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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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Related in: MedlinePlus

Diindolylmethane (DIM) potentiates the effect of cisplatin in ovarian cancer cells. (A) SKOV-3 cells were exposed to 20 or 50 μM DIM for 24 h, followed by exposure to 10 μM cisplatin for another 24 h. Survival of control and treated cells was evaluated by sulforhodamine B assay. (B) Combination effect of cisplatin and DIM on the signal transducer and activator of transcription 3 (STAT3) pathway. Representative blots of control and treated cells were examined for Tyr-705 and Ser-727 STAT3, STAT3, Mcl-1, survivin, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase 3. Blots were stripped and probed with actin. (C) Effect of combination treatment on nuclear localization of STAT3. Nuclear fractions from cells treated with or without 50 μM DIM or 10 μM cisplatin or both were subjected to western blotting. Representative blots of Tyr-705 STAT3 and STAT3 are shown. Lamin B was used as loading control. (D) Luciferase activity in combination treatment was measured in SKOV-3. Whole cell lysates were collected and firefly luciferase activities were corrected for Renilla luciferase levels and then normalized relative to the control, which was considered as 100%. (E) Aortic rings (1 mm) were harvested from Sprague-Dawley rats, immersed in matrigel, and treated with interleukin (IL)-6 (50 ng/ml) in the absence or presence of cisplatin (10 μM) with or without DIM (50 μM) for 4 days, and then photographed under a microscope (4 ×). Representative photographs are shown. (F) DIM inhibits the invasion of SKOV-3 cells. Invasion assay was performed using Boyden's chamber (BD Sciences) according to manufacturer's instructions. (G) DIM inhibits vascular epithelial growth factor (VEGF) secretion. Cells were plated, stimulated with VEGF, and treated with DIM for 24 h. Media was collected and assayed for VEGF by ELISA (Invitrogen) kit according to manufacturer's instructions. All experiments were performed independently three times. The differences between all the groups were compared by non-parametric analysis of variance with Bonferroni post hoc comparisons.
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Figure 5: Diindolylmethane (DIM) potentiates the effect of cisplatin in ovarian cancer cells. (A) SKOV-3 cells were exposed to 20 or 50 μM DIM for 24 h, followed by exposure to 10 μM cisplatin for another 24 h. Survival of control and treated cells was evaluated by sulforhodamine B assay. (B) Combination effect of cisplatin and DIM on the signal transducer and activator of transcription 3 (STAT3) pathway. Representative blots of control and treated cells were examined for Tyr-705 and Ser-727 STAT3, STAT3, Mcl-1, survivin, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase 3. Blots were stripped and probed with actin. (C) Effect of combination treatment on nuclear localization of STAT3. Nuclear fractions from cells treated with or without 50 μM DIM or 10 μM cisplatin or both were subjected to western blotting. Representative blots of Tyr-705 STAT3 and STAT3 are shown. Lamin B was used as loading control. (D) Luciferase activity in combination treatment was measured in SKOV-3. Whole cell lysates were collected and firefly luciferase activities were corrected for Renilla luciferase levels and then normalized relative to the control, which was considered as 100%. (E) Aortic rings (1 mm) were harvested from Sprague-Dawley rats, immersed in matrigel, and treated with interleukin (IL)-6 (50 ng/ml) in the absence or presence of cisplatin (10 μM) with or without DIM (50 μM) for 4 days, and then photographed under a microscope (4 ×). Representative photographs are shown. (F) DIM inhibits the invasion of SKOV-3 cells. Invasion assay was performed using Boyden's chamber (BD Sciences) according to manufacturer's instructions. (G) DIM inhibits vascular epithelial growth factor (VEGF) secretion. Cells were plated, stimulated with VEGF, and treated with DIM for 24 h. Media was collected and assayed for VEGF by ELISA (Invitrogen) kit according to manufacturer's instructions. All experiments were performed independently three times. The differences between all the groups were compared by non-parametric analysis of variance with Bonferroni post hoc comparisons.

Mentions: We wanted to determine whether DIM can potentiate the effect of cisplatin, a drug used to treat ovarian cancer patients. Cisplatin usually is associated with several side effects such as renal toxicity and reduction of white and red blood cells. Moreover, tumors often become resistant to cisplatin therapy. Hence, enhancing the effect of cisplatin at low doses by agents that are non-toxic may be a better strategy, not only for effective treatment but also to reduce harmful side effects. To test whether or not DIM potentiates the effect of cisplatin, SKOV-3 cells were treated with 20 μM or 50 μM DIM for 24 h followed by exposure to 10 μM cisplatin for an additional 24 h. It is important to mention that the IC50 of cisplatin in SKOV-3 cells is 40 μM; we used one-quarter of the IC50 concentration. Growth inhibition in combination treatment was significantly higher as compared to either treatment alone (Figure 5A). We observed around 50% to 70% reduction in the survival of SKOV-3 cells treated with a combination of DIM and cisplatin as compared to 28% by cisplatin alone (P < 0.001). We calculated the combination index (CI) for DIM and cisplatin combination treatment in SKOV-3 cells. CI values for the combination of cisplatin with 20 μM and 50 μM DIM treatment were 0.45 and 0.56, respectively, indicating the synergistic effect of DIM with cisplatin.


