In this study, we tested the cytostatic and cytotoxic properties of Mito-CP11 and CP in MCF-7 human breast cancer cells and MCF-10A normal mammary epithelial cells. At sub-micromolar concentrations, Mito-CP11, a mitochondria-targeted five-membered ring nitroxide, exerted greater cytostatic effects in MCF-7 than MCF-10A cells. CP, a five-membered nitroxide, was not found to be cytostatic or cytotoxic over a wide range of concentrations in these cells.
and mitochondria-targeted compounds like Mito-Q are known to be preferentially taken up by the mitochondria.27,30
Lipophilic cations distribute their charge over a large surface area, allowing them to easily penetrate the lipid bilayers. The large negative membrane potential of 150–180 mV across the mitochondrial inner membrane is responsible for mitochondrial accumulation of the lipophilic triphenylphosphonium cations. Mito-Q was shown to accumulate 50- to 100-fold within mitochondria.31
Nevertheless, the difference in ΔΨm
detectable between normal and malignant cancerous cells is at least 60 mV. Thus, mitochondria-targeted compounds are sensitive to the higher ΔΨm
in malignant cells, and selectively accumulate in their mitochondria. The increased toxicity of lipophilic cationic probes (e.g., rhodamine 123) was linked to enhanced accumulation of these probes into cancer cells. However, it was not clear whether the mitochondrial accumulation of rhodamine 123 was significantly higher in cancer cells as compared with noncancerous control cells. Several synthetic alkyl-lysophospholipid analogs (e.g., edelfosine) exhibited selective antitumor action that is attributed to accumulation of the phospholipid ether in tumor cells.32
In this study, we measured the steady-state concentrations of Mito-CP11
in mitochondrial fractions of MCF-7 and MCF10A cells. As shown in , the intensity of the EPR signal in both mitochondrial fractions (i.e., from MCF-7 and MCF-10A cells) was nearly identical. Based on this result, we conclude that Mito-CP11
-induced differential toxicity is not simply due to differential uptake of Mito-CP11
into MCF-7 and MCF-10A mitochondria, and that other factors may be involved.
Recently, we reported Mito-CP11
acting as an antioxidant in bovine aortic endothelial cells treated with H2
Due to selective uptake of Mito-CP11
in the mitochondria, it inhibited the cytochrome c
release and caspase-3 activation in cells treated with peroxides. In SOD1G93A
and Mito-Q at nanomolar concentrations prevented mitochondrial dysfunction, diminished superoxide production and restored motor neuron survival.33
Nitroxides are known to react with various types of ROS like peroxyl, hydroxyl and superoxide radicals. Nitroxides catalyze the superoxide dismutation in a pH-dependent manner.26
During this reaction, two molecules of superoxide dismutate to form O2
. Recent reports have shown that traditional antioxidants such as ascorbic acid, exert strong pro-oxidant properties in response to the tumor microenvironment, increasing the levels of ROS like H2
which lead to cancer cell death.34
Paradoxically, in normal cells, antioxidants protect from ROS-mediated cell damage.11,12
The proposed use of statins in breast cancer therapy remains controversial.35–37
Results from a meta analysis showed that statins had no effect on breast cancer. Previously, we and others showed that hydrophilic statins such as pravastatin did not induce cytotoxicity in breast cancer cells.1,2
Results from a new study conducted on a large population of postmenopausal women with breast cancer lead to the conclusion that the use of lipophilic or hydrophobic statins exhibit beneficial effects in breast cancer therapy.37
However, when both hydrophobic and hydrophilic statins (e.g., pravastatin) were considered together as a class, no statistically significant beneficial effect was found.37
Also, when pravastatin data were excluded in the randomized clinical trials used in the meta analysis, the results of the meta analysis were not statistically significant. These results clearly indicate that hydrophobic or lipophilic statins but not hydrophilic statins (pravastatin) should be considered in breast cancer therapy. Another important consideration is that statins, at concentrations that can be achieved in plasma (sub-micromolar levels) may not effectively act as a chemotherapeutic. The present cell culture shows that Mito-CP11
could sensitize breast cancer cells to fluvastatin-induced cytotoxicity. Thus, mitochondria-targeted nitroxides such as Mito-CP11
could act as an effective antitumor adjuvant in augmenting the chemotherapeutic or chemopreventive potential of statins.
