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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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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.
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fig5: 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.

Mentions: As described in the previous sections, NB7M is a cytotoxic agent that activates apoptotic processes in SKOV-3 ovarian cancer cells. To investigate if NB7M affects the proliferation of SKOV-3 cells (particularly at drug concentrations when viability is not affected or partially reduced), we performed a BrdU incorporation assay. In this assay, BrdU incorporation into replicating DNA is detected by an antibody–peroxidase conjugate allowing a colorimetric reaction with the colour intensity directly representing cell proliferation. NB7M dose-dependently reduced SKOV-3 proliferation (Figure 5A). At a drug concentration of 1.5 μM (for 48 h) proliferation of treated SKOV-3 was inhibited by 70% as compared to untreated cells. Even at drug concentrations in the nano-molar range BrdU incorporation into the DNA was reduced. Thus, NB7M is a potent pro-apoptotic agent as well as an antiproliferative agent.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2600706&req=5

fig5: 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.
Mentions: As described in the previous sections, NB7M is a cytotoxic agent that activates apoptotic processes in SKOV-3 ovarian cancer cells. To investigate if NB7M affects the proliferation of SKOV-3 cells (particularly at drug concentrations when viability is not affected or partially reduced), we performed a BrdU incorporation assay. In this assay, BrdU incorporation into replicating DNA is detected by an antibody–peroxidase conjugate allowing a colorimetric reaction with the colour intensity directly representing cell proliferation. NB7M dose-dependently reduced SKOV-3 proliferation (Figure 5A). At a drug concentration of 1.5 μM (for 48 h) proliferation of treated SKOV-3 was inhibited by 70% as compared to untreated cells. Even at drug concentrations in the nano-molar range BrdU incorporation into the DNA was reduced. Thus, NB7M is a potent pro-apoptotic agent as well as an antiproliferative agent.

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