Targeting Mitochondrial DNA with a Platinum-Based Anticancer Agent

An analog of the anticancer drug cisplatin (mtPt) was delivered to mitochondria of human cells using a peptide specifically targeting this organelle. mtPt induces apoptosis without damaging nuclear DNA, indicating that mtDNA damage is sufficient to mediate the activity of a platinum-based chemotherapeutic. This study demonstrates the specific delivery of a platinum drug to mitochondria and investigates the effects of directing this agent outside the nucleus.National Cancer Institute (U.S.) (Grant CA034992)David H. Koch Graduate Fellowshi

Mitochondria-penetrating peptides conjugated to desferrioxamine as chelators for mitochondrial labile iron

<div><p>Desferrioxamine (DFO) is a bacterial siderophore with a high affinity for iron, but low cell penetration. As part of our ongoing project focused on DFO-conjugates, we synthesized, purified, characterized and studied new mtDFOs (DFO conjugated to the Mitochondria Penetrating Peptides TAT_49-57, 1A, SS02 and SS20) using a succinic linker. These new conjugates retained their strong iron binding ability and antioxidant capacity. They were relatively non toxic to A2780 cells (IC50 40–100 μM) and had good mitochondrial localization (Rr +0.45 –+0.68) as observed when labeled with carboxy-tetramethylrhodamine (TAMRA) In general, mtDFO caused only modest levels of mitochondrial DNA (mtDNA) damage. DFO-SS02 retained the antioxidant ability of the parent peptide, shown by the inhibition of mitochondrial superoxide formation. None of the compounds displayed cell cycle arrest or enhanced apoptosis. Taken together, these results indicate that mtDFO could be promising compounds for amelioration of the disease symptoms of iron overload in mitochondria.</p></div

Re-directing an alkylating agent to mitochondria alters drug target and cell death mechanism.

We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential

Antioxidant activity of mtDFO (20 μM).

<p>Their effects on the oxidation rate of the probe DHR treated with (a) increasing amounts of Fe(NTA) or (b) serum of iron-overloaded myelodysplastic patients are displayed. For n = 4, p < 0.05. a.u. = arbitrary units of fluorescence. Results are the average of quadruplicates and representative of at least two isolated experiments. Different letters indicate significant differences with control (no chelator) after one way ANOVA (P < 0.05).</p

Cellular effects on A2780 cells treated with ½ IC₅₀ of the mtDFO.

<p>(a) mtDNA amplification. (b) Production of mitochondrial superoxide. (c) Cell cycle arrest. (d) Apoptosis tested by the Annexin V method. r.u. = relative units; *: statistically significant differences in relation to untreated controls (P < 0.01). Different letters indicate significant differences in relation to untreated controls after one way ANOVA (P < 0.05).</p

IC₅₀ (μM) of mtDFO, DFO and TAMRA-tagged mtDFO in A2780 cells after 24 h.

<p>IC₅₀ (μM) of mtDFO, DFO and TAMRA-tagged mtDFO in A2780 cells after 24 h.</p

Structures of DFO and mtDFO in this study.

<p>The succinyl linker is grayed. Dmt: 2’,6’-dimethyltyrosine; F_X: cyclohexylalanine; r: <i>d</i>-arginine.</p

Colocalization of mtDFO(TAMRA) in mitochondria of A2780 cells.

<p>Rr = Pearson correlation coefficient.</p

<i>In vivo</i> profiles of mt-Cbl and Cbl.

<p>(a<i>) </i><b><i>Pharmacokinetic profile of mt-Cbl and Cbl.</i></b> 10 mg/kg of Cbl or mt-Cbl was administered to mice and plasma levels of both compounds were measured via HPLC-MS/MS. Mt-Cbl is eliminated more gradually than Cbl. Mean values plotted, n = 3, error bars are standard deviation. (b<i>) </i><b><i>Biodistribution of mt-Cbl and Cbl.</i></b> Both compounds show similar profiles suggesting that the MPP peptide does not alter distribution of Cbl. Mean values plotted, n = 3, error bars are standard deviation. (c) <b><i>Liver enzyme levels following treatment.</i></b> Mice were treated as above and their plasma was assessed for levels of bilirubin, alkaline phosphatase and aspartate transaminase. No significant increase was noted in any of the enzyme levels following treatment with Cbl or mt-Cbl. Mean values plotted, n = 4, error bars are s.e.m.</p