A recent report suggests that long-term administration of the statins, atorvastatin and lovastatin, failed to inhibit rodent mammary carcinogenesis in Sprague-Dawley rats.38
In this model, mammary tumors were induced by intravenous injection of methylnitrososourea (MNU). Statins were given in the diet. The results are, however, not in agreement with the inhibitory effects of fluvastatin in DMBA-induced mammary carcinogenesis as reported in the present study. We do not yet have a clear-cut explanation for the observed discrepancies between the two studies; however, it must be pointed out that both studies significantly differed in several aspects of experimentation design, most notably in rat strain and the type of carcinogen used to induce breast tumors.
However, the present data show that fluvastatin, when administered chronically at 4 mg/ml to DMBA-treated rats, can inhibit tumor formation in a breast cancer prevention animal model. Future studies should test the scope of the combined administration of fluvastatin and Mito-CP as potential chemotherapeutics in a xenograft animal model of breast carcinogenesis.
NFκB is a very plausible participant in the signaling pathways that are disrupted by Mito-CP11
in breast cancer cells. Numerous studies indicate that NFκB activity promotes breast cancer development and progression, due to the NFκB-dependent expression of proteins that promote cancer cell survival, proliferation and metastasis.15–17
Many of these proteins are induced by TNFα, which is a well-known activator of NFκB in breast cancer and other types of cancer.11,18
The importance of NFκB in promoting the malignant phenotype is further indicated by reports that NFκB activity is higher in breast cancer cell lines, including MCF-7 cells, than in the mammary epithelial MCF-10A cells.16,17,39,40
Based on these pro-proliferative functions of NFκB in breast cancer, our finding that Mito-CP11
inhibits NFκB activity in MCF-7 cells provides a plausible mechanism to account for the antiproliferative effects of Mito-CP11
in MCF-7 cells. It is likely that the antioxidant properties of Mito-CP11
are responsible for this loss of NFκB activity, since antioxidants have been found to inhibit NFκB activity in multiple cell types.13
accumulates mainly in the mitochondria, it is also present in the cytoplasm,31
where redox-sensitive kinases that regulate NFκB are located.13,14
Thus, the antioxidant effects of Mito-CP in the cytoplasm, and potentially in other organelles, could disrupt NFκB signaling pathways. Intriguingly, we found that Mito-CP11
inhibits basal NFκB activity more in MCF-7 than MCF-10A cells, which might account in part for the ability of Mito-CP11
to inhibit proliferation more in MCF-7 than MCF-10A cells. We also found that Mito-CP11
potently inhibits TNFα-induced activation of NFκB in MCF-7 cells. This effect of Mito-CP11
might contribute to its cytotoxic actions, since agents that inhibit the TNFα-induced activation of NFκB decrease the survival of cancer cells.13
Taken together, these findings provide evidence that inactivation of NFκB by Mito-CP11
, mostly likely due to its antioxidant properties, contributes to antiproliferative effects of this drug.
