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Diindolylmethane suppresses ovarian cancer growth and potentiates the effect of cisplatin in tumor mouse model by targeting signal transducer and activator of transcription 3 (STAT3).

Kandala PK, Srivastava SK - BMC Med (2012)

Bottom Line: Phosphorylation of STAT3 at Tyr-705 and Ser-727 was reduced by DIM in a concentration-dependent manner.Importantly, diindolylmethane treatment potentiated the effects of cisplatin in SKOV-3 cells by targeting STAT3.Western blotting analysis of tumor lysates indicated increased apoptosis and reduced STAT3 activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences and Cancer Biology Center, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.

ABSTRACT

Background: Signal transducer and activator of transcription 3 (STAT3) is activated in majority of ovarian tumors and confers resistance to cisplatin treatment in patients with ovarian cancer. We have reported previously that diindolylmethane (DIM) inhibits the growth of ovarian cancer cells. However, to date the exact mechanism by which DIM induces growth suppressive effects has not been clear. In this report the mode of action of DIM is investigated.

Methods: Six human ovarian cancer cell lines and an ovarian tumor xenograft animal model were used to study the effect of diindolylmethane alone or in combination with cisplatin.

Results: Diindolylmethane treatment induced apoptosis in all six ovarian cancer cell lines. Phosphorylation of STAT3 at Tyr-705 and Ser-727 was reduced by DIM in a concentration-dependent manner. In addition, diindolylmethane treatment inhibited nuclear translocation, DNA binding, and transcriptional activity of STAT3. Interleukin (IL)-6-induced phosphorylation of STAT3 at Tyr-705 was significantly blocked by DIM. Overexpression of STAT3 by gene transfection blocked DIM-induced apoptosis. In addition, DIM treatment reduced the levels of IL-6 in ovarian cancer cells and in the tumors. DIM treatment also inhibited cell invasion and angiogenesis by suppressing hypoxia-inducible factor 1α (HIF-1α) and vascular epithelial growth factor (VEGF). Importantly, diindolylmethane treatment potentiated the effects of cisplatin in SKOV-3 cells by targeting STAT3. Oral administration of 3 mg diindolylmethane per day and subsequent administration of cisplatin substantially inhibited in vivo tumor growth. Western blotting analysis of tumor lysates indicated increased apoptosis and reduced STAT3 activation.

Conclusions: These findings provide a rationale for further clinical investigation of DIM alone or in combination for chemoprevention and/or chemotherapy of ovarian cancer.

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Diindolylmethane (DIM) inhibits signal transducer and activator of transcription 3 (STAT3) pathway in ovarian cancer cells. (A) Representative blots showing the concentration-dependent effect of DIM on phosphorylated (p)-STAT3 (Tyr-705), p-STAT3 (Ser-727), STAT3, Mcl-1, survivin in (i) SKOV-3, (ii) OVCAR-3, (iii) TOV-21G and (iv) A2780 ovarian cancer cells. Concentration-dependent effect of DIM on p-STAT3 (Tyr-705) and STAT3 in (v) OVCAR-429 and (vi) OVCAR-433 cells. Actin was used as loading control. (B) Effect of DIM on nuclear translocation of p-STAT3 and STAT3. Representative blots of nuclear lysates of (i) SKOV-3, (ii) OVCAR-3, (iii) TOV21G and (iv) A2780 cells treated with DIM. Lamin B was used as loading control. (C) Effect of DIM on STAT3 DNA binding. (i) SKOV-3 cells or (ii) OVCAR-3 cells were treated for 24 h with or without 75 μM DIM and nuclear cell extracts were tested for STAT3 DNA-binding activity as measured by the Universal EZ-TFA transcription factor colorimetric assay. (D) Effect of DIM on STAT3-regulated luciferase reporter activity. STAT3 luciferase transcriptional activity was determined in (i) SKOV-3, (ii) OVCAR-3, (iii) TOV21G or (iv) A2780 cells. Firefly luciferase activities were corrected for Renilla luciferase levels and then normalized relative to the control, which was considered as 100%. Means and SD of two independent experiments performed in triplicate are shown. The Student's t test was used for statistical analysis to compare control and DIM treatment.
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Figure 2: Diindolylmethane (DIM) inhibits signal transducer and activator of transcription 3 (STAT3) pathway in ovarian cancer cells. (A) Representative blots showing the concentration-dependent effect of DIM on phosphorylated (p)-STAT3 (Tyr-705), p-STAT3 (Ser-727), STAT3, Mcl-1, survivin in (i) SKOV-3, (ii) OVCAR-3, (iii) TOV-21G and (iv) A2780 ovarian cancer cells. Concentration-dependent effect of DIM on p-STAT3 (Tyr-705) and STAT3 in (v) OVCAR-429 and (vi) OVCAR-433 cells. Actin was used as loading control. (B) Effect of DIM on nuclear translocation of p-STAT3 and STAT3. Representative blots of nuclear lysates of (i) SKOV-3, (ii) OVCAR-3, (iii) TOV21G and (iv) A2780 cells treated with DIM. Lamin B was used as loading control. (C) Effect of DIM on STAT3 DNA binding. (i) SKOV-3 cells or (ii) OVCAR-3 cells were treated for 24 h with or without 75 μM DIM and nuclear cell extracts were tested for STAT3 DNA-binding activity as measured by the Universal EZ-TFA transcription factor colorimetric assay. (D) Effect of DIM on STAT3-regulated luciferase reporter activity. STAT3 luciferase transcriptional activity was determined in (i) SKOV-3, (ii) OVCAR-3, (iii) TOV21G or (iv) A2780 cells. Firefly luciferase activities were corrected for Renilla luciferase levels and then normalized relative to the control, which was considered as 100%. Means and SD of two independent experiments performed in triplicate are shown. The Student's t test was used for statistical analysis to compare control and DIM treatment.

