Nano-fenretinide demonstrates remarkable activity in acute promyeloid leukemia cells

Abstract

Acute promyelocytic leukemia (APL) is characterized by rearrangements of the retinoic acid receptor, RARα, which makes all-trans retinoic acid (ATRA) highly effective in the treatment of this disease, inducing promyelocytes differentiation. Current therapy, based on ATRA in combination with arsenic trioxide, with or without chemotherapy, provides high rates of event-free survival and overall survival. However, a decline in the drug activity, due to increased ATRA metabolism and RARα mutations, is often observed over long-term treatments. Furthermore, dedifferentiation can occur providing relapse of the disease. In this study we evaluated fenretinide, a semisynthetic ATRA derivative, encapsulated in nanomicelles (nano-fenretinide) as an alternative treatment to ATRA in APL. Nano-fenretinide was prepared by fenretinide encapsulation in a self-assembling phospholipid mixture. Physico-chemical characterization was carried out by dinamic light scattering and spectrophotometry. The biological activity was evaluated by MTT assay, flow cytometry and confocal laser-scanning fluorescence microscopy. Nano-fenretinide induced apoptosis in acute promyelocytic leukemia cells (HL60) by an early increase of reactive oxygen species and a mitochondrial potential decrease. The fenretinide concentration that induced 90-100% decrease in cell viability was about 2.0 µM at 24 h, a concentration easily achievable in vivo when nano-fenretinide is administered by oral or intravenous route, as demonstrated in previous studies. Nano-fenretinide was effective, albeit at slightly higher concentrations, also in doxorubicin-resistant HL60 cells, while a comparison with TK6 lymphoblasts indicated a lack of toxicity on normal cells. The results indicate that nano-fenretinide can be considered an alternative therapy to ATRA in acute promyelocytic leukemia when decreased efficacy, resistance or recurrence of disease emerge after protracted treatments with ATRA.

Keywords: All-trans retinoic acid; 4-Hydroxyphenyl retinamide; APL; Antitumor activity; HL60; HL60R; Mitochondrial membrane potential; Reactive oxygen species; TK6.

Figure 1

Schematic representation of nanomicelles entrapping FEN or ATRA. The nanomicelles were obtained by an amphiphilic mixture based on phospholipids, glyceryl tributyrate and 2-hydroxypropyl beta cyclodextrin.

Figure 2

Relative viability of HL60 cells treated with increasing concentrations of free FEN (dark green bar), NF (light green bar), free ATRA (dark blue bar) or NA (light blue bar) for 24, 48 and 72 h. Viability was extimated by MTT assay and expressed as percentage versus control (100%) (mean ± SD, n = 6).

Figure 3

Cytofluorimetric data. Effect of (a) FEN and NF, (b) ATRA and NA on cell cycle evaluated by cytofluorimetric assay. The percentage of HL60 cells in the different phases are reported for different treatment concentrations after 72 h. Dark blue, dim blue and light blue bars indicate the cell percentages in the G0/G1, S, G2/M phase respectively. (c) ROS production in HL60 treated with different concentrations of free FEN or NF for 4 h. Increment in ROS production was evaluated by DCF assay and expressed as fold increase with respect to control taken as 1 (mean ± SD, n = 6) (*p < 0.05). (d) Mitochondrial potential of HL60 cells treated with free FEN or NF for 24 h, 48 h, 72 h. Dark green and light green bars indicate cell percentage with high potential and low potential respectively (mean ± SD, n = 6) (d).

Figure 4

Confocal microscopy to evaluate the nuclear morphology of control HL60 cells (CTR) and treated cells for 24 h with free FEN, NF, ATRA and NA. Apoptotic nuclei are highlighted by arrows. Images were recorded at 40 × magnification, bar = 10 µm.

Figure 5

Relative viability of HL60 R cells treated with increasing concentrations of free FEN and NF for 24 h, 48 h and 72 h. Viability was evaluated by MTT assay and expressed as percentage versus control (100%) (mean ± SD, n = 6).

Figure 6

Confocal microscopy of HL60, HL60R, TK6 cells after 4 h treatment with free FEN (FEN) or NF at 8 µM at 40 × magnification, bar = 10 µm.