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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) suppresses the growth of ovarian tumors alone and in combination with cisplatin by inhibiting signal transducer and activator of transcription 3 (STAT3) in nude mice. SKOV-3 tumor cells were implanted into athymic nude mice. Once each mouse had a palpable tumor, mice received 3 mg/day DIM by oral gavage every day or 5 mg/kg cisplatin intraperitoneally twice a week or both. (A) (i) Effect of DIM on tumor growth. *P < 0.05 when compared to control. (ii) Tumor weight of mice from different groups during the course of in vivo study. (B) Inhibition of STAT3 signaling in the tumors of mice administered with DIM alone or in combination with cisplatin. Tumors from control and treated mice were excised on day 48 after implantation, lysed and analyzed by western blotting for Tyr-705 STAT3, Ser-727 STAT3, total STAT3, Mcl-1, survivin, cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase 3. Blots were stripped and reprobed with actin antibody to verify equal protein loading. Each lane represents a different tumor sample. (C) Densitometric quantitation of western blotting represented above. Legends on the bars indicate (a) statistically significant compared to control, (b) statistically significant compared to DIM, (c) statistically significant compared to cisplatin. (D) Levels of interleukin (IL)-6 in tumors from control mice and DIM treated mice. The differences between all the groups were compared by non-parametric analysis of variance with Bonferroni post hoc comparisons.
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Figure 6: Diindolylmethane (DIM) suppresses the growth of ovarian tumors alone and in combination with cisplatin by inhibiting signal transducer and activator of transcription 3 (STAT3) in nude mice. SKOV-3 tumor cells were implanted into athymic nude mice. Once each mouse had a palpable tumor, mice received 3 mg/day DIM by oral gavage every day or 5 mg/kg cisplatin intraperitoneally twice a week or both. (A) (i) Effect of DIM on tumor growth. *P < 0.05 when compared to control. (ii) Tumor weight of mice from different groups during the course of in vivo study. (B) Inhibition of STAT3 signaling in the tumors of mice administered with DIM alone or in combination with cisplatin. Tumors from control and treated mice were excised on day 48 after implantation, lysed and analyzed by western blotting for Tyr-705 STAT3, Ser-727 STAT3, total STAT3, Mcl-1, survivin, cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase 3. Blots were stripped and reprobed with actin antibody to verify equal protein loading. Each lane represents a different tumor sample. (C) Densitometric quantitation of western blotting represented above. Legends on the bars indicate (a) statistically significant compared to control, (b) statistically significant compared to DIM, (c) statistically significant compared to cisplatin. (D) Levels of interleukin (IL)-6 in tumors from control mice and DIM treated mice. The differences between all the groups were compared by non-parametric analysis of variance with Bonferroni post hoc comparisons.

Mentions: We demonstrated that DIM induces apoptosis in ovarian cancer cells and enhances the effect of cisplatin by inhibiting STAT3 pathway in culture models. To validate these effects in vivo, we performed a tumor xenograft assay. About 5 × 106 SKOV-3 cells were injected subcutaneously into both the right and left flanks of female athymic nude mice. Once each mouse had a tumor of about 90 mm3, they were randomized into four groups. DIM treatment started 24 days after tumor implantation, and cisplatin treatment began 10 days later. Our results demonstrated that DIM alone and in combination treatment substantially retarded the growth of SKOV-3 tumors as compared to control or cisplatin treatment. For example, at day 48 the average tumor volume in control mice and cisplatin-treated mice was around 400 mm3 and 300 mm3, respectively, whereas the average tumor volume in mice that received DIM alone or in combination with cisplatin was 210 mm3 and 138 mm3, respectively (Figure 6A). The combination treatment suppressed tumors by 65% as compared to controls. Interestingly, there was no significant change in the weight of mice treated with DIM as compared to mice in the control group. The weight of mice declined significantly in response to cisplatin treatment. However, the decline in weight in the combination treatment group was not as significant as in the mice receiving cisplatin treatment alone (Figure 6A ii). To determine whether DIM-mediated tumor growth suppression was due to inhibition of STAT3, tumor lysates were subjected to western blotting. As shown in Figure 6B, p-STAT3 (Tyr-705), STAT3, and Mcl-1 were downregulated in the tumors of mice treated with DIM alone or in combination with cisplatin as compared to control or cisplatin treatment. Furthermore, cleavage of caspase 3 and PARP increased significantly in the tumors of mice treated with combination, yet again confirming that DIM suppressed tumor growth by inducing apoptosis in vivo. Furthermore, IL-6 levels in tumor lysates from DIM treated mice were significantly less as compared to levels in the tumor lysates from control mice (Figure 6D). Taken together, our results clearly establish that DIM induces apoptosis in ovarian cancer cells in vitro and in vivo by targeting the STAT3 pathway and synergistically enhancing the effects of 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) suppresses the growth of ovarian tumors alone and in combination with cisplatin by inhibiting signal transducer and activator of transcription 3 (STAT3) in nude mice. SKOV-3 tumor cells were implanted into athymic nude mice. Once each mouse had a palpable tumor, mice received 3 mg/day DIM by oral gavage every day or 5 mg/kg cisplatin intraperitoneally twice a week or both. (A) (i) Effect of DIM on tumor growth. *P < 0.05 when compared to control. (ii) Tumor weight of mice from different groups during the course of in vivo study. (B) Inhibition of STAT3 signaling in the tumors of mice administered with DIM alone or in combination with cisplatin. Tumors from control and treated mice were excised on day 48 after implantation, lysed and analyzed by western blotting for Tyr-705 STAT3, Ser-727 STAT3, total STAT3, Mcl-1, survivin, cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase 3. Blots were stripped and reprobed with actin antibody to verify equal protein loading. Each lane represents a different tumor sample. (C) Densitometric quantitation of western blotting represented above. Legends on the bars indicate (a) statistically significant compared to control, (b) statistically significant compared to DIM, (c) statistically significant compared to cisplatin. (D) Levels of interleukin (IL)-6 in tumors from control mice and DIM treated mice. The differences between all the groups were compared by non-parametric analysis of variance with Bonferroni post hoc comparisons.