Mentions: Our next step was to investigate the mechanism by which DIM induces apoptosis. STAT3 is activated in almost 90% of ovarian cancers. Overexpression of STAT3 was reported in stage III and IV ovarian tumors [14]. We hypothesized that DIM induces apoptosis in ovarian cancer cells by inhibiting STAT3, and then systematically tested our hypothesis. SKOV-3, OVCAR-3, TOV-21G, A2780, OVCAR-429 and OVCAR-433 cells treated with varying concentrations of DIM were subjected to western blotting. Our results clearly show that DIM substantially inhibits the activation of STAT3 by suppressing phosphorylation at Tyr-705 and Ser-727 (Figure 2A i-vi). The protein levels of STAT3 decreased modestly with DIM treatment. Our results further show that the expression of Mcl-1 and survivin were drastically decreased by DIM treatment in a concentration-dependent manner in all four cell lines (Figure 2A i-iv). Regulated by STAT3, both Mcl-1 and survivin have been implicated in cancer growth. These results demonstrate that DIM targets STAT3 pathway in ovarian cancer cells.


Diindolylmethane suppresses ovarian cancer growth and potentiates the effect of cisplatin in tumor mouse model by targeting signal transducer and activator of transcription 3 (STAT3).

Kandala PK, Srivastava SK - BMC Med (2012)

Diindolylmethane (DIM) inhibits signal transducer and activator of transcription 3 (STAT3) pathway in ovarian cancer cells. (A) Representative blots showing the concentration-dependent effect of DIM on phosphorylated (p)-STAT3 (Tyr-705), p-STAT3 (Ser-727), STAT3, Mcl-1, survivin in (i) SKOV-3, (ii) OVCAR-3, (iii) TOV-21G and (iv) A2780 ovarian cancer cells. Concentration-dependent effect of DIM on p-STAT3 (Tyr-705) and STAT3 in (v) OVCAR-429 and (vi) OVCAR-433 cells. Actin was used as loading control. (B) Effect of DIM on nuclear translocation of p-STAT3 and STAT3. Representative blots of nuclear lysates of (i) SKOV-3, (ii) OVCAR-3, (iii) TOV21G and (iv) A2780 cells treated with DIM. Lamin B was used as loading control. (C) Effect of DIM on STAT3 DNA binding. (i) SKOV-3 cells or (ii) OVCAR-3 cells were treated for 24 h with or without 75 μM DIM and nuclear cell extracts were tested for STAT3 DNA-binding activity as measured by the Universal EZ-TFA transcription factor colorimetric assay. (D) Effect of DIM on STAT3-regulated luciferase reporter activity. STAT3 luciferase transcriptional activity was determined in (i) SKOV-3, (ii) OVCAR-3, (iii) TOV21G or (iv) A2780 cells. Firefly luciferase activities were corrected for Renilla luciferase levels and then normalized relative to the control, which was considered as 100%. Means and SD of two independent experiments performed in triplicate are shown. The Student's t test was used for statistical analysis to compare control and DIM treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3298725&req=5

