Nitrooxy-Doxorubicin: A Mitochondrial-Targeting Anthracycline Derivative That Overcomes Drug-Resistance

C Riganti1,2, B Rolando3, J Kopecka1, I Campia1, K Chegaev3, L Lazzarato3, R Fruttero3, D Ghigo1,2. 1Department of Oncology, University of Turin, via Santena 5/bis, 10126, Turin, Italy, 2Center for Experimental Research and Medical Studies (CeRMS), University of Turin, via Santena 5/bis, 10126, Turin, Italy, 3Department of Science and Drug Technology, University of Turin, via Pietro Giuria 9, 10125, Turin, Italy

The major limitations of doxorubicin (DOX) are the onset of drug resistance and the cardiotoxicity. In previous studies we have shown that nitric oxide (NO) donors associated with DOX overcome the resistance. NO can nitrate critical tyrosines on ATP-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp/ABCB1), multidrug resistance related proteins (MRPs/ABCCs) and breast cancer resistance protein (BCRP/ABCG2). Following the nitration, the activity of ABC transporters was inhibited and DOX was less effluxed [1].

In order to improve the effects of NO in reversing drug resistance, we designed new polyvalent drugs that were at the same time anti-cancer agents and NO-delivery systems. We produced synthetic DOXs conjugated with NO-releasing groups, namely nitrooxy-DOX (NitDOX) and 3-phenylsulfonylfuroxan-DOX (FurDOX) [2]. We tested them in a panel of human drug-sensitive (HT29, A549, K562) and drug-resistant (HT29-dx, A549-dx, K562-dx) cancer cells, created by selecting the parental cell lines in a DOX-containing medium [1].

Nit-DOX and Fur-DOX released NO in a dose- and time-dependent manner and nitrated ABC transported (measured as reported in [1]) in the drug-resistant cells. They were more accumulated than DOX (p < 0.002) and had a lower IC50 (< 15 µM) than DOX (> 50 µM) in all the drug-resistant cells analyzed. Nit-DOX was not more cytotoxic than DOX in cardiomyocytes and in non transformed colon epithelial cells in vitro, and was further investigated.

Surprisingly NitDOX showed properties far different from DOX: it did not inhibit the purified topoisomerase II enzyme in the range 1-50 μM, was uptaken with a faster kinetics, produced different metabolites, showed an extranuclear distribution and a preferential accumulation in mitochondria. The mitochondrial delivery was due to the higher hydrophobicity (log D7.4 for DOX: 0.47 + 0.08; for NitDOX: 2.72 + 0.09; p < 0.001) and to the lower efflux of NitDOX that nitrated MRP1 and BCRP transporters of mitochondrial membranes. Following nitration, the drug was more retained within mitochondria (p < 0.002), suggesting that its efflux through MRP1 and BCRP was inhibited. In mitochondria NitDOX inhibited the tricarboxylic acid cycle (p < 0.001), the electron flux through Complex I (p < 0.005) and the synthesis of ATP (p < 0.001), induced the release of cytosolic cytochrome c and the activation of caspase-9-dependent apoptosis at 5 μM. All these events were dependent on the release of NO, since they were reduced by NO scavengers. Of note, these effects were equally achieved in drug-sensitive and drug-resistant cells.

We propose nitrooxy-doxorubicin as a new polyvalent lead compound, characterized by different mechanisms from anthracyclines and able to overcome drug resistance.

References

[1] Riganti C, Miraglia E, Viarisio D, Costamagna C, Pescarmona G, Ghigo D, Bosia A (2005). Nitric oxide reverts the resistance to doxorubicin in human colon cancer cells by inhibiting the drug efflux. Cancer Res 65: 516-525.

[2] Chegaev K, Riganti C, Lazzarato L, Rolando B, Guglielmo S, Campia I, Fruttero R, Bosia A, Gasco A (2011). Nitric oxide donor – doxorubicin conjugates accumulate into doxorubicin resistant human colon cancer cells inducing cytotoxicity. ACS Med Chem Lett 2: 494-497.