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Oleanane triterpenoid CDDO-Me induces apoptosis in multidrug resistant osteosarcoma cells through inhibition of Stat3 pathway.

Ryu K, Susa M, Choy E, Yang C, Hornicek FJ, Mankin HJ, Duan Z - BMC Cancer (2010)

Bottom Line: Treatment with CDDO-Me significantly decreased the level of nuclear translocation and phosphorylation of Stat3.Furthermore, CDDO-Me increased the cytotoxic effects of doxorubicin in the MDR osteosarcoma cell lines.This study provides the framework for the clinical evaluation of CDDO-Me, either as monotherapy or perhaps even more effectively in combination with doxorubicin to treat osteosarcoma and overcome drug resistance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedic Surgery, Massachusetts General Hospital, Boston, MA 02114, USA.

ABSTRACT

Background: The activation of signal transducer and activator of transcription 3 (Stat3) pathway correlates with tumor growth, survival, drug resistance and poor prognosis in osteosarcoma. To explore the potential therapeutic values of this pathway, we assessed both the expression and the activation of Stat3 pathway in several pairs of multidrug resistant (MDR) osteosarcoma cell lines, and tissues. To explore the potential therapeutic values of this pathway, we analyzed the ability of the synthetic oleanane triterpenoid, C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me), to inhibit Stat3 expression and activation as well as its effects on doxorubicin sensitivity in osteosarcoma cells.

Methods: Expression of Stat3, phosphorylated Stat3 (pStat3) and Stat3 targeted proteins, including Bcl-XL, Survivin and MCL-1 were determined in drug sensitive and MDR osteosarcoma cell lines and tissues by Western blot analysis. The effect of CDDO-Me on osteosarcoma cell growth was evaluated by MTT and apoptosis by PARP cleavage assay and caspase-3/7 activity.

Results: Stat3 pathway was activated in osteosarcoma tissues and in MDR cell lines. CDDO-Me inhibited growth and induced apoptosis in osteosarcoma cell lines. Treatment with CDDO-Me significantly decreased the level of nuclear translocation and phosphorylation of Stat3. The inhibition of Stat3 pathway correlated with the suppression of the anti-apoptotic Stat3 targeted genes Bcl-XL, survivin, and MCL-1. Furthermore, CDDO-Me increased the cytotoxic effects of doxorubicin in the MDR osteosarcoma cell lines.

Conclusions: Stat3 pathway is overexpressed in MDR osteosarcoma cells. CDDO-Me significantly inhibited Stat3 phosphorylation, Stat3 nuclear translocation and induced apoptosis in osteosarcoma. This study provides the framework for the clinical evaluation of CDDO-Me, either as monotherapy or perhaps even more effectively in combination with doxorubicin to treat osteosarcoma and overcome drug resistance.

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Related in: MedlinePlus

CDDO-Me inhibits EGFP-Stat3 nuclear translocation in osteosarcoma cell line U-2OS. U-2OS cells which stably express the EGFP-Stat3 fusion protein were incubated for 4 h with either IL-6 alone (A-D) or CDDO-Me (1 μM) followed immediately thereafter with the addition of IL-6 to a final concentration of 30 ng/ml (A'-D'). To counterstain the nuclei, the cells were incubated with 1 μg/ml Hoechst 33342 (Invitrogen, Carlsbad, CA) for 1 minute. Cells were photographed 1 h later. Subcellular localization of the fusion protein was assessed by fluorescence microscopy.
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Figure 4: CDDO-Me inhibits EGFP-Stat3 nuclear translocation in osteosarcoma cell line U-2OS. U-2OS cells which stably express the EGFP-Stat3 fusion protein were incubated for 4 h with either IL-6 alone (A-D) or CDDO-Me (1 μM) followed immediately thereafter with the addition of IL-6 to a final concentration of 30 ng/ml (A'-D'). To counterstain the nuclei, the cells were incubated with 1 μg/ml Hoechst 33342 (Invitrogen, Carlsbad, CA) for 1 minute. Cells were photographed 1 h later. Subcellular localization of the fusion protein was assessed by fluorescence microscopy.

Mentions: To identify the interruption of IL-6 dependent Stat3 nuclear translocation by CDDO-Me, a novel real-time cell-based method was developed to image the EGFP-Stat3 chimera in the nucleus and cytoplasm in human osteosarcoma cell line U-2OS. Resting cells demonstrated that the majority of EGFP-Stat3 was cytoplasmic (Fig. 4A, A') until the addition of IL-6, which then promptly induced translocation of fluorescent Stat3 to the nucleus in U-2OS cells (Fig. 4B). Pretreatment of the cells with CDDO-Me (1 μM) blocked IL-6 dependent EGFP-Stat3 nuclear translocation (Fig. 4B').


