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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 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).
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fig2: 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).

Mentions: NB7M proved to be highly and dose-dependently cytotoxic for all five cancer cell lines studied, including ovarian cancer cell lines SKOV-3 and OVCAR-3 (60–70% cytotoxicity at 2.5 μM) (Figure 1B). In contrast, TCL-1 and HTR-8 (trophoblasts) were not affected at 5 μM NB7M. As these control cell lines possess a similar metabolic rate as cancer cells in this screen, NB7M apparently showed selective cytotoxicity against cancer-derived cells. To further evaluate the tumour-type selectivity of NB7M, the effect of this compound at 10 μM was screened in an NCI60 cell line growth/viability assay (http://dtp.nci.nih.gov/screening.html, Figure 2A). NB7M at 10 μM proved to significantly reduce the viability of colon cancer cells (COLO205, HCT-116, HCT-15, KM12 and SW620), breast cancer (NCI/ADR-RES, MCF7), ovarian cancer (IGROV1, OVCAR-3, OVCAR-8), leukaemia (CCRF-CEM, HL-60, K-562, MOLT-4), renal cancer (ACHN, CAKI-1, SN12C, TK-10), melanoma (LOX IMVI, M14, MALME-3M, SK-MEL-2, SK-MEL-28), prostate (DU145) and non-small lung cancer cells (NCI-H23) cells. In contrast, several non-small lung cancer cell subtypes (EKVX, NCI-H322M and NCI-H226) and a breast cancer cell line (BT-549) were resistant to NB7M treatment (Figure 2A). In the same assay the dose-dependent effect of various concentrations of NB7M (10 nM–100 μM) on a panel of ovarian cancer cell lines was compared (Figure 2B). The growth of IGROV1, OVCAR-3, OVCAR-5 was highly affected by 10 μM of the drug (as shown for OVCAR-3 in the cytotoxicity assay performed in our laboratory) and OVCAR-8 and OVCAR-4 responded strongly to treatment, whereas SKOV-3 revealed less but still significant reduction in growth compared to the other five lines tested by the National Cancer Institute (NCI). Taken together, the NCI screen (http://dtp.nci.nih.gov/screening.html) and our cytotoxicity assays suggest NB7M to be highly and specifically detrimental to cell lines derived from certain tumour types, including ovarian cancer, but less effective against other tumour types and marginally cytotoxic for control cell lines (e.g., TCL-1, HTR-8; Figure 1B and MRC-5 fibroblasts, Singh et al, 2008).


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 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).
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fig2: 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).
Mentions: NB7M proved to be highly and dose-dependently cytotoxic for all five cancer cell lines studied, including ovarian cancer cell lines SKOV-3 and OVCAR-3 (60–70% cytotoxicity at 2.5 μM) (Figure 1B). In contrast, TCL-1 and HTR-8 (trophoblasts) were not affected at 5 μM NB7M. As these control cell lines possess a similar metabolic rate as cancer cells in this screen, NB7M apparently showed selective cytotoxicity against cancer-derived cells. To further evaluate the tumour-type selectivity of NB7M, the effect of this compound at 10 μM was screened in an NCI60 cell line growth/viability assay (http://dtp.nci.nih.gov/screening.html, Figure 2A). NB7M at 10 μM proved to significantly reduce the viability of colon cancer cells (COLO205, HCT-116, HCT-15, KM12 and SW620), breast cancer (NCI/ADR-RES, MCF7), ovarian cancer (IGROV1, OVCAR-3, OVCAR-8), leukaemia (CCRF-CEM, HL-60, K-562, MOLT-4), renal cancer (ACHN, CAKI-1, SN12C, TK-10), melanoma (LOX IMVI, M14, MALME-3M, SK-MEL-2, SK-MEL-28), prostate (DU145) and non-small lung cancer cells (NCI-H23) cells. In contrast, several non-small lung cancer cell subtypes (EKVX, NCI-H322M and NCI-H226) and a breast cancer cell line (BT-549) were resistant to NB7M treatment (Figure 2A). In the same assay the dose-dependent effect of various concentrations of NB7M (10 nM–100 μM) on a panel of ovarian cancer cell lines was compared (Figure 2B). The growth of IGROV1, OVCAR-3, OVCAR-5 was highly affected by 10 μM of the drug (as shown for OVCAR-3 in the cytotoxicity assay performed in our laboratory) and OVCAR-8 and OVCAR-4 responded strongly to treatment, whereas SKOV-3 revealed less but still significant reduction in growth compared to the other five lines tested by the National Cancer Institute (NCI). Taken together, the NCI screen (http://dtp.nci.nih.gov/screening.html) and our cytotoxicity assays suggest NB7M to be highly and specifically detrimental to cell lines derived from certain tumour types, including ovarian cancer, but less effective against other tumour types and marginally cytotoxic for control cell lines (e.g., TCL-1, HTR-8; Figure 1B and MRC-5 fibroblasts, Singh et al, 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.

Show MeSH
Related in: MedlinePlus