Figure 2: Diindolylmethane (DIM) inhibits signal transducer and activator of transcription 3 (STAT3) pathway in ovarian cancer cells. (A) Representative blots showing the concentration-dependent effect of DIM on phosphorylated (p)-STAT3 (Tyr-705), p-STAT3 (Ser-727), STAT3, Mcl-1, survivin in (i) SKOV-3, (ii) OVCAR-3, (iii) TOV-21G and (iv) A2780 ovarian cancer cells. Concentration-dependent effect of DIM on p-STAT3 (Tyr-705) and STAT3 in (v) OVCAR-429 and (vi) OVCAR-433 cells. Actin was used as loading control. (B) Effect of DIM on nuclear translocation of p-STAT3 and STAT3. Representative blots of nuclear lysates of (i) SKOV-3, (ii) OVCAR-3, (iii) TOV21G and (iv) A2780 cells treated with DIM. Lamin B was used as loading control. (C) Effect of DIM on STAT3 DNA binding. (i) SKOV-3 cells or (ii) OVCAR-3 cells were treated for 24 h with or without 75 μM DIM and nuclear cell extracts were tested for STAT3 DNA-binding activity as measured by the Universal EZ-TFA transcription factor colorimetric assay. (D) Effect of DIM on STAT3-regulated luciferase reporter activity. STAT3 luciferase transcriptional activity was determined in (i) SKOV-3, (ii) OVCAR-3, (iii) TOV21G or (iv) A2780 cells. Firefly luciferase activities were corrected for Renilla luciferase levels and then normalized relative to the control, which was considered as 100%. Means and SD of two independent experiments performed in triplicate are shown. The Student's t test was used for statistical analysis to compare control and DIM treatment.
Mentions: Our next step was to investigate the mechanism by which DIM induces apoptosis. STAT3 is activated in almost 90% of ovarian cancers. Overexpression of STAT3 was reported in stage III and IV ovarian tumors [14]. We hypothesized that DIM induces apoptosis in ovarian cancer cells by inhibiting STAT3, and then systematically tested our hypothesis. SKOV-3, OVCAR-3, TOV-21G, A2780, OVCAR-429 and OVCAR-433 cells treated with varying concentrations of DIM were subjected to western blotting. Our results clearly show that DIM substantially inhibits the activation of STAT3 by suppressing phosphorylation at Tyr-705 and Ser-727 (Figure 2A i-vi). The protein levels of STAT3 decreased modestly with DIM treatment. Our results further show that the expression of Mcl-1 and survivin were drastically decreased by DIM treatment in a concentration-dependent manner in all four cell lines (Figure 2A i-iv). Regulated by STAT3, both Mcl-1 and survivin have been implicated in cancer growth. These results demonstrate that DIM targets STAT3 pathway in ovarian cancer cells.

Bottom Line: Phosphorylation of STAT3 at Tyr-705 and Ser-727 was reduced by DIM in a concentration-dependent manner.Importantly, diindolylmethane treatment potentiated the effects of cisplatin in SKOV-3 cells by targeting STAT3.Western blotting analysis of tumor lysates indicated increased apoptosis and reduced STAT3 activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biomedical Sciences and Cancer Biology Center, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.

ABSTRACT

Background: Signal transducer and activator of transcription 3 (STAT3) is activated in majority of ovarian tumors and confers resistance to cisplatin treatment in patients with ovarian cancer. We have reported previously that diindolylmethane (DIM) inhibits the growth of ovarian cancer cells. However, to date the exact mechanism by which DIM induces growth suppressive effects has not been clear. In this report the mode of action of DIM is investigated.

Methods: Six human ovarian cancer cell lines and an ovarian tumor xenograft animal model were used to study the effect of diindolylmethane alone or in combination with cisplatin.

Results: Diindolylmethane treatment induced apoptosis in all six ovarian cancer cell lines. Phosphorylation of STAT3 at Tyr-705 and Ser-727 was reduced by DIM in a concentration-dependent manner. In addition, diindolylmethane treatment inhibited nuclear translocation, DNA binding, and transcriptional activity of STAT3. Interleukin (IL)-6-induced phosphorylation of STAT3 at Tyr-705 was significantly blocked by DIM. Overexpression of STAT3 by gene transfection blocked DIM-induced apoptosis. In addition, DIM treatment reduced the levels of IL-6 in ovarian cancer cells and in the tumors. DIM treatment also inhibited cell invasion and angiogenesis by suppressing hypoxia-inducible factor 1α (HIF-1α) and vascular epithelial growth factor (VEGF). Importantly, diindolylmethane treatment potentiated the effects of cisplatin in SKOV-3 cells by targeting STAT3. Oral administration of 3 mg diindolylmethane per day and subsequent administration of cisplatin substantially inhibited in vivo tumor growth. Western blotting analysis of tumor lysates indicated increased apoptosis and reduced STAT3 activation.

Conclusions: These findings provide a rationale for further clinical investigation of DIM alone or in combination for chemoprevention and/or chemotherapy of ovarian cancer.

Show MeSH
Related in: MedlinePlus