Nitric oxide inhibits ATPase activity and induces resistance to topoisomerase II-poisons in human MCF-7 breast tumor cells

Abstract

Background: Topoisomerase poisons are important drugs for the management of human malignancies. Nitric oxide (^•NO), a physiological signaling molecule, induces nitrosylation (or nitrosation) of many cellular proteins containing cysteine thiol groups, altering their cellular functions. Topoisomerases contain several thiol groups which are important for their activity and are also targets for nitrosation by nitric oxide.

Methods: Here, we have evaluated the roles of ^• NO/ ^• NO-derived species in the stability and activity of topo II (α and β) both in vitro and in human MCF-7 breast tumor cells. Furthermore, we have examined the effects of ^• NO on the ATPase activity of topo II.

Results: Treatment of purified topo IIα and β with propylamine propylamine nonoate (PPNO), an NO donor, resulted in inhibition of the catalytic activity of topo II. Furthermore, PPNO significantly inhibited topo II-dependent ATP hydrolysis. ^• NO-induced inhibition of these topo II (α and β) functions resulted in a decrease in cleavable complex formation in MCF-7 cells in the presence of m-AMSA and XK469 and induced significant resistance to both drugs in MCF-7 cells.

Conclusion: PPNO treatment resulted in the nitrosation of the topo II protein in MCF-7 cancer cells and inhibited both catalytic-, and ATPase activities of topo II. Furthermore, PPNO significantly affected the DNA damage and cytotoxicity of m-AMSA and XK469 in MCF-7 tumor cells.

General significance: As tumors express nitric oxide synthase and generate ^• NO, inhibition of topo II functions by ^• NO/ ^• NO-derived species could render tumors resistant to certain topo II-poisons in the clinic.

Keywords: ATPase inhibition; Nitric oxide; Resistance; Topoisomerase; XK469; m-AMSA.

Fig. 1

Effect of PPNO (100 µM) on topoisomerase II-induced decatenation of kDNA. The decatenation of topo II α (A) was carried out using kDNA as described in the Section 3. Lane 1, control kDNA; Lane 2, with 100 µM PPNO; Lane 3 with topo IIα 2 U; lane 4, with topo IIα 2 U and 25 µM PPNO; Lane 5, 50 µM PPNO and Lane 6, 100 µM PPNO. (B) Decatenation induced by topo IIβ Lane 1, control kDNA; lane 2, with 100 µM PPNO; Lane 3, topo IIβ 2 U; Lane 4 with topo IIα 2 and 25 µM PPNO; Lane 5, with 50 µM PPNO and Lane 6, with 100 µM PPNO. NC, nicked open circular kDNA; CC, closed circular kDNA; ORI, origin. (C) and (D) Quantifications of DNA following decatenation with purified topoisomerases IIα and β, respectively.

Fig. 2

Effects of PPNO on topoisomerase-induced ATP hydrolysis. ATP hydrolysis was assayed calorimetrically using malachite green as described in the Section 3 (A) effects of topo IIα concentrations on ATP hydrolysis; (B) effects of various concentrations of PPNO on topo IIα-induced ATP hydrolysis; (C) effects of topo IIβ concentrations of ATP hydrolysis; (D) effects of various concentrations of PPNO on topo IIβ-induced ATP hydrolysis.

Fig. 3

(A) Effects of PPNO on DNA damage induced by m-AMSA (2.0 µM) and XK469 (50 µM) in MCF-7 cells as detected by ϒH2AX signals. MCF-7 (400–500,000 cells/ml) were treated with drug in the presence or absence of 100 µM PPNO for 2 h. Cells were preincubated with PPNO in 1% FBS media before adding drugs and incubating mixtures in the complete media for an additional 2 h. The cells were washed with ice-cold PBS and collected, lysed and analyzed by the standard Western blots as described in the Section 3. (B) Quantifications of DNA damage (γ-H2AX band) shown in (A).

Fig. 4

S-Nitrosation of -SH groups of topoisomerase IIα (A) and topoisomerase IIβ (B) in MCF-7 cells by PPNO (100 µM) and GSNO (500 µM). Cells were treated with NO-donors for 18 h as described in the Section 3 and processed for confocal microscopy studies. (C) Effects of ^•NO on topo IIβ protein levels in MCF-7 cells. Cells were treated with 100 µM PPNO for 24 h as described in the Section 3. Cells were collected, lysed and analyzed by Western blots for topo IIβ. (D) Quantifications of topo IIβ protein shown in (C).

Fig. 5

Cytotoxicity of m-AMSA in MCF-7 cells. (A) Cells were seeded in 6-well plates in triplicate and allowed to attach for 18 h. PPNO (100 µM) treatment was carried out in a medium containing 1% FBS without antibiotics for 2 h. m-AMSA alone (□); m-AMSA in the presence of PPNO (■). Formation of cleavage complexes (B) in MCF-7 cells in the presence of m-AMSA (□) and m-AMSA in the presence of PPNO (■). Cells were treated with 100 µM PPNO for 2 h in a medium containing 1% FBS without antibiotics before treating with m-AMSA for 1 h in the complete medium and carrying out SDS-KCl precipitation assays as described in the Section 3. Data represent at least three independent experiments. ^** and * p values≤0.005 and ≤0.05 compared with concentration-matched samples.

Fig. 6

Cytotoxicity of XK469 in MCF-7 cells. (A) Cells were seeded in 6-well plates in triplicates and allowed to attach for 18 h. PPNO (100 µM) treatment was carried out in a medium containing 1% FBS without antibiotics for 2 h as described in the Section 3. XK469 alone (□); XK469 in the presence of PPNO (■). Formation of cleavage complexes (B) in MCF-7 cells by XK469 (□) alone and XK469 in the presence of PPNO (■). Cells were treated with 100 µM PPNO for 2 h in a medium containing 1% FBS without antibiotics before treating with XK469 for 1 h in the complete medium and carrying out SDS-KCl precipitation assays as described in the Methods section. Data represent at least three independent experiments. ^** and ^* p values≤0.005 and ≤0.05 compared with concentration-matched samples.