Oleanane triterpenoid CDDO-Me induces apoptosis in multidrug resistant osteosarcoma cells through inhibition of Stat3 pathway.

Ryu K, Susa M, Choy E, Yang C, Hornicek FJ, Mankin HJ, Duan Z - BMC Cancer (2010)

CDDO-Me inhibits EGFP-Stat3 nuclear translocation in osteosarcoma cell line U-2OS. U-2OS cells which stably express the EGFP-Stat3 fusion protein were incubated for 4 h with either IL-6 alone (A-D) or CDDO-Me (1 μM) followed immediately thereafter with the addition of IL-6 to a final concentration of 30 ng/ml (A'-D'). To counterstain the nuclei, the cells were incubated with 1 μg/ml Hoechst 33342 (Invitrogen, Carlsbad, CA) for 1 minute. Cells were photographed 1 h later. Subcellular localization of the fusion protein was assessed by fluorescence microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874784&req=5

Figure 4: CDDO-Me inhibits EGFP-Stat3 nuclear translocation in osteosarcoma cell line U-2OS. U-2OS cells which stably express the EGFP-Stat3 fusion protein were incubated for 4 h with either IL-6 alone (A-D) or CDDO-Me (1 μM) followed immediately thereafter with the addition of IL-6 to a final concentration of 30 ng/ml (A'-D'). To counterstain the nuclei, the cells were incubated with 1 μg/ml Hoechst 33342 (Invitrogen, Carlsbad, CA) for 1 minute. Cells were photographed 1 h later. Subcellular localization of the fusion protein was assessed by fluorescence microscopy.
Mentions: To identify the interruption of IL-6 dependent Stat3 nuclear translocation by CDDO-Me, a novel real-time cell-based method was developed to image the EGFP-Stat3 chimera in the nucleus and cytoplasm in human osteosarcoma cell line U-2OS. Resting cells demonstrated that the majority of EGFP-Stat3 was cytoplasmic (Fig. 4A, A') until the addition of IL-6, which then promptly induced translocation of fluorescent Stat3 to the nucleus in U-2OS cells (Fig. 4B). Pretreatment of the cells with CDDO-Me (1 μM) blocked IL-6 dependent EGFP-Stat3 nuclear translocation (Fig. 4B').

Bottom Line: Treatment with CDDO-Me significantly decreased the level of nuclear translocation and phosphorylation of Stat3.Furthermore, CDDO-Me increased the cytotoxic effects of doxorubicin in the MDR osteosarcoma cell lines.This study provides the framework for the clinical evaluation of CDDO-Me, either as monotherapy or perhaps even more effectively in combination with doxorubicin to treat osteosarcoma and overcome drug resistance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedic Surgery, Massachusetts General Hospital, Boston, MA 02114, USA.

ABSTRACT

Background: The activation of signal transducer and activator of transcription 3 (Stat3) pathway correlates with tumor growth, survival, drug resistance and poor prognosis in osteosarcoma. To explore the potential therapeutic values of this pathway, we assessed both the expression and the activation of Stat3 pathway in several pairs of multidrug resistant (MDR) osteosarcoma cell lines, and tissues. To explore the potential therapeutic values of this pathway, we analyzed the ability of the synthetic oleanane triterpenoid, C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me), to inhibit Stat3 expression and activation as well as its effects on doxorubicin sensitivity in osteosarcoma cells.

Methods: Expression of Stat3, phosphorylated Stat3 (pStat3) and Stat3 targeted proteins, including Bcl-XL, Survivin and MCL-1 were determined in drug sensitive and MDR osteosarcoma cell lines and tissues by Western blot analysis. The effect of CDDO-Me on osteosarcoma cell growth was evaluated by MTT and apoptosis by PARP cleavage assay and caspase-3/7 activity.

Results: Stat3 pathway was activated in osteosarcoma tissues and in MDR cell lines. CDDO-Me inhibited growth and induced apoptosis in osteosarcoma cell lines. Treatment with CDDO-Me significantly decreased the level of nuclear translocation and phosphorylation of Stat3. The inhibition of Stat3 pathway correlated with the suppression of the anti-apoptotic Stat3 targeted genes Bcl-XL, survivin, and MCL-1. Furthermore, CDDO-Me increased the cytotoxic effects of doxorubicin in the MDR osteosarcoma cell lines.

Conclusions: Stat3 pathway is overexpressed in MDR osteosarcoma cells. CDDO-Me significantly inhibited Stat3 phosphorylation, Stat3 nuclear translocation and induced apoptosis in osteosarcoma. This study provides the framework for the clinical evaluation of CDDO-Me, either as monotherapy or perhaps even more effectively in combination with doxorubicin to treat osteosarcoma and overcome drug resistance.

Show MeSH
Related in: MedlinePlus