NB7M effect on cell growth in NCI₆₀ cancer cell line screen. (A) NCI₆₀ cell line in vitro screening at 10 μM NB7M. Cells (see Supplementary Information) were treated in 96-well plates with 10 μM of NB7M or vehicle and cell viability of the TCA fixed treated and untreated cells assessed after 48 h with sulphorhodamine-B (SRB) solution and absorbance read at 515 nM. (B) Dose-dependent effect of NB7M on ovarian cancer cells in NCI₆₀ screen (see (A) and Supplementary Information).

Expression of prosurvival markers and MAPKs in SKOV-3 following NB7M treatment; effect of MAPK inactivation on cell viability. (A) Activation of MAPKs. SKOV-3 cells were treated with 2 μM of NB7M for 6, 18 or 36 h. Analysis of the expression of proteins in the lysates of treated and untreated cells by PAGE and western blot analysis was carried out as described (Material and Methods), using primary antibodies against pro- and activated/phosphorylated (P-) SAP/JNK, p38 and ERK1/2. As an internal standard for equal loading, the blots were probed with an anti-β-actin antibody. (B) Effect of p38 MAPK inactivation on cell viability. SKOV-3 cells were pre-incubated with specific inhibitors (40 μM) against p38 MAPK for 2 h and treated with NB7M (0, 1 or 2 μM) in the continued presence of the inhibitors for an additional 48 h. The MTS viability assay was carried out as described (Materials and Methods). Experiments were performed in triplicates; data are expressed as the mean of the triplicate determinations (X±s.d.) of a representative experiment in % cell viability of samples with untreated cells. (C) Effect of NB7M on DNA-pK and Axl. SKOV-3 cells were treated with 2 μM of NB7M for 0, 6, 18 or 36 h. Analysis of the expression of proteins in the lysates of treated and untreated cells by PAGE/western blot analysis was carried out using primary antibodies against DNA-pK and Axl proteins. (D) Inactivation of survival signalling proteins and transcription factor proteins: SKOV-3 cells were treated with 2 μM of NB7M for 6, 18 or 36 h. Analyses of the expression of proteins in the lysates of treated and untreated cells by PAGE/western blotting were carried out using primary antibodies against pro- and activated/phosphorylated PI-3K, STAT-3, IKKα or NF-κB.

Design concept and cytotoxicity of indolyl ethyl isothiocyanate NB7M. (A) Synthesis and structure of 7Me-IEITC derivative NB7M. (see Materials and Methods; Supplementary Information). (B) Comparative analysis of the cytotoxic effect of NB7M in various human cancer and control cell lines. SKOV-3, OVCAR-3 (ovarian epithelial adenocarcinomas), PC-3 (prostate adenocarcinoma), BxPC-3 (pancreatic adenocarcinoma), A-431 (epidermoid carcinoma), HeLa (endometrial), TCL-1 (trophoblasts) and HTR-8 (first-trimester cytotrophoblasts) human cell lines were treated with various concentrations (0–20 μM) of NB7M for 48 h. The MTS viability assay was carried out as described (Materials and Methods). Experiments were performed in triplicates; data are expressed as the mean of the triplicate determinations (X±s.d.) of a representative experiment in % cell viability of samples with untreated cells (100%).

NB7M causes apoptosis in SKOV-3 platinum-resistant ovarian cancer cells. (A) Membrane depolarisation analysis after NB7M treatment. SKOV-3 cells were treated for 12 or 24 h with 2 μM NB7M, fixed and stained with DiOC6(3) as described (Materials and Methods). Fluorescence of the single-cell population was measured by flow cytometry and the transmembrane depolarisation potential of the single-cell populations plotted. Ten thousand cells were analysed in each sample. (B) Morphological changes following NB7M treatment. SKOV-3 cells were treated for 24 h with 2 μM NB7M, fixed and stained with 4′-6-diamidino-2-phenylindole (DAPI) as described (Materials and Methods) before mounting. Microscopy was carried out (Nikon Eclipse TE2000-E inverted microscope, × 20 objective), and representative images were taken. Bar=10 μM. (C) Caspase activation following NB7M treatment. SKOV-3 cells were treated with 2 μM of NB7M for 1, 6, 18 or 36 h. Analysis of the expression of proteins in the lysates of treated and untreated cells was carried out by PAGE and western blot analysis as described (Material and Methods). Primary antibodies against activated caspases-3, -8, -9, and inactivated/cleaved PARP-1 were used. As an internal standard for equal loading, the blots were probed with an anti-β-actin antibody. (D) Effect of caspase inhibitors on SKOV-3 viability following NB7M treatment. SKOV-3 cells were pre-incubated with specific inhibitor (40 μM) against caspase-3 for 2 h and treated with NB7M (0, 1 or 2 μM) in the continued presence of the inhibitors (40 μM) for an additional 48 h. The MTS viability assay was carried out as described (Materials and Methods). Experiments were performed in triplicates; data are expressed as the mean of the triplicate determinations (X±s.d.) of a representative experiment in % cell viability of samples with untreated cells. (E) TUNEL assay. SKOV-3 cells were treated with either 2 μM of NB7M or 25 μM of actinomycin D for 48 h. Labelling of DNA nicks with fluorescein-12-dUTP and chromatin counterstaining with propidium iodide was carried out as described (Materials and Methods). Representative images were taken, apoptotic stain (FL-dUTP, green) and nuclear stain (Pi, red) overlaid; TUNEL-positive nuclei because of DNA fragmentation appear as yellow areas. Bar=10 μM.

Effect of NB7M on proliferation and cell-cycle progression of SKOV-3 cells. (A) Cell Proliferation/BrdU incorporation. SKOV-3 cells were treated with NB7M (0, 0.187, 0.375, 0.75, 1.5, 3 μM) for 48 h. The proliferation assay was carried out as described (Materials and Methods). Experiments were performed in triplicates; data are expressed as the mean of the triplicate determinations (X±s.d.) in % cell proliferation of untreated cells. (B and C) Cell-cycle analysis by FACS. SKOV-3 cells were treated with various NB7M (250 or 500 nM) for 6, 18 or 36 h. Cell-cycle analysis of treated and untreated cells was carried out as described (Materials and Methods). Data are presented as the relative fluorescence intensity of cell subpopulations in a bar chart (B) or table (C). (D) Expression of cyclin-dependent kinase inhibitors in NB7M-treated SKOV-3 cells. Expression of p21 and p27 inhibitors in NB7M and vehicle-treated SKOV-3 cells were analysed by western blotting of lysates and probed with the appropriate primary and secondary antibodies.