Delocalized lipophilic cations selectively target the mitochondria of carcinoma cells.

Traditional chemotherapies, aimed at DNA replication in rapidly dividing cells, have achieved only limited success in the treatment of carcinomas due largely to their lack of specificity for cells of tumorigenic origin. It is important, therefore, to investigate treatment strategies aimed at novel cellular targets that are sufficiently different between normal cells and cancer cells so as to provide a basis for selective tumor cell killing. Delocalized lipophilic cations (DLCs) are concentrated by cells and into mitochondria in response to negative inside transmembrane potentials. The higher plasma and/or mitochondrial membrane potentials of carcinoma cells compared to normal epithelial cells account for the selective accumulation of DLCs in carcinoma mitochondria. Since most DLCs are toxic to mitochondria at high concentrations, their selective accumulation in carcinoma mitochondria and consequent mitochondrial toxicity provide a basis for selective carcinoma cell killing. Several of these compounds have already displayed some degree of efficacy as chemotherapeutic agents in vitro and in vivo. The effectiveness of DLCs can also be enhanced by their use in photochemotherapy or combination drug therapy. Discovery of the biochemical differences that account for the higher membrane potentials in carcinoma cells is expected to lead to the design of new DLCs targeted specifically to those differences, resulting in even greater selectivity and efficacy for tumor cell killing.

Photoactivation enhances the mitochondrial toxicity of the cationic rhodacyanine MKT-077.

In this study, the mitochondrial phototoxicity of the cationic rhodacyanine MKT-077 was investigated by comparing its effects on the inhibition of mitochondrial respiration and the structural integrity of mitochondrial DNA (mtDNA) in the presence and absence of added high-intensity visible light (7.5 J/cm2). Results indicate that photoirradiation significantly enhances the mitochondrial toxicity of MKT-077 at both the biochemical and DNA levels. For example, the concentration of MKT-077 required to achieve one-half maximal inhibition of ADP-stimulated respiration was observed to be 6-fold lower in the presence versus absence of high-intensity light (one-half maximal inhibition at 2.5 versus 15 microg MKT-077/ mg, respectively). In addition, photoirradiation produced a 25-fold increase in inhibition of succinate-cytochrome c reductase activity by MKT-077 (one-half maximal inhibition at 2 versus 50 microg MKT-077/ml, +/-light, respectively) and a 6-fold increase in inhibition of cytochrome oxidase activity (one-half maximal inhibition at 5 versus 30 microg MKT-077/ml, +/-light, respectively). Furthermore, the combination of 25 microg/ml MKT-077 and 7.5 J/cm2 visible light caused significant degradation of mtDNA in isolated rat liver mitochondria, whereas the same concentration of dye in the absence of light had only a modest effect on mtDNA. Evaluation of light-induced MKT-077 lipid peroxidation in mitochondrial membrane fragments by the thiobarbituric acid test and by measurement of nonrespiratory-linked oxygen uptake suggests that mitochondrial phototoxicity by MKT-077 may be the result of lipid peroxidation via reactive oxygen species. These results have important implications with regard to the potential use of MKT-077 in photochemotherapy.

Selective damage to carcinoma mitochondria by the rhodacyanine MKT-077.

We investigated the mitochondrial toxicity of the lipophilic cation, MKT-077, and the role of mitochondria in selective malignant cell killing by this compound by examining the effect of MKT-077 on mitochondrial structure and function in treated cells and in isolated organelles. Results of this study demonstrate changes in mitochondrial ultrastructure that are induced by MKT-077 treatment in carcinoma cells but not in similarly treated normal epithelial cells. In addition, MKT-077 was found to inhibit respiratory activity in isolated intact mitochondria and electron transport activity in freeze-thawed mitochondrial membrane fragments in a dose-dependent manner. The concentration of MKT-077 necessary to obtain half-maximal inhibition of ADP-stimulated respiration was approximately 4-fold greater in mitochondria isolated from cells of the normal epithelial cell line, CV-1 (15 micrograms MKT-077/mg protein), as compared to the human colon carcinoma cell line, CX-1 (4 micrograms MKT-077/mg protein). Further, the data show a selective loss of mitochondrial DNA in CX-1 and CRL1420 cells (carcinoma) but not CV-1 cells (normal epithelial) treated with 3 microgram/ml MKT-077 for up to 3 days. Under the same conditions, nuclear DNA was unaffected in all three cell lines. The sensitivity of the cell lines tested to mitochondrial damage by MKT-077 correlates well with their sensitivity to cytotoxicity by MKT-077. These results demonstrate selective mitochondrial damage by MKT-077 at the cellular, biochemical, and molecular levels and suggest that selective effects on mitochondrial structure and function may provide a basis for the selective malignant cell killing exhibited by this compound.

