Organometallic iron(III)-salophene exerts cytotoxic properties in neuroblastoma cells via MAPK activation and ROS generation - PubMed (original) (raw)

Organometallic iron(III)-salophene exerts cytotoxic properties in neuroblastoma cells via MAPK activation and ROS generation

Kyu Kwang Kim et al. PLoS One. 2011.

Abstract

The objective of the present study was to investigate the specific effects of Iron(III)-salophene (Fe-SP) on viability, morphology, proliferation, cell cycle progression, ROS generation and pro-apoptotic MAPK activation in neuroblastoma (NB) cells. A NCI-DTP cancer screen revealed that Fe-SP displayed high toxicity against cell lines of different tumor origin but not tumor type-specificity. In a viability screen Fe-SP exhibited high cytotoxicity against all three NB cell lines tested. The compound caused cell cycle arrest in G1 phase, suppression of cells progressing through S phase, morphological changes, disruption of the mitochondrial membrane depolarization potential, induction of apoptotic markers as well as p38 and JNK MAPK activation, DNA degradation, and elevated generation of reactive oxygen species (ROS) in SMS-KCNR NB cells. In contrast to Fe-SP, non-complexed salophene or Cu(II)-SP did not raise ROS levels in NB or SKOV-3 ovarian cancer control cells. Cytotoxicity of Fe-SP and activation of caspase-3, -7, PARP, pro-apoptotic p38 and JNK MAPK could be prevented by co-treatment with antioxidants suggesting ROS generation is the primary mechanism of cytotoxic action. We report here that Fe-SP is a potent growth-suppressing and cytotoxic agent for in vitro NB cell lines and, due to its high tolerance in previous animal toxicity studies, a potential therapeutic drug to treat NB tumors in vivo.

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Conflict of interest statement

Competing Interests: There is a pending patent application that is related to this manuscript (for RKS, RMS, LB). This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1

Figure 1. Comparative analysis of the cytotoxic effect of Fe-SP on NB and other cancer cell lines.

(A) Viability of NB cell lines upon Fe-SP treatment. The cytotoxic effect of Fe-SP (0–3 µM) on human NB cell lines (SK-N-SH, SH-SY5Y, SMS-KCNR) was compared to a human cancer cell line of different origin (PC-3) and endothelial cells (HUVEC). Treatment with SP served as a negative control. The MTS viability assay was carried out as described (Materials and Methods). Data are expressed as the mean of the triplicate determinations (X±SD) of a representative experiment in % cell viability of untreated cells [100%]. (B,C) Differential effect of Fe-SP on cell growth in a NCI60 cancer cell line screen. Fe-SP effects were screened in a NCI60 cell line growth assay (

http://dtp.nci.nih.gov/screening.html

). Cells were treated in 96 well plates and cell growth of the TCA fixed treated and untreated cells assessed after 48 h.

Figure 2

Figure 2. Morphology changes, mitochondrial membrane depolarization potential, apoptotic and necrotic effects and DNA fragmentation in NB cells after Fe-SP treatment.

(A) Morphological appearance/DAPI staining. SMS-KCNR NB cells were treated for 24 h with Fe-SP at a concentration of 0.4 µM before microscopic analysis by DIC or fluorescence analysis after chromatin staining (DAPI) as described (Materials and Methods). Images obtained from a representative experiment are shown. Bar = 10 µm. (B) Mitochondrial membrane depolarization potential (ΔΨm) analysis. SMS-KCNR NB cells were treated for 24 h with 0.8 µM Fe-SP or SP control, fixed and stained with PI and Rhodamine 123 as described (Materials and Methods). Fluorescence of the single cell population was measured by flow cytometry (right panel) and the transmembrane depolarization potential of the single cell populations plotted. Intact cells = Q4, loss of ΔΨm = Q3, ruptured cell membrane (and loss of ΔΨm) = Q1 and Q2. (C) Apoptotic and necrotic cell population. SMS-KCNR NB cells were treated with 0.2 or 0.8 µM Fe-SP or for 24 h and floating and attached cells collected and combined. The quantification of apoptotic cells (Annexin V plasma membrane staining) and necrotic cells (7-AAD DNA staining) of SKOV-3 cells was carried out by flow cytometry as described (Materials and Methods). Viable cells = Q3, necrosis = Q1, early apoptosis = Q4, late apoptosis/necrosis = Q2. (D) Analysis of DNA fragmentation in a TUNEL Assay. SMS-KCNR NB cells were treated with Fe-SP (0.8, 1.6 µM) for 24 h. A TUNEL assay was carried out by co-staining with fluorescein-12-dUTP (labeling of DNA nicks in apoptotic cells) and of chromatin with propidium iodide (Materials and Methods). During fluorescent microscopy, representative images were taken, apoptotic stain (green) and nuclear stain (red) overlaid. TUNEL positive nuclei due to DNA fragmentation appear as yellow areas. Bar = 10 µM.

