolated mitochondria, LND causes the dissipation of the mitochondrial inner transmembrane potential and the release of apoptogenic factors capable of inducing nuclear apoptosis in vitro. Thus the mitochondrion is the subcellular target of LND. All eects of LND on isolated mitochondria are counteracted by cyclosporin A, an inhibitor of the mitochondrial PT pore. We therefore tested the eect of LND on the puri®ed PT pore reconstituted into liposomes. LND permeabilizes liposomal membranes containing the PT pore. This eect is prevented by addition of recombinant Bcl-2 protein but not by a mutant Bcl-2 protein that has lost its apoptosisinhibitory function. Altogether these data indicate that LND represents a novel type of anti-cancer agent which induces apoptosis via a direct eect on the mitochondrial PT pore.
Summary Loss of chromosome 10 was observed in 10 out of 12 xenografted glioblastomas studied. Chromosome 10 carries the gene coding the hexokinase type I isoenzyme (HK-I), which catalyses the first step of glycolysis, which is essential in brain tissue and glioblastomas. We investigated the relationships between the relative chromosome 10 number, the amount of HK-I mRNA, HK-I activity and its intracellular distribution, and glycolysis-related parameters such as the lactate-pyruvate ratio, lactate dehydrogenase (LDH) and ATP contents. Individual tumour HK-I mRNA amounts were 23 -65% lower than that of normal human brain and reflected the relative decrease of chromosome 10 number (ax<0.01). Total HK activities of individual glioblastomas varied considerably but were constantly (a mean of seven times) lower than that of normal brain tissue. The mitochondria-bound HK-I fraction of individual tumours was generally over 50%, compared with that of normal brain tissue. As shown by lactate-pyruvate ratios, in all the gliomas, glycolysis was elevated to an average of 3-fold that measured in normal brain. An elevated ATP content was also constantly noted. Adaptation of glioblastoma metabolism to the chromosome 10 loss and to the HK-I transcription unit emphasises the critical role of glycolysis in their survival. We hypothesise that HK-I, the enzyme responsible for initiating glycolysis necessary for brain function, may approach its lowest limit in gliomas, thereby opening therapeutic access to pharmacological anti-metabolites affecting energy metabolism and tumour growth.
Hexokinase plays a key role in regulating cell energy metabolism. Hexokinase is mainly particulate, bound to the mitochondrial outer membrane in brain and tumour cells. We hypothesized that the intracellular pH (pH1) controls the intracellular distribution of hexokinase. Using the SNB-19 glioma cell line, pH1 variations were imposed by incubating cells in a high-K+ medium at different pH values containing specific ionophores (nigericin and valinomycin), without affecting cell viability. Subcellular fractions of cell homogenates were analysed for hexokinase activity. Imposed pH1 changes were verified microspectrofluorimetrically by using the pH1-sensitive probe SNARF-1-AM (seminaphtho-rhodafluor-1-acetoxymethyl ester). Imposition of an acidic pH1 for 30 min strongly decreased the particulate/total hexokinase ratio, from 63% in the control sample to 31%. Conversely, when a basic pH1, was imposed, the particulate/total hexokinase ratio increased to 80%. The glycolytic parameters, namely lactate/pyruvate ratio, glucose 6-phosphate and ATP levels, were measured concomitantly. Lactate/pyruvate ratio and ATP level were both markedly decreased by acidic pH1 and increased by basic pH1. Conversely, the glucose 6-phosphate level was increased by acidic pH1 and decreased by basic pH1. To demonstrate that the change of hexokinase distribution was not due to altered metabolite levels of glycolysis, a pH1 was imposed for a 5 min incubation time. Modification of the hexokinase distribution was similar to that noted after a 30 min incubation, whereas metabolite levels of glycolysis were not affected. These results provide evidence that the intracellular distribution of hexokinase is highly sensitive to variations of the pH1, and regulates hexokinase activity.
The human DNA-binding HSA kin17 protein cross-reacts with antibodies raised against the stress-activated Escherichia coli RecA protein. We show here that HSA kin17 protein is directly associated with chromosomal DNA as judged by cross-linking experiments on living cells. We detected increased amounts of DNAbound HSA kin17 protein 24 h after ␥ irradiation, with 2.6-fold more HSA kin17 molecules after 6 Gy of irradiation (46,000 -117,000 molecules). At this time we observed that highly proliferating RKO cells displayed the concentration and co-localization of HSA kin17 and replication protein A in nucleoplasmic foci. Our results suggest that 24 h post-irradiation HSA kin17 protein may localize at the sites of unrepaired DNA damages. RKO clones expressing an HSA KIN17 antisense transcript (RASK.5 and RASK.13 cells) revealed that reduced HSA kin17 protein levels are correlated with a decrease in clonogenic cell growth and cell proliferation, as well as an accumulation of cells in early and mid-S phase. Taken together our observations support the idea that HSA kin17 protein is a DNA maintenance protein involved in the cellular response to the presence of DNA damage and suggest that it helps to overcome the perturbation of DNA replication produced by unrepaired lesions. Ionizing radiation (IR)1 induces a large range of DNA damage, including DNA double-strand breaks (DSBs), which represent a major threat to the integrity of mammalian genomes through chromosomal breakages and rearrangements (1). In mammalian cells, DSBs are repaired either by the homologous recombinational repair or by nonhomologous end joining (2, 3). DSB repair pathways are usually characterized by the sequestration of many factors into discrete nuclear foci at the sites of DNA lesions and until completion of DSB repair (4). Some of the proteins belonging to these pathways act as a sensor for DNA damage or are involved in cell cycle checkpoints. This is the case for the histone H2AX, 53BP1, RPA, Rad51, BRCA1, or the Mre11-Rad50-Nbs1 nuclease complex (4 -8). For instance, the tumor suppressor gene BRCA1, previously involved in the regulation of the replication checkpoint and transcription-coupled repair, forms a multiprotein complex with Mre11-Rad50-Nbs1 and other proteins following irradiation, termed as BASC (BRCA1-associated surveillance complex), which may serve as a sensor of DNA lesions (8,9).In this cascade of IR-induced proteins forming nuclear foci, we characterized here the HSA kin17 protein. Murine MMU kin17 protein was identified on the basis of a cross-reactivity with antibodies raised against the Escherichia coli RecA protein, a key enzyme in homologous recombination and recombinational repair of damaged DNA (10, 11). This cross-reactivity stemmed from a sequence homology stretching over 39 amino acids highly conserved during evolution (12). This domain is located in the carboxyl-terminal region of the E. coli RecA protein, a region involved in the regulation of DNA binding (13). Recent data show that kin17 proteins are highly conserved d...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.