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Figure 7 | BMC Cancer

Figure 7

From: Lipophilic aroylhydrazone chelator HNTMB and its multiple effects on ovarian cancer cells

Figure 7

Effect of iron/ HNTMB and copper/ HNTMB complexes on viability, ROS generation, and induction of apoptosis. (A) Cytotoxicity of HNTMB in SKOV-3 cells when complexed with Fe(II), Fe(III), Cu(I) or Cu(II) Complexes were formed by combining HNTMB and respective metal salts to 1000× [0.4 mM HNTMB; 1.6 or 0.8 mM Fe(II), Fe(III), Cu(I) or Cu(II)] the assay concentration before addition (diluted 1:1000) to the cells (48 h treatment). As controls served cells left untreated, incubated with metal salts (1.6 μM) or non-complexed HNTMB (at IC50 concentration, 0.4 μM). Data are expressed as mean of triplicate determinations (X ± SD) in % cell viability of untreated cells [100%]. (B) Combinational effect of HNTMB iron- and copper-complexes on cell viability in SKOV-3 cells. Group 1: HNTMB complex formation was performed under competing conditions for Fe and Cu-chelation at 1000× the desired assay concentration before addition of this mixture (diluted 1:1000) to the cells (48 h treatment). Group 2:Stock solutions (1000×) of pre-formed complexes of Fe(II) or Fe(III)/- and Cu(II)/HNTNB were individually diluted before simultaneous addition to the cells (48 h treatment). (C) Detection of intracellular ROS in SKOV-3 cells Generation of ROS following treatment with non-complexed or complexed HNTMB was measured by flow cytometry. Data are presented as relative-fluorescence-intensity in a 2-dimensional FACS profile (standardized gating; 10.000 events). (D) Effect of HNTMB complexation with Fe(III) or Cu(II) on the viability of various ovarian cancer cell lines SKOV-3, OVCAR-3 or NUTU-19 cells were treated with 0.4 μM non-complexed or complexed (Cu(II)-upper panel; Fe(III)-lower panel) HNTMB for 48 h and the assay carried out. (E) Effect of HNTMB complexation with Fe(III) or Cu(II) on expression of apoptotic markers in various ovarian cancer cell lines SKOV-3, OVCAR-3 or NUTU-19 cells were treated with 0.4 μM non-complexed or complexed HNTMB for 48 h. Western blot analysis of cell lysates was carried with primary antibodies against PARP-1, caspase-7 and GAPDH (control).

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