Realgar-induced apoptosis and differentiation in all-trans retinoic acid (ATRA)-sensitive NB4 and ATRA-resistant MR2 cells

Abstract

Realgar has been used in Western medicine and Chinese traditional medicine since ancient times, and its promising anticancer activity has attracted much attention in recent years, especially for acute promyelocytic leukemia (APL). However, the therapeutic action of realgar treatment for APL remains to be fully elucidated. Cellular cytotoxicity, proliferation, apoptosis and differentiation were comprehensively investigated in realgar-treated cell lines derived from PML-RARα+ APL patient, including the all-trans retinoic acid (ATRA)-sensitive NB4 and ATRA-resistant MR2 cell lines. For analysis of key regulators of apoptosis and differentiation, gene expression profiles were performed in NB4 cells. Realgar was found to induce apoptosis and differentiation in both cell lines, and these effects were exerted simultaneously. Gene expression profiles indicated that genes influenced by realgar treatment were involved in the modulation of signal transduction, translation, transcription, metabolism and the immune response. Given its low toxicity, realgar is a promising alternative reagent for the therapy of APL. Our data contribute to an understanding of the underlying mechanism responsible for the therapeutic effects of realgar in the clinical treatment of APL.

Figure 1

The inhibition rates following realgar treatment in NB₄ and MR₂ cells from the MTT assay. (A) NB₄ and (B) MR₂ were treated with three different concentrations of realgar: 177 μg/l, 355 μg/l and 711 μg/l.

Figure 2

Cell morphology of NB₄ and MR₂ under microscopy. (A) Untreated NB₄ and (C) MR₂ cells with H&E stain by light microscopy. (B) Treated NB₄ and (D) MR₂ cells with H&E stain by light microscopy treated with 177 μg/l realgar for 60 h. (E) Untreated NB₄ and (J) MR₂ cells by transmission electron microscopy. (F-I) Treated NB₄ and (K-N) MR₂ cells by transmission electron microscopy treated with 355 μg/l realgar for 48 and 65 h, respectively.

Figure 3

The results of DNA content analyses following realgar treatment. NB₄ cells (I): untreated (A), 355 μg/l realgar (B), 711 μg/l realgar (C) and 1422 μg/l realgar (D) for 24 h, MR2 cells (J): untreated (E), 177 μg/l realgar for 60 h (F), 355 μg/l realgar for 36 h (G), 355 μg/l realgar for 60 h (H).

Figure 4

The results of NBT reduction assays. (A) Absorbance rates at the wavelength of 568.5 nm in NB₄ and (B) MR₂ cell lines untreated or treated with 177 μg/l, 355 μg/l realgar and 1 μM ATRA for 24 h, 48 h and 72 h, respectively.

Figure 5

The result of CD11b and CD33 expression. The expression rate of CD11b and CD33 in (A) NB₄ and (B) MR₂.

Figure 6

Immunofluorescence analysis of PML-RARα in NB₄. (A) Untreated NB₄ cells as blank control; (B) 1 μM As₂S₂; (C) 1 μM (300 μg/l) realgar; (D) 1 μM As₂O₃; (E) 1 μM As₂S₃ and (F) 1 μM ATRA.