AZT causes tissue-specific inhibition of mitochondrial bioenergetic function.

The mitochondrial myopathy associated with long-term AZT therapy is a factor that limits the clinical efficacy of this compound in the treatment of AIDS. The biochemical basis for this tissue-specific pathology was investigated by measuring the effect of AZT on various aspects of bioenergetic function in mitochondria isolated from rat skeletal muscle, brain, and liver. AZT induced a dose-dependent inhibition of both NADH-linked respiration in intact mitochondria and NADH-cytochrome c reductase activity (but not succinate-cytochrome c reductase activity) in freeze-thawed mitochondrial preparations isolated from all three tissue types (1/2 maximal inhibition was obtained at 2 mg/ml and between 0.3 and 0.8 mg/ml AZT, respectively). These data demonstrate that high concentrations of AZT inhibit electron transfer through respiratory enzyme complex I. Moreover, AZT was shown to induce a tissue-specific inhibition of succinate-linked respiration in intact mitochondria isolated from rat skeletal muscle (1/2 maximal inhibition at 0.5 mg/ml AZT) and possibly brain, but not liver. The data suggest that this inhibition possibly occurs at the level of succinate transport. These results may help to explain the tissue-specific mitochondrial effects that are induced by long-term zidovudine treatment of AIDS patients and suggest that the anti-retroviral activity exhibited by AZT may be distinct from its mechanism of mitochondrial toxicity.

Denaturing gradient gel method for mapping single base changes in human mitochondrial DNA.

A denaturing gradient gel electrophoresis (DGGE) method is described that detects even single base pair changes in mitochondrial DNA (mtDNA). In this method, restriction fragments of mtDNA are electrophoresed in a urea/formamide gradient gel at 60 degrees C. Migration distance of each mtDNA fragment in the gel depends on melting behavior which reflects base composition. Fragments are located by Southern blotting with specific mtDNA probes. With just four carefully chosen restriction enzymes and as little as 50-100 ng of mtDNA, the method covers almost the entire human mitochondrial genome. To demonstrate the method, human mtDNA was analyzed. In six normal individuals, DGGE revealed melting behavior polymorphisms (MBPs) in mtDNA fragments that were not detected by restriction fragment length polymorphism (RFLP) analysis in agarose gels. Another individual, shown to have a melting behavior polymorphism in the cytochrome b coding region, was studied in detail. By mapping, the mutation was deduced to lie between nt 14905 and 15370. The affected fragment was amplified by PCR and sequenced. Specific base changes were identified in the region predicted by the gel result. This method will be especially useful as a diagnostic tool in mitochondrial disease for rapid localization of mtDNA mutations to specific regions of the genome, but DGGE also could complement RFLP analysis as a more sensitive method to follow maternal lineage in human and animal populations in a variety of research fields.

Mitochondrial toxicity of cationic photosensitizers for photochemotherapy.

The triarylmethane derivative Victoria Blue-BO (VB-BO) and the chalcogenapyrylium (CP) dyes have potential for use in photochemotherapy, because they are taken up by the mitochondria of malignant cells and cause cell death. To clarify the mechanism of cell killing we examined the phototoxic effects of VB-BO and a series of three CP dyes on bioenergetic function in isolated rat liver mitochondria. Without photoirradiation, and irrespective of the respiratory substrate used, each of the compounds tested induced some uncoupling of oxidative phosphorylation. Visible irradiation of VB-BO produced an inhibition of mitochondrial respiration when glutamate plus malate, but not succinate, was used as the respiratory substrate. With photoirradiation VB-BO was also shown to inhibit rotenone-sensitive NADH-cytochrome c reductase activity, but it had no effect on succinate-cytochrome c reductase activity. These data indicate that photoactivation of VB-BO produces selective inhibition of mitochondrial respiratory complex I. Photoirradiation of the CP dyes inhibited both complex I and complex II initiated respiratory activity. With photoirradiation, the CP dyes also inhibited both NADH- and succinate-cytochrome c reductase activities, as well as other membrane-bound enzymes, cytochrome c oxidase and succinate dehydrogenase, but not the mitochondrial matrix enzyme, citrate synthetase, or the cytosolic enzyme, lactate dehydrogenase. alpha-Tocopherol protected bioenergetic activities against CP dye photodamage. These results suggest that mitochondrial photosensitization by CP compounds is mediated by the production of membrane-damaging singlet oxygen which causes nonspecific damage to membranes and membrane-bound enzymes.

