A copper chelate of thiosemicarbazone NSC 689534 induces oxidative/ER stress and inhibits tumor growth in vitro and in vivo

Abstract

In this study, a Cu(2+) chelate of the novel thiosemicarbazone NSC 689534 was evaluated for in vitro and in vivo anti-cancer activity. Results demonstrated that NSC 689534 activity (low micromolar range) was enhanced four- to fivefold by copper chelation and completely attenuated by iron. Importantly, once formed, the NSC 689534/Cu(2+) complex retained activity in the presence of additional iron or iron-containing biomolecules. NSC 689534/Cu(2+) mediated its effects primarily through the induction of ROS, with depletion of cellular glutathione and protein thiols. Pretreatment of cells with the antioxidant N-acetyl-l-cysteine impaired activity, whereas NSC 689534/Cu(2+) effectively synergized with the glutathione biosynthesis inhibitor buthionine sulfoximine. Microarray analysis of NSC 689534/Cu(2+)-treated cells highlighted activation of pathways involved in oxidative and ER stress/UPR, autophagy, and metal metabolism. Further scrutiny of the role of ER stress and autophagy indicated that NSC 689534/Cu(2+)-induced cell death was ER-stress dependent and autophagy independent. Last, NSC 689534/Cu(2+) was shown to have activity in an HL60 xenograft model. These data suggest that NSC 689534/Cu(2+) is a potent oxidative stress inducer worthy of further preclinical investigation.

Fig. 1

Copper chelation enhances the anti-proliferative activity of NSC 689534. A) Structure of NSC 689534. B) HL60 and PC3 cells were treated with the indicated concentrations of NSC 689534 (68) or NSC 689534 pre-incubated with 2 molar equivalents of Fe(II), Fe(III), or Cu(II) for 48 hours before viability was determined. C) Plot of the IC₅₀ values as % of control obtained for dose response curves. IC₅₀s were determined for NSC 689534 containing 0, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0 and 4.0 mols of copper per mol thiosemicarbazone. The IC₅₀s are plotted as a % of no copper (control) versus molar ratio of Cu²⁺ to NCS 689534. D) HL60 cells were treated with increasing concentrations of NSC 689534 alone or in combination with 2.5µM hemoglobin (Hb), 100 µg/ml transferrin (Tf), or 10 µM hemin (Hm) (left panel) or with increasing concentrations of NSC 689534/Cu²⁺ alone or in combination with 20 µM Fe (II), 20 µM Fe (III), or 10 µM Hemin (Hm) (right panel) for 48 hours and viability determined. IC₅₀ values obtained are plotted versus each treatment condition. 68[Cu] refers to NSC 689534 in the presence of a 2 fold molar ratio of Cu²⁺. Assays were performed at least twice with triplicate determinations for each point and the data pooled. The SEM of six determinations is shown when they are greater than the symbol or bar

Fig. 2

NSC 689534/Cu²⁺ induces apoptosis in HL60 and mainly necrosis in PC3 cells. (A) HL60 and PC3 cells were treated for 48 h with either NSC 689534 or NSC 689534/Cu²⁺ at the indicated concentrations. This was followed by fixation in 70% ethanol, labeling with propidium iodide (PI) and analysis by flow cytometry for cell cycle perturbations. *HL60 cells shown were treated with a 10 fold lower dose than indicated on axis (0.1, 0.25 and 0.5 µM). B) Analysis of apoptosis induction for HL60 and PC3 cells treated with NSC 689534 or NSC 689534/Cu²⁺ by flow cytometry using Annexin V/PI staining. (C) HL60 or PC3 cells were treated with either DMSO (vehicle) or the indicated concentration of NSC 689534/Cu²⁺, and caspase cleavage assessed after 24 or 48 hours, respectively. The SEM is shown when greater than the bar. (D) HL60 and PC3 cells were treated with either vehicle or the indicated concentration of NSC 689534 or NSC 689534/Cu²⁺ for 24 or 48 hrs, respectively and cleavage of PARP-1 assessed using immunoblotting as described in Materials and Methods. The arrow denotes the predominant 89 kDa PARP cleavage product. Results are representative of three separate experiments.

Fig. 3

NSC 689534/Cu²⁺ anti-cancer activity is ROS-mediated. A) PC3 cells were treated with 2.5 µM NSC 689534 or 2.5 µM NSC 689534/Cu²⁺ for 24 hours and then analyzed for expression of HSPA6 and HMOX1 by immunocytochemistry and immunoblotting. B) Cell lines were labeled for 2h with the fluorescent ROS sensor DCFDA and then treated for 2h with the indicated concentration of NSC 689534 or NSC 689534/Cu²⁺ followed by analysis by flow cytometry (FL1 channel). DCF fluorescence of the indicated treatment (solid profile) is shown compared to vehicle control (open profile). Inset values indicate fold increase in DCFDA fluorescence versus control. C) HL60 cells were treated with the indicated concentration of NSC 689534/Cu²⁺ alone or in the presence of 12 mM L-NAC (top panel) or 1µM BSO (bottom panel) for 24 h, followed by viability determination. Results are representative of three separate experiments. D) HL60 (black bar) or PC3 cells (open bar) were incubated for 2 or 16 hrs with vehicle, NSC 689534, or NSC 689534/Cu²⁺. Following this, GSH and total cellular thiol levels were determined as described in Materials and Methods and results plotted as % control.

Fig. 4

NSC 689534/Cu²⁺ treatment is associated with macroautophagy and ER-stress induction. A) (Upper panel) PC3 cells were plated onto coverslips and treated with DMSO, 2.5 µM NSC 689534 or 2.5 µM NSC 689534/Cu²⁺ for 24 hours followed by labeling with acridine orange for 2 hours and visualization by microscopy. (Lower panel) A second set of samples were also prepared and analyzed by flow cytometry (treated samples in gray, control samples in black). B) PC3 cells on glass coverslips were treated with either vehicle (DMSO), 2.5 µM of NSC 689534 or NSC 689534/Cu²⁺, or a combination of NSC 689534/Cu²⁺ and 200 µM wortmannin for 48 hours, fixed and stained with an anti-LC3 antibody and visualized by microscopy at 60× magnification. C) Cells were treated with either 2.5 µM NSC 689534/Cu²⁺, 25 nM of thapsigargin, 50 µM of rapamycin, or serum starved either alone or in combination with 10 µM of Pepstatin A and E64d for 48 hours, then harvested and immunoblotted for conversion of LC3-I (upper band) to LC3-II (lower band). Densitometry (lower panel) was included to assess fold expression of LC3-II in the Pepstatin A/E64d treated versus drug alone. D) (upper panel) PC3 cells were treated with 1, 2 or 5 µM NSC 689534 or NSC 689534/Cu²⁺ for 24 hours, harvested and immunoblotted for expression of the ER-stress markers GRP78 and CHOP. Lysate from thapsigargin (T) treated PC3 cells was included as a positive control. V refers to vehicle treated cells (DMSO) and C indicates treatment with 100µM Cu²⁺. (lower panel) PC3 cells were treated with the indicated concentration of NSC 689534/Cu2+ alone or in combination with 1 µg/ml cycloheximide for 48 h. Cell viability was determined using crystal violet staining as described in Materials and Methods. Each value represents the mean +/− SEM of three independent measurements. The experiment was performed twice.

Fig. 5

NSC 689534/Cu²⁺ has in vivo activity in an HL60 xenograft model. (A) Graph of effects of once or twice daily doses of 689534 or 689534[Cu] on growth rates in an HL60 xenograft. ** represents statistically significant differences from control at p < 0.05. B) Photograph of representative animals at termination of the experiment.