Submitted on September 15, 2006
Accepted on November 25, 2006
Phosphorothioate Oligonucleotides Reduce Mitochondrial Outer Membrane Permeability to ADP
Wenzhi Tan1, Johnathan C. Lai2, Paul Miller3, C. A. Stein4, and Marco Colombini1*
1 Biology, University of Maryland, 20742, Maryland, United States
2 Biomedical Engineering, Columbia University, New York, New York, United States
3 Biochemistry & Molecular Biology, Johns Hopkins University, Baltimore, Maryland, United States
4 Oncology, Albert Einstein-Montefiore Cancer Center, Bronx, New York, United States
* To whom correspondence should be addressed. E-mail: colombini{at}umd.edu.
G3139, an antisense Bcl-2 phosphorothioate oligodeoxyribonucleotide, induces apoptosis in melanoma and other cancer cells. This apoptosis happens prior to and in the absence of the downregulation of Bcl-2 and thus seems to be Bcl-2-independent. Binding of G3139 to mitochondria and its ability to close VDAC have led to the hypothesis that G3139 acts, in part, by interacting with VDAC channels in the mitochondrial outer membrane (PNAS 103: 7494, 2006). In this study, we demonstrate the G3139 is able to reduce the mitochondrial outer membrane permeability to ADP by a factor of 6-7 with a KI between 0.2 and 0.5 µM. Since VDAC is responsible for this permeability, this result strengthens the aforesaid hypothesis. Other mitochondrial respiration components are not affected by [G3139] up to 1 µM. Higher levels begin to inhibit respiration rates, decrease light scattering and increase uncoupled respiration. These results agree with accumulating evidence that VDAC closure favors cytochrome c release. The speed of this effect (within 10 minutes) places it early in the metabolic cascade with cytochrome c release occurring at later times. Other phosphorothioate oligonucleotides are also able to induce VDAC closure and there is some length dependence. The phosphorothioate linkages are required to induce the reduction of outer membrane permeability. At levels below 1 µM, phosphorothioate oligonucleotides are the first specific tools to restrict mitochondrial outer membrane permeability.
