Trisenox disrupts MDM2-DAXX-HAUSP complex and activates p53, cell cycle regulation and apoptosis in acute leukemia cells

Abstract

Trisenox (TX) has been used in the treatment of both de novo and relapsed acute promyelocytic leukemia (APL) patients. Using in vitro APL cell lines model in this research, we report on a new target of TX action through disruption of MDM2-DAXX-HAUSP complex, degradation of MDM2, and activation of p53 expression. TX-induced stress signal was transmitted by protein kinase (ATM & ATR) and phosphorylation of its downstream targets CHK1, CHK2, ATM, and ATR, respectively at the Ser 345, Thr68, Ser1981 and Ser 428 residues involved in complex disruption and p53 up-regulation. TX-activated p53 led to cell cycle arrest and apoptosis in APL cells. Our results showed that TX inhibited cell proliferation, disrupted complex molecules expression and association in APL cells. Our functional studies indicated that TX-induced down-regulation of complex molecules expression was mostly neutralized in both p53 knockdown NB4 cells and nutilin-3 treated KG1a cells. Hence our findings provide a functional evidence of TX action on cell cycle regulation and apoptosis in APL cells. This novel target of TX activity may be useful for designing new APL drugs.

Keywords: APL; MDM2-DAXX-HAUSP complex; apoptosis; p53; trisenox.

Figure 1. TX inhibits proliferation of APL cells

APL cell lines were treated with different concentrations of TX for 24 hours and 21 hours with tritium labeled thymidine. After incubation, cells were harvested, counted, and ³H-methyl thymidine incorporation expressed as cpm/dish. Highly statistically significant decreases (p < 0.01) in cell proliferation were observed in HL-60 (A), KG-1a (B) and KG-1a (C).

Figure 2. TX activates p53 leading to cell cycle arrest

TX activated p53 and p21 through down regulation of expression of cyclins, cdks, and ki67 in HL-60 (A), KG-1a (C), NB4 (D), and U937 (E). TX induced cell cycle arrest at S and G2/M phase prominently (F) in APL cells.

Figure 3. TX induces intrinsic pathway of apoptosis

TX upregulated the expression levels of Bax, Cytochrome C, PARP, and caspase 3 through down regulation of Bcl-2 expression in HL-60 (A), KG-1a (B), and NB4 (C). Mitochondria were isolated from samples and mitochondrial membrane potentials [MMPs] were measured by spectrofluoremetry. TX was reduced the MMPs in HL-60 (D), KG-1a (E) and NB4 (F) cells.

Figure 4. TX disrupts complex molecules expression and association

TX effect on both expression and association of complex molecules in APL cell lines was analyzed by Western blotting and immunoprecipitation (IP) method. TX disrupted/changed the expression level of complex molecules in a concentration dependent manner and changed association level of complex molecules in HL-60 (A and C) and NB4 (B and D) cells.

Figure 5. Functional mechanism of TX in complex disruption

TX induced the phosphorylation of CHK1, CHK2, ATM, and ATR at different residues and changed the expression levels of ATM and ATR in both KG1a cells and HL-60 cells (A, B). Nutilin-3, an antagonist of MDM2 neutralized TX repression of MDM2 without effecting DAXX and HAUSP expression (C). TX treatment did not significantly reduce the expression of complex molecules in p53-knockout NB4 cells (D).

Figure 6. Summary of TX new target of action in APL cells

TX enters in APL cells, and induces oxidative stress and DNA damage, leading to a reduced expression of complex molecules, MDM2 degradation through stress signal coordinated by protein kinases (ATM & ATR) and their downstream targets CHK1 and CHK2 residues, and subsequent activation of p53. TX-activated p53 induces cell cycle arrest in APL cells and forces them to undergo apoptosis.