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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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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.
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fig4: 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.

Mentions: To define key signalling responses of SKOV-3 cells to treatment with NB7M, we analysed the expression and activation/phosphorylation of cellular markers involved in prosurvival or pro-apoptotic signalling. Immunoblotting of PAGE-separated cellular lysates revealed that NB7M (at 2 μM) caused a rapid, strong, and sustained activation of p38 and JNK MAPK (Figure 4A). Both MAPKs are crucial factors in signalling cascades responding to inflammatory cytokines, stress, UV light, osmotic shock, cytotoxic drugs and diverse pro-apoptotic stimuli (Pearson et al, 2001). Upregulation of activated p38 and JNK MAPKs resulted in slight downregulation of the basal level of inactive JNK and p38 (Figure 4A). In addition, expression of the phosphorylated/activated form of ERK1/2 in SKOV-3 was downregulated upon NB7M treatment (2 μM), whereas inactive ERK1/2 remained at high level in untreated or treated SKOV-3 cells. Both ERK 1 and 2 (p44 and p42 MAPKs) generally participate in a protein kinase cascade that plays a critical role in the regulation of cell growth and differentiation and can be found activated in their role as survival factors as well as in apoptotic events (Pearson et al, 2001; Ahmed-Choudhury et al, 2006).


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)

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.
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Related In: Results  -  Collection

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

fig4: 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.
Mentions: To define key signalling responses of SKOV-3 cells to treatment with NB7M, we analysed the expression and activation/phosphorylation of cellular markers involved in prosurvival or pro-apoptotic signalling. Immunoblotting of PAGE-separated cellular lysates revealed that NB7M (at 2 μM) caused a rapid, strong, and sustained activation of p38 and JNK MAPK (Figure 4A). Both MAPKs are crucial factors in signalling cascades responding to inflammatory cytokines, stress, UV light, osmotic shock, cytotoxic drugs and diverse pro-apoptotic stimuli (Pearson et al, 2001). Upregulation of activated p38 and JNK MAPKs resulted in slight downregulation of the basal level of inactive JNK and p38 (Figure 4A). In addition, expression of the phosphorylated/activated form of ERK1/2 in SKOV-3 was downregulated upon NB7M treatment (2 μM), whereas inactive ERK1/2 remained at high level in untreated or treated SKOV-3 cells. Both ERK 1 and 2 (p44 and p42 MAPKs) generally participate in a protein kinase cascade that plays a critical role in the regulation of cell growth and differentiation and can be found activated in their role as survival factors as well as in apoptotic events (Pearson et al, 2001; Ahmed-Choudhury et al, 2006).

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