Figure 3

Figure 3. Fe-SP inhibits proliferation of NB cells.

(A) BrdU incorporation assay. SMS-KCNR NB cells were treated with various concentrations (0.1–3 µM) of Fe-SP for 24 h. A colorimetric assay (based on BrdU incorporation) was carried out as described (Materials and Methods). Data are expressed as the mean of the triplicate determinations (X±SD) in % of absorbance by triplicate samples of untreated cells [ = 100%]. (B,C) Fe-SP blocks cell cycle progression in G1 phase. SMS-KCNR NB cells were treated with 0.2, 0.4 and 0.8 µM Fe-SP for 24. Cell cycle analysis by FACS based on propidium-iodide intercalation into the cellular chromatin was carried out as described (Materials and Methods). Data are presented as (A) relative fluorescence intensity in a 2-dimensional FACS profile (ModFit LT software; black lines = data line and model fit line of entire population; shaded areas = model components/subpopulations of G0/G1, S, G2/M, apoptotic cells or in (B) a table. Standardized gating was used for all samples. Ten thousand events were analyzed for each sample.

Figure 4

Figure 4. Effect of Fe-SP or Cu-SP on viability, ROS generation, and induction of apoptic markers and MAPK expression.

(A) Cytotoxicity of Fe-SP or Cu-SP in NB cells. The viability assay was carried out after 24 h treatment of SMS-KCNR NB cells with 0–3 µM of Fe-SP, Cu-SP or respective metal salts. Experiments were performed in triplicates; data are expressed as the mean of the triplicate determinations (X±SD) of a representative experiment in % cell viability of untreated cells [ = 100%]. (B) Generation of intracellular Reactive Oxygen Species (ROS) after Fe-SP treatment. Generation of intracellular ROS following SMS-KCNR NB cells (large panels) or SKOV-3 OC control cells (small panels) after treatment for 4 h with 1.6 µM of Fe-SP or Cu-SP was measured by flow cytometry (see Materials and Methods). Data are presented as relative fluorescence intensity in a 2-dimensional FACS profile. Standardized gating was used for all samples. (C) Fe-SP cytotoxicity is blocked by antioxidant ascorbic acid. Cells were treated with with1.6 µM Fe-SP alone or in combination with antioxidant ascorbic acid. Viability of SMS-KCNR NB cells is presented as bar diagram and in percentages and of SKOV-3 OC control cells in percentages (insert). (D) Inhibiton of ROS generation in NB cells after Fe-SP treatment. Generation of intracellular ROS in SMS-KCNR NB was measured after treatment with 1.6 µM Fe-SP alone or in combination with antioxidant ascorbic acid (200 µM). (E) Expression of apoptotic markers and MAPK in NB cells after Fe-SP treatment with and without inhibiton of ROS generation. SMS-KCNR NB cells were treated with 0.8 µM Fe-SP in the absence (3, 6, 14, 24 h treatment, left panel) or presence of ascorbic acid (200 µM, 24 h treatment, right panel). Immunoblotting was carried out with primary antibodies against PARP-1, caspase-3, -7, pro-survival marker XIAP (inhibitor of effector caspases), and pro-apoptotic MAPK SAP/JNK or p38 in the active (phosphorylated) or inactive form. As an internal standard for equal loading blots were probed with an anti-GAPDH.

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