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) potentiates the effect of cisplatin in ovarian cancer cells. (A) SKOV-3 cells were exposed to 20 or 50 μM DIM for 24 h, followed by exposure to 10 μM cisplatin for another 24 h. Survival of control and treated cells was evaluated by sulforhodamine B assay. (B) Combination effect of cisplatin and DIM on the signal transducer and activator of transcription 3 (STAT3) pathway. Representative blots of control and treated cells were examined for Tyr-705 and Ser-727 STAT3, STAT3, Mcl-1, survivin, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase 3. Blots were stripped and probed with actin. (C) Effect of combination treatment on nuclear localization of STAT3. Nuclear fractions from cells treated with or without 50 μM DIM or 10 μM cisplatin or both were subjected to western blotting. Representative blots of Tyr-705 STAT3 and STAT3 are shown. Lamin B was used as loading control. (D) Luciferase activity in combination treatment was measured in SKOV-3. Whole cell lysates were collected and firefly luciferase activities were corrected for Renilla luciferase levels and then normalized relative to the control, which was considered as 100%. (E) Aortic rings (1 mm) were harvested from Sprague-Dawley rats, immersed in matrigel, and treated with interleukin (IL)-6 (50 ng/ml) in the absence or presence of cisplatin (10 μM) with or without DIM (50 μM) for 4 days, and then photographed under a microscope (4 ×). Representative photographs are shown. (F) DIM inhibits the invasion of SKOV-3 cells. Invasion assay was performed using Boyden's chamber (BD Sciences) according to manufacturer's instructions. (G) DIM inhibits vascular epithelial growth factor (VEGF) secretion. Cells were plated, stimulated with VEGF, and treated with DIM for 24 h. Media was collected and assayed for VEGF by ELISA (Invitrogen) kit according to manufacturer's instructions. All experiments were performed independently three times. The differences between all the groups were compared by non-parametric analysis of variance with Bonferroni post hoc comparisons.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Diindolylmethane (DIM) potentiates the effect of cisplatin in ovarian cancer cells. (A) SKOV-3 cells were exposed to 20 or 50 μM DIM for 24 h, followed by exposure to 10 μM cisplatin for another 24 h. Survival of control and treated cells was evaluated by sulforhodamine B assay. (B) Combination effect of cisplatin and DIM on the signal transducer and activator of transcription 3 (STAT3) pathway. Representative blots of control and treated cells were examined for Tyr-705 and Ser-727 STAT3, STAT3, Mcl-1, survivin, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase 3. Blots were stripped and probed with actin. (C) Effect of combination treatment on nuclear localization of STAT3. Nuclear fractions from cells treated with or without 50 μM DIM or 10 μM cisplatin or both were subjected to western blotting. Representative blots of Tyr-705 STAT3 and STAT3 are shown. Lamin B was used as loading control. (D) Luciferase activity in combination treatment was measured in SKOV-3. Whole cell lysates were collected and firefly luciferase activities were corrected for Renilla luciferase levels and then normalized relative to the control, which was considered as 100%. (E) Aortic rings (1 mm) were harvested from Sprague-Dawley rats, immersed in matrigel, and treated with interleukin (IL)-6 (50 ng/ml) in the absence or presence of cisplatin (10 μM) with or without DIM (50 μM) for 4 days, and then photographed under a microscope (4 ×). Representative photographs are shown. (F) DIM inhibits the invasion of SKOV-3 cells. Invasion assay was performed using Boyden's chamber (BD Sciences) according to manufacturer's instructions. (G) DIM inhibits vascular epithelial growth factor (VEGF) secretion. Cells were plated, stimulated with VEGF, and treated with DIM for 24 h. Media was collected and assayed for VEGF by ELISA (Invitrogen) kit according to manufacturer's instructions. All experiments were performed independently three times. The differences between all the groups were compared by non-parametric analysis of variance with Bonferroni post hoc comparisons.
Mentions: We wanted to determine whether DIM can potentiate the effect of cisplatin, a drug used to treat ovarian cancer patients. Cisplatin usually is associated with several side effects such as renal toxicity and reduction of white and red blood cells. Moreover, tumors often become resistant to cisplatin therapy. Hence, enhancing the effect of cisplatin at low doses by agents that are non-toxic may be a better strategy, not only for effective treatment but also to reduce harmful side effects. To test whether or not DIM potentiates the effect of cisplatin, SKOV-3 cells were treated with 20 μM or 50 μM DIM for 24 h followed by exposure to 10 μM cisplatin for an additional 24 h. It is important to mention that the IC50 of cisplatin in SKOV-3 cells is 40 μM; we used one-quarter of the IC50 concentration. Growth inhibition in combination treatment was significantly higher as compared to either treatment alone (Figure 5A). We observed around 50% to 70% reduction in the survival of SKOV-3 cells treated with a combination of DIM and cisplatin as compared to 28% by cisplatin alone (P < 0.001). We calculated the combination index (CI) for DIM and cisplatin combination treatment in SKOV-3 cells. CI values for the combination of cisplatin with 20 μM and 50 μM DIM treatment were 0.45 and 0.56, respectively, indicating the synergistic effect of DIM with cisplatin.

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