Aberrant mitochondria in two human colon carcinoma cell lines.

Electron micrographs of CCL237 and FET cells (two slowly growing, differentiated human colon carcinoma lines) revealed enlarged mitochondria with few cristae. Polarographic measurement of respiratory activity in mitochondria isolated from these cell lines was compared to that of CV-1 cells (a normal monkey kidney epithelial line) and MIP101 cells (another human colon carcinoma line), both of which have mitochondria with a "normal" appearance. The respiratory control ratios of CCL237 and FET mitochondria were found to be considerably lower than those of CV-1 and MIP101 mitochondria (approximately 3 as compared to greater than 10, respectively), indicating that in CCL237 and FET mitochondria the processes of substrate oxidation and phosphorylation of ADP are only loosely coupled. In intact cells, differences in radiolabeled tetraphenylphosphonium uptake showed that the mitochondrial membrane potential in CCL237 and FET cells was less than that in CV-1 and MIP101 cells, and that nigericin failed to hyperpolarize the mitochondria of CCL237 and FET cells. In addition, FET mitochondria exhibited significantly lower ADP-stimulated and uncoupled respiratory rates than mitochondria isolated from the other cell types, indicating that in the former, the capacity for oxidative phosphorylation is somehow impaired. Selective toxicity of FET cells was obtained by treatment with 2-deoxyglucose, an inhibitor of glycolysis, suggesting the possibility of exploiting the phenotype of impaired oxidative metabolism for chemotherapy.

Basis for the selective cytotoxicity of rhodamine 123.

Using rat liver mitochondria we determined that the primary biochemical target for inhibition of mitochondrial bioenergetic function by rhodamine 123 (Rh123) was FoF1-ATPase and that the amount of Rh123 associated with mitochondria is proportional to the mitochondrial membrane potential. Inhibition of coupled respiration by Rh123 in mitochondria isolated from CX-1, a Rh123-sensitive carcinoma cell type, and CV-1, a Rh123-insensitive normal epithelial cell type, was linearly related to the amount of Rh123 added (micrograms/mg protein) with CX-1 mitochondria exhibiting 2-fold greater inhibition compared to CV-1 mitochondria at any given amount of dye. The inhibition pattern for mitochondria isolated from MIP101, a Rh123-insensitive carcinoma cell type, was nonlinear, exhibiting greater sensitivity than CV-1 mitochondria at very low amounts of Rh123 but becoming less sensitive than either CV-1 or CX-1 at higher amounts. Rh123 inhibited FoF1-ATPase activity to a similar extent and in a concentration-dependent manner in both CV-1 and CX-1 mitochondria, but a different and complex pattern of inhibition was apparent for MIP101 mitochondria. Moreover, mitochondria from the 2 carcinoma cell types, CX-1 and MIP101, had higher membrane potentials (163 +/- 7 and 158 +/- 8 mV, respectively) than did mitochondria from the normal epithelial cell type, CV-1 (104 +/- 9 mV). It was concluded that differences in both mitochondrial membrane potential and sensitivity of FoF1-ATPase contribute to the selective cytotoxicity exhibited by Rh123 for certain cell types in vitro.

Rhodamine 123 inhibits bioenergetic function in isolated rat liver mitochondria.

Rhodamine 123 accumulates in the mitochondria of living cells and exhibits selective anticarcinoma activity. The biochemical basis of toxicity was investigated by testing the effect of the dye on isolated rat liver mitochondria. Much lower concentrations of rhodamine 123 were required to inhibit ADP-stimulated respiration and ATP synthesis in well-coupled energized mitochondria than were required to inhibit uncoupled respiration and uncoupler-stimulated ATP hydrolysis. The amount of rhodamine 123 associated with the mitochondria was several-fold greater under energized as compared to non-energized conditions, which may explain why coupled functions appeared to be more sensitive than uncoupled functions to inhibition at low concentrations of rhodamine 123. It was concluded that the site of rhodamine 123 inhibition is most likely the F0F1 ATPase complex and possibly electron transfer reactions as well.