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Isothiocyanate NB7M causes selective cytotoxicity, pro-apoptotic signalling and cell-cycle regression in ovarian cancer cells.

Singh RK, Lange TS, Kim KK, Singh AP, Vorsa N, Brard L - Br. J. Cancer (2008)

Bottom Line: In a screen performed by the National Cancer Institute (NCI) (NCI(60) cancer cell-line assay) NB7M (NSC746077) reduced growth up to 100% with an IC(50) between 0.1 and 10 microM depending on the cell line studied.Subcytotoxic doses of NB7M inhibited DNA synthesis, caused G1-phase cell-cycle arrest and upregulated p27 expression.In addition, NB7M acts as a growth/cell-cycle-suppressing agent and may be developed as a potential therapeutic drug to treat ovarian cancer.

View Article: PubMed Central - PubMed

Affiliation: Molecular Therapeutics Laboratory, Program in Women's Oncology, Department of Obstetrics and Gynecology, Women and Infants' Hospital, Brown University, Providence, RI 02905, USA.

ABSTRACT
The present report identifies indole-3-ethyl isothiocyanate NB7M as a potent cytotoxic agent with selective activity against cell lines derived from various tumour types. Ovarian cancer cell lines showed sensitivity to NB7M (60-70% cytotoxicity at 2.5 microM), in contrast to control cells (TCL-1 and HTR-8; IC(50) approximately 15 microM). In a screen performed by the National Cancer Institute (NCI) (NCI(60) cancer cell-line assay) NB7M (NSC746077) reduced growth up to 100% with an IC(50) between 0.1 and 10 microM depending on the cell line studied. Using SKOV-3 ovarian cancer cells as a model, mechanisms of cytotoxicity were analysed. NB7M caused hallmarks of apoptosis such as PARP-1 deactivation, chromatin condensation, DNA nicks, activation of caspases-9, -8, -3, loss of mitochondrial transmembrane depolarisation potential and upregulation of pro-apoptotic mitogen activated protein kinases (p38, SAP/JNK). NB7M downregulated phosphorylation of prosurvival kinases (PI-3K, AKT, IKK alpha), transcription factor NF-kappaB, and expression of DNA-Pk and AXL receptor tyrosine kinase. Subcytotoxic doses of NB7M inhibited DNA synthesis, caused G1-phase cell-cycle arrest and upregulated p27 expression. The present report suggests that NB7M is a selective cytotoxic agent in vitro for cell lines derived from ovarian and certain other tumours. In addition, NB7M acts as a growth/cell-cycle-suppressing agent and may be developed as a potential therapeutic drug to treat ovarian cancer.

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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.
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fig3: 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.

Mentions: To understand the mechanism involved in the cellular response to NB7M treatment, we examined the mitochondrial transmembrane depolarisation potential (ΔYm) of SKOV-3 cells by flow cytometry. NB7M at a concentration of 2 μM caused a rapid loss of ΔYm (29% loss within 12 h; 44% within 24 h) in SKOV-3 cells (Figure 3A). NB7M showed similar reductions in the ΔYm of SMS-KCNR, a chemotherapy-resistant neuroblastoma cancer cell line (Brard et al, 2008). Loss of ΔYm due to chemical agents has been reported to be an indicator of onset of early apoptotic events (Petit et al, 1995).


Isothiocyanate NB7M causes selective cytotoxicity, pro-apoptotic signalling and cell-cycle regression in ovarian cancer cells.

Singh RK, Lange TS, Kim KK, Singh AP, Vorsa N, Brard L - Br. J. Cancer (2008)

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.
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Related In: Results  -  Collection

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fig3: 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.
Mentions: To understand the mechanism involved in the cellular response to NB7M treatment, we examined the mitochondrial transmembrane depolarisation potential (ΔYm) of SKOV-3 cells by flow cytometry. NB7M at a concentration of 2 μM caused a rapid loss of ΔYm (29% loss within 12 h; 44% within 24 h) in SKOV-3 cells (Figure 3A). NB7M showed similar reductions in the ΔYm of SMS-KCNR, a chemotherapy-resistant neuroblastoma cancer cell line (Brard et al, 2008). Loss of ΔYm due to chemical agents has been reported to be an indicator of onset of early apoptotic events (Petit et al, 1995).

Bottom Line: In a screen performed by the National Cancer Institute (NCI) (NCI(60) cancer cell-line assay) NB7M (NSC746077) reduced growth up to 100% with an IC(50) between 0.1 and 10 microM depending on the cell line studied.Subcytotoxic doses of NB7M inhibited DNA synthesis, caused G1-phase cell-cycle arrest and upregulated p27 expression.In addition, NB7M acts as a growth/cell-cycle-suppressing agent and may be developed as a potential therapeutic drug to treat ovarian cancer.

View Article: PubMed Central - PubMed

Affiliation: Molecular Therapeutics Laboratory, Program in Women's Oncology, Department of Obstetrics and Gynecology, Women and Infants' Hospital, Brown University, Providence, RI 02905, USA.

ABSTRACT
The present report identifies indole-3-ethyl isothiocyanate NB7M as a potent cytotoxic agent with selective activity against cell lines derived from various tumour types. Ovarian cancer cell lines showed sensitivity to NB7M (60-70% cytotoxicity at 2.5 microM), in contrast to control cells (TCL-1 and HTR-8; IC(50) approximately 15 microM). In a screen performed by the National Cancer Institute (NCI) (NCI(60) cancer cell-line assay) NB7M (NSC746077) reduced growth up to 100% with an IC(50) between 0.1 and 10 microM depending on the cell line studied. Using SKOV-3 ovarian cancer cells as a model, mechanisms of cytotoxicity were analysed. NB7M caused hallmarks of apoptosis such as PARP-1 deactivation, chromatin condensation, DNA nicks, activation of caspases-9, -8, -3, loss of mitochondrial transmembrane depolarisation potential and upregulation of pro-apoptotic mitogen activated protein kinases (p38, SAP/JNK). NB7M downregulated phosphorylation of prosurvival kinases (PI-3K, AKT, IKK alpha), transcription factor NF-kappaB, and expression of DNA-Pk and AXL receptor tyrosine kinase. Subcytotoxic doses of NB7M inhibited DNA synthesis, caused G1-phase cell-cycle arrest and upregulated p27 expression. The present report suggests that NB7M is a selective cytotoxic agent in vitro for cell lines derived from ovarian and certain other tumours. In addition, NB7M acts as a growth/cell-cycle-suppressing agent and may be developed as a potential therapeutic drug to treat ovarian cancer.

Show MeSH
Related in: MedlinePlus