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Figure 6: Diindolylmethane (DIM) suppresses the growth of ovarian tumors alone and in combination with cisplatin by inhibiting signal transducer and activator of transcription 3 (STAT3) in nude mice. SKOV-3 tumor cells were implanted into athymic nude mice. Once each mouse had a palpable tumor, mice received 3 mg/day DIM by oral gavage every day or 5 mg/kg cisplatin intraperitoneally twice a week or both. (A) (i) Effect of DIM on tumor growth. *P < 0.05 when compared to control. (ii) Tumor weight of mice from different groups during the course of in vivo study. (B) Inhibition of STAT3 signaling in the tumors of mice administered with DIM alone or in combination with cisplatin. Tumors from control and treated mice were excised on day 48 after implantation, lysed and analyzed by western blotting for Tyr-705 STAT3, Ser-727 STAT3, total STAT3, Mcl-1, survivin, cleaved poly(ADP-ribose) polymerase (PARP) and cleaved caspase 3. Blots were stripped and reprobed with actin antibody to verify equal protein loading. Each lane represents a different tumor sample. (C) Densitometric quantitation of western blotting represented above. Legends on the bars indicate (a) statistically significant compared to control, (b) statistically significant compared to DIM, (c) statistically significant compared to cisplatin. (D) Levels of interleukin (IL)-6 in tumors from control mice and DIM treated mice. The differences between all the groups were compared by non-parametric analysis of variance with Bonferroni post hoc comparisons.
Mentions: We demonstrated that DIM induces apoptosis in ovarian cancer cells and enhances the effect of cisplatin by inhibiting STAT3 pathway in culture models. To validate these effects in vivo, we performed a tumor xenograft assay. About 5 × 106 SKOV-3 cells were injected subcutaneously into both the right and left flanks of female athymic nude mice. Once each mouse had a tumor of about 90 mm3, they were randomized into four groups. DIM treatment started 24 days after tumor implantation, and cisplatin treatment began 10 days later. Our results demonstrated that DIM alone and in combination treatment substantially retarded the growth of SKOV-3 tumors as compared to control or cisplatin treatment. For example, at day 48 the average tumor volume in control mice and cisplatin-treated mice was around 400 mm3 and 300 mm3, respectively, whereas the average tumor volume in mice that received DIM alone or in combination with cisplatin was 210 mm3 and 138 mm3, respectively (Figure 6A). The combination treatment suppressed tumors by 65% as compared to controls. Interestingly, there was no significant change in the weight of mice treated with DIM as compared to mice in the control group. The weight of mice declined significantly in response to cisplatin treatment. However, the decline in weight in the combination treatment group was not as significant as in the mice receiving cisplatin treatment alone (Figure 6A ii). To determine whether DIM-mediated tumor growth suppression was due to inhibition of STAT3, tumor lysates were subjected to western blotting. As shown in Figure 6B, p-STAT3 (Tyr-705), STAT3, and Mcl-1 were downregulated in the tumors of mice treated with DIM alone or in combination with cisplatin as compared to control or cisplatin treatment. Furthermore, cleavage of caspase 3 and PARP increased significantly in the tumors of mice treated with combination, yet again confirming that DIM suppressed tumor growth by inducing apoptosis in vivo. Furthermore, IL-6 levels in tumor lysates from DIM treated mice were significantly less as compared to levels in the tumor lysates from control mice (Figure 6D). Taken together, our results clearly establish that DIM induces apoptosis in ovarian cancer cells in vitro and in vivo by targeting the STAT3 pathway and synergistically enhancing the effects of 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