Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia

Abstract

To enhance therapeutic efficacy and reduce adverse effects, practitioners of traditional Chinese medicine (TCM) prescribe a combination of plant species/minerals, called formulae, based on clinical experience. Nearly 100,000 formulae have been recorded, but the working mechanisms of most remain unknown. In trying to address the possible beneficial effects of formulae with current biomedical approaches, we use Realgar-Indigo naturalis formula (RIF), which has been proven to be very effective in treating human acute promyelocytic leukemia (APL) as a model. The main components of RIF are realgar, Indigo naturalis, and Salvia miltiorrhiza, with tetraarsenic tetrasulfide (A), indirubin (I), and tanshinone IIA (T) as major active ingredients, respectively. Here, we report that the ATI combination yields synergy in the treatment of a murine APL model in vivo and in the induction of APL cell differentiation in vitro. ATI causes intensified ubiquitination/degradation of promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) oncoprotein, stronger reprogramming of myeloid differentiation regulators, and enhanced G(1)/G(0) arrest in APL cells through hitting multiple targets compared with the effects of mono- or biagents. Furthermore, ATI intensifies the expression of Aquaglyceroporin 9 and facilitates the transportation of A into APL cells, which in turn enhances A-mediated PML-RARalpha degradation and therapeutic efficacy. Our data also indicate A as the principal component of the formula, whereas T and I serve as adjuvant ingredients. We therefore suggest that dissecting the mode of action of clinically effective formulae at the molecular, cellular, and organism levels may be a good strategy in exploring the value of traditional medicine.

Fig. 1.

In vivo therapeutic efficacies of ATI on an APL murine model. (A) ATI significantly prolongs the life span of mice bearing PML-RARα-positive leukemic cells compared with those treated with mono- or bitreatment of A, T, and I (P < 0.001). (B) ATI does not cause loss of body weight, suggesting that ATI might probably not cause severe toxicity. (C) Treatment with ATI results in cell maturation revealed by an accumulation of Gr-1 and Mac-1-positive cells in BM and peripheral blood (PB). (Dosage of agents used: A, 10 mg/kg; T, 50 mg/kg; I, 50 mg/kg. A is administrated by i.v. injection, whereas T and I are given orally.)

Fig. 2.

ATI induces terminal differentiation of NB4, NB4-R2, and primary leukemic cells harvested from APL patients. (A) Morphological examination of APL cells treated with ATI. (B) Detection of NBT reduction activity and CD11b/CD14 expression of cells treated with ATI. (C) Expression of CD11b in primary leukemic cells treated with ATI in vitro. Concentration of agents used: A, 0.375 μM; T, 0.75 μM; I, 0.75 μM. (D) Combination of A, T, and I exerts synergic effects on NB4 and NB4-R2 cells, as reflected by the median-effect method of Chou and Talalay (23).

Fig. 3.

ATI triggers degradation of PML-RARα oncoprotein and reprogramming of differentiation regulators. (A) Enhanced degradation of PML-RARα is seen in NB4 (Left) and NB4-R2 (Right) cells treated with the ATI combination compared with those treated with mono- or bitreatment of A, T, and I. (B) TI intensifies matrix transfer from the nucleoplasm and ubiquitination of PML-RARα caused by A. Cells are pretreated with MG-132 for 1 h and then with the protocols indicated for 6 h. Western blot is performed as described (24). (C) Effects of ATI on differentiation regulators of NB4 cells at the protein level. (D) Effects of the ATI combination on differentiation regulators at the mRNA level, assessed by semiquantitative RT-PCR. (E) ATI treatment up-regulates the expression of RARβ2 at the mRNA level; this might be due to the dissociation of RARβ2 from HDAC1 upon ATI treatment.

Fig. 4.

Effects of ATI on cell cycle progression and cell cycle modulators. (A) ATI treatment leads to the G₁/G₀ arrest of NB4 and NB4-R2 cells. (B) Effects of the ATI combination on important cell cycle regulators in NB4 (Left) and NB4-R2 (Right) cells. (C) Down-regulation of CDK2 by ATI treatment on NB4 (Upper) and NB4-R2 (Lower) cells results in decrease of phosphorylated H1. Cells are treated with A, T, I, and the combined drugs for 24 h. CDK2 is immunoprecipitated (IP) from cell lysates and assayed for kinase activity using Histone H1 as a substrate. The efficiency of IP is confirmed and quantitated by Western blot analysis, and CDK2 at each treatment group is adjusted to the same level.

Fig. 5.

T and I facilitate arsenic uptake by malignant promyelocytes via up-regulation of AQP9. (A) Combinatory use of T and/or I increases intracellular arsenic concentration ([As]_i) in NB4 cells (*, P = 0.002; #, P = 0.004; **, P = 0.002). (B and C) T and/or I in combination with A up-regulate AQP9 expression at both the mRNA (B) and protein (C) levels (Coom, stained with Coomassie blue). (D) Immunofluorescence analysis of AQP9 expression in NB4 cells treated with the ATI combination. (E) AQP9-specific siRNA down-regulates AQP9 expression by approximately one-half in NB4 cells (NB4-AQP9-Si) compared with cells treated with nonsilencing siRNA control (NC). (F) Treatment with AQP9-specific siRNA reduces [As]_i in NB4-AQP9-Si cells upon ATI compared with NB4-NC cells (*, P = 0.018; #, P = 0.037; **, P = 0.009). (G) Treatment with AQP9-specific siRNA inhibits differentiation of NB4 cells on ATI, revealed by analysis of CD11b expression.