Effects of nitric oxide donors on cybrids harbouring the mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) A3243G mitochondrial DNA mutation

Abstract

Reactive nitrogen and oxygen species (O2*-, H2O2, NO* and ONOO-) have been strongly implicated in the pathophysiology of neurodegenerative and mitochondrial diseases. In the present study, we examined the effects of nitrosative and/or nitrative stress generated by DETA-NO {(Z)-1-[2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate}, SIN-1 (3-morpholinosydnonimine hydrochloride) and SNP (sodium nitroprusside) on U87MG glioblastoma cybrids carrying wt (wild-type) and mutant [A3243G (Ala3243-->Gly)] mtDNA (mitochondrial genome) from a patient suffering from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). The mutant cybrids had reduced activity of cytochrome c oxidase, significantly lower ATP level and decreased mitochondrial membrane potential. However, endogenous levels of reactive oxygen species were very similar in all cybrids regardless of whether they carried the mtDNA defects or not. Furthermore, the cybrids were insensitive to the nitrosative and/or nitrative stress produced by either DETA-NO or SIN-1 alone. Cytotoxicity, however, was observed in response to SNP treatment and a combination of SIN-1 and glucose-deprivation. The mutant cybrids were significantly more sensitive to these insults compared with the wt controls. Ultrastructural examination of dying cells revealed several characteristic features of autophagic cell death. We concluded that nitrosative and/or nitrative stress alone were insufficient to trigger cytotoxicity in these cells, but cell death was observed with a combination of metabolic and nitrative stress. The vulnerability of the cybrids to these types of injury correlated with the cellular energy status, which were compromised by the MELAS mutation.

Pancratistatin Selectively Targets Cancer Cell Mitochondria and Reduces Growth of Human Colon Tumor Xenografts

“…These findings indicated that either pancratistatin does not directly compete with compounds, or that oxidative insult is not the primary cause of pancratistatininduced cell death. Moreover, we report that mtDNAdeficient r 0 cancer cells resisted pancratistatin-induced cell death, which indicated that pancratistatin may be targeting a component of MRC complexes, as the parental cell line was sensitive to treatment (18,32).…”

“…HT-29 cells were grown in McCoy's 5A media and CCD-18Co cells were grown in Eagle's minimum essential medium (MEM); both were supplemented as recommended by ATCC. Parental stock glioblastoma (U87MG) and the counterpart mtDNA-depleted cell line (U87MG r 0 ) were a generous gift from Dr. M. Sikorska at NRC (18). These cells were grown in MEM supplemented with 10% fetal bovine serum and 10 mmol/L gentamycin.…”

Diabetes-associated mitochondrial DNA mutation A3243G impairs cellular metabolic pathways necessary for beta cell function

“…Addition of 10 mmol/l glucose lowered ROS levels in mutant M12 cells. Interestingly, cytotoxicity has been reported in the absence of glucose in cells carrying the A3243G mtDNA mutation [29]. Upon glucose deprivation, ATP synthesis, normally compensated by oxidative phosphorylation, is inefficient in mtDNA mutants (Fig.…”

“…The imbalance between excessive formation of ROS and limited antioxidant defences causes oxidative stress [27]. Only few studies have looked at ROS generation in cells with mtDNA mutations [28][29][30]. It is debatable whether mtDNA-depletion in rho°cells generates more ROS than control cells would [31][32][33][34].…”

Increased ROS generation and SOD activity in heteroplasmic tissues of transmitochondrial mice with A3243G mitochondrial DNA mutation

“…For example, it has been shown that mice with mutant mtDNA do not exhibit increased ROS levels in spite of carrying a high mutational burden (Meissner, 2007). Another report has demonstrated that mutant cybrids carrying A3243G mtDNA from a patient suffering from MELAS have reduced activity of cytochrome c oxidase significantly, lower ATP level and decreased mitochondrial membrane potential, but the endogenous levels of ROS are very similar in all cybrids regardless of whether they carry the mtDNA defects or not (Sandhu et al, 2005). Therefore, up to date the interplay between A3243G mtDNA mutation and ROS has not been fully clarified.…”