In addition to its antioxidant property, Mito-CP11
might also have unique functions due to the presence of the alkyl chain linked to the triphenylphosphonium group. Alkylphosphonium salts have previously been shown to exert antitumor activity in a variety of human tumor cell lines.41
Several alkylphosphonium cations also suppressed tumor growth in animal xenograft models.41
Some phosphonium cations exhibited cytotoxicity in drug-resistant ovarian cancer cell lines and other multidrug-resistant experimental tumors.42
The proposed mechanism involves selective accumulation of lipophilic, cationic phosphonium compounds in mitochondria of neoplastic cells, leading to inhibition of mitochondrial respiration. The molecular target involved in enhanced cytostatic and cytotoxic effects of phosphonium compounds in tumor cells still remains to be established. In our experiments, however, the methyltriphenylphosphonium cation, at the concentrations used, did not affect cell proliferation. Recently, it was reported that mitochondrial ROS regulate cell proliferation via the extracellular signal-regulated kinase (ERK1/2) pathway.43
Mitochondria-targeted antioxidants (MTAs) (Mito-CP11
) but not “untargeted” nitroxides induced an increase in phosphorylated ERK1/2 in tumor cells.43
The upstream MEK kinase (MAPK kinase) induced cell proliferation or cell death, depending upon the levels.44
Previous data also suggest that oncogene-induced mitochondrial ROS serve as signaling molecules to dampen the ERK1/2 MAPK pathway to levels that promote cellular proliferation.30
More recently, it was shown that Mito-Q (Co-enzyme Q10
conjugated to a triphenylphosphonium moiety) potently induces antiproliferative mechanism in breast cancer cells but not in healthy mammary cells.45
It was proposed that Mito-Q, being a weak electrophile, induced cell cycle arrest and autophagy via Nrf2-regulated enzyme NQO1.45
Thus, the molecular target involved in MTA-mediated cytostatic and cytotoxic effects in cancer cells is dependent on the MTA structure.
An alternative mechanism by which Mito-CP enhanced fluvastatin-induced breast cancer cell cytotoxicity may be related to statin's ability to inhibit glycolytic activity in breast cancer cells.46
Using NMR metabonomic analysis, the investigators showed that incubation of MDA-MB-468 cells with lovastatin for 48 h strongly inhibited the glycolytic activity and decreased the de novo formation of 13
C-alanine and 13
Mitochondria-targeted cationic compounds exacerbated the anti-proliferative effects of 2-DG in pancreatic cancer cells.47
We propose a similar type of mechanism for the cationic antioxidant, Mito-CP. Blocking tumor cell mitochondrial function with targeted lipophilic cations hypersensitized tumor cells to glycolytic inhibitors.47
As L-NAME did not have any effect on Mito-CP11
/fluvastatin-enhanced breast cancer cell cytotoxicity, we can rule out nitric oxide and/or peroxynitrite involvement.
Another caveat is that the half-life of fluvastatin in humans is nearly 3 h, and can reach the steady-state concentration in the plasma up to 102 ng/ml (0.24 µM) with administration of extended release capsules (80 mg). Although the clinical relevance of the combined therapeutic efficacy of MTAs (0.5 µM) and statins (1 µM) remains uncertain, additional investigation in a preclinical animal model using modified dosing schedules is necessary.
Finally, MTAs may enhance the efficacy of conventional chemotherapeutics and act as radioprotectors in radiation therapy.48
With most conventional chemotherapy, the cumulative dose of an antitumor agent that causes tumor cell killing often stimulates toxic side effects. The combination chemotherapy typically involves the use of two or more chemotherapeutic agents, all of which exhibit toxic side effects in normal cells. MTAs can potentially decrease the levels of the conventional chemotherapeutic agent used in cancer treatment. Another potential application for MTA-mediated adjuvant chemotherapy may be related to targeting and killing breast cancer with erbB2 expression that is associated with nearly 30% of breast cancers.
In summary, the present study demonstrates that mitochondria-targeted nitroxides induce antiproliferative effects in breast cancer cells but not in normal mammary epithelial cells. Mito-CP11 exacerbated the cytotoxic effects of fluvastatin in breast cancer cells. Unlike fluvastatin, the effects due to Mito-CP11 were not dependent on mevalonate. Mito-CP11 significantly inhibits NFκB activity in MCF-7 cells, which might contribute to the antiproliferative effects of Mito-CP11 in these cells. Future studies will investigate the chemotherapeutic effects of Mito-CP11 alone and in combination with fluvastatin in a xenograft animal model of breast carcinogenesis.