Antitumoral synergistic effect of the combination of uncoupler of oxidative phosphorylation and mitochondrial ribosome inhibitor in human breast cancer: in vitro and in vivo evaluation

[Speaker] Sebastian A. Fuentes-Retamal:1
[Co-author] Liliana A. Peredo-Silva:1, Cristian A. Sandoval-Acuna:2, Ana I. Liempi:3, Daniela I. Guzman-Rivera:1, Mario A. Pavani:1, Vicente E. Castro-Castillo:4, Mabel E. Catalan:1, Ulrike Kemmerling:3, Jorge F. Ferreira:1
1:Clinical and Molecular Pharmacology Program, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, University of Chile, Chile, 2:Institute of Biotechnology, Czech Academy of Sciences, Prague, Czech Republic, 3:Program of Anatomy and Developmental Biology, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, University of Chile, Chile, 4:Department of Organic and Physical Chemistry, Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Chile

Breast cancer has the highest incidence and mortality in women. Mitochondrion is recognized as an essential organelle in the initial tumor process. It is also accepted as a molecular target in the treatment of cancer for its essential role in metabolic, death, calcium and ROS control. Also, the higher transmembrane potential in mitochondrial cancer cells make them susceptible to be efficiently targeted with delocalized lipophilic cations such as triphenylphosphonium (TPP+) derivatives, which serve as a vehicle to selectively guide pharmacophores.
We use Gentisic acid attached to TPP+ through a saturated chain of ten carbons (GA-TPP+C10) as uncoupler of OXPHOS plus doxycycline as mitochondrial ribosome inhibitor to get a synergistic interaction. The interaction was assayed in vitro using the human breast cancer cell lines MCF7 and MDA-MB-231, and the in vivo effect on tumor growth was analyzed by a xenographic model using NOD.CB17-Prkdcscid/J strain and MDA-MB-231 cell line.
It was shown the effect GA-TPP+C10 on cellular metabolism: simultaneous induction of uncoupling of OXPHOS and decrease of glycolysis, which triggers an energy crisis evidenced by the activation of AMPK. This effect was demonstrated in both cell lines, characterized by a highly differentiated phenotype and metabolism. Doxycycline, demonstrated an inhibition in the expression of mitochondrial proteins synthesized from the mitochondrial genome, without affecting those of nuclear origin. When performing the combined treatment with GA-TPP+C10 and doxycycline, it was observed that a synergistic interaction develops on the cytotoxic effect, triggering an increased apoptotic cell death: Phosphatidylserine exposure, activation of caspases, cleavage of PARP. Then, the compounds were tested using a xenographic model, in which GA-TPP+C10 was able to inhibit tumor growth and decrease proliferation markers Ki-67 and PCNA. This effect was significantly enhanced when co-administered with doxycycline, corroborating the data obtained in in vitro assays.
We show that the combination of two drugs that present a different mechanism of action on the mitochondria, can exert a synergistic cytotoxic interaction, which is verified by the in vitro and in vivo data. Therefore, it is concluded that this combination is a potential tool in the search for new treatments for breast cancer.

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