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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) blocks cell migration and neovascularization by inhibiting hypoxia-inducible factor 1α (HIF-1α) and vascular epithelial growth factor (VEGF). (A) SKOV-3, (B) OVCAR-3 or (C) TOV21G cells were plated, scratched with a pipette tip and incubated in the absence or presence of 50 μM DIM. Photographs were taken at 0 h and 24 h using an inverted microscope. The wound area in DIM treated and control cells were quantified by ImageJ software. Results are presented as means ± SD of triplicates. (D) DIM inhibits interleukin (IL)-6-induced vessel sprouting ex vivo. Representative photographs are presented. (E) Effect of DIM on proangiogenic proteins was analyzed by western blotting. SKOV-3 or OVCAR-3 cells were treated with 10 μM MG132 for 1 h and then exposed to 75 μM DIM for 6 h. (F) Cells were treated as above and then treated with IL-6 for 15 minutes. Representative blots of HIF-1α and VEGF are shown. Blots were further stripped and probed with actin. Experiments were performed independently three times. The Student's t test was used for statistical analysis to compare control and DIM treatment.
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Figure 4: Diindolylmethane (DIM) blocks cell migration and neovascularization by inhibiting hypoxia-inducible factor 1α (HIF-1α) and vascular epithelial growth factor (VEGF). (A) SKOV-3, (B) OVCAR-3 or (C) TOV21G cells were plated, scratched with a pipette tip and incubated in the absence or presence of 50 μM DIM. Photographs were taken at 0 h and 24 h using an inverted microscope. The wound area in DIM treated and control cells were quantified by ImageJ software. Results are presented as means ± SD of triplicates. (D) DIM inhibits interleukin (IL)-6-induced vessel sprouting ex vivo. Representative photographs are presented. (E) Effect of DIM on proangiogenic proteins was analyzed by western blotting. SKOV-3 or OVCAR-3 cells were treated with 10 μM MG132 for 1 h and then exposed to 75 μM DIM for 6 h. (F) Cells were treated as above and then treated with IL-6 for 15 minutes. Representative blots of HIF-1α and VEGF are shown. Blots were further stripped and probed with actin. Experiments were performed independently three times. The Student's t test was used for statistical analysis to compare control and DIM treatment.

Mentions: Recent literature suggested a novel role of STAT3 in angiogenesis and metastasis. Since DIM suppressed the activation of STAT3 in ovarian cancer cells, we wanted to test whether DIM can inhibit invasion and angiogenesis. We first determined the anti-invasive potential of DIM by evaluating cell migration in a wound healing assay. A wound was made on confluent monolayer cells with a pipette tip and, after washing with fresh medium, cells were treated with or without 50 μM DIM. Our results revealed that after 24 h DIM treatment, SKOV-3 cells migrated into 20% of the wounded area, whereas control cells migrated into 80% of the wounded area, showing the potential anti-invasive property of DIM (Figure 4A). Similar observations were made in OVCAR-3 and TOV-21G cells (Figure 4B, C).


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) blocks cell migration and neovascularization by inhibiting hypoxia-inducible factor 1α (HIF-1α) and vascular epithelial growth factor (VEGF). (A) SKOV-3, (B) OVCAR-3 or (C) TOV21G cells were plated, scratched with a pipette tip and incubated in the absence or presence of 50 μM DIM. Photographs were taken at 0 h and 24 h using an inverted microscope. The wound area in DIM treated and control cells were quantified by ImageJ software. Results are presented as means ± SD of triplicates. (D) DIM inhibits interleukin (IL)-6-induced vessel sprouting ex vivo. Representative photographs are presented. (E) Effect of DIM on proangiogenic proteins was analyzed by western blotting. SKOV-3 or OVCAR-3 cells were treated with 10 μM MG132 for 1 h and then exposed to 75 μM DIM for 6 h. (F) Cells were treated as above and then treated with IL-6 for 15 minutes. Representative blots of HIF-1α and VEGF are shown. Blots were further stripped and probed with actin. Experiments were performed independently three times. 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 4: Diindolylmethane (DIM) blocks cell migration and neovascularization by inhibiting hypoxia-inducible factor 1α (HIF-1α) and vascular epithelial growth factor (VEGF). (A) SKOV-3, (B) OVCAR-3 or (C) TOV21G cells were plated, scratched with a pipette tip and incubated in the absence or presence of 50 μM DIM. Photographs were taken at 0 h and 24 h using an inverted microscope. The wound area in DIM treated and control cells were quantified by ImageJ software. Results are presented as means ± SD of triplicates. (D) DIM inhibits interleukin (IL)-6-induced vessel sprouting ex vivo. Representative photographs are presented. (E) Effect of DIM on proangiogenic proteins was analyzed by western blotting. SKOV-3 or OVCAR-3 cells were treated with 10 μM MG132 for 1 h and then exposed to 75 μM DIM for 6 h. (F) Cells were treated as above and then treated with IL-6 for 15 minutes. Representative blots of HIF-1α and VEGF are shown. Blots were further stripped and probed with actin. Experiments were performed independently three times. The Student's t test was used for statistical analysis to compare control and DIM treatment.
Mentions: Recent literature suggested a novel role of STAT3 in angiogenesis and metastasis. Since DIM suppressed the activation of STAT3 in ovarian cancer cells, we wanted to test whether DIM can inhibit invasion and angiogenesis. We first determined the anti-invasive potential of DIM by evaluating cell migration in a wound healing assay. A wound was made on confluent monolayer cells with a pipette tip and, after washing with fresh medium, cells were treated with or without 50 μM DIM. Our results revealed that after 24 h DIM treatment, SKOV-3 cells migrated into 20% of the wounded area, whereas control cells migrated into 80% of the wounded area, showing the potential anti-invasive property of DIM (Figure 4A). Similar observations were made in OVCAR-3 and TOV-21G cells (Figure 4B, C).

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