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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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Time-dependent inhibition of Stat3 phosphorylation and down-regulated Stat3-mediated antiapoptotic proteins by CDDO-Me. Parental cells KHOS, U-2OS (A) and MDR cells KHOSR2, U-2OSTR (B) were treated for 24 h with CDDO-Me (1 μM), and then harvested and processed for Western blotting. For Western blot analysis, 30 μg of total cellular proteins were subjected to immunoblotting with specific antibodies.
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Figure 6: Time-dependent inhibition of Stat3 phosphorylation and down-regulated Stat3-mediated antiapoptotic proteins by CDDO-Me. Parental cells KHOS, U-2OS (A) and MDR cells KHOSR2, U-2OSTR (B) were treated for 24 h with CDDO-Me (1 μM), and then harvested and processed for Western blotting. For Western blot analysis, 30 μg of total cellular proteins were subjected to immunoblotting with specific antibodies.

Mentions: To evaluate the time-dependent inhibition of Stat3 activation, the cell lines were treated with 1 μM of CDDO-Me for varying time periods for 24 h. In a time-dependent manner, the pStat3 level decreased as early as 4 hours after the addition of 1 μM of CDDO-Me treatment (Fig. 6). We next examined whether exposure of cell lines to CDDO-Me resulted in decreased expression of the antiapoptotic proteins Bcl-XL, survivin and MCL-1 by time-dependent inhibition (Fig. 6). Results showed incubation of CDDO-Me for 24 h caused significant down-regulation of Bcl-XL, survivin and MCL-1 expression in both drug sensitive (Fig. 6A) and MDR (Fig. 6B) cell lines in a time dependent manner. The effect of CDDO-Me on the induction of apoptosis was assessed by evaluating PARP cleavage in a time-dependent manner. PARP cleavage was detected in all cells after 12 hours of treatment with 1 μM of CDDO-Me (Fig. 6).


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)

Time-dependent inhibition of Stat3 phosphorylation and down-regulated Stat3-mediated antiapoptotic proteins by CDDO-Me. Parental cells KHOS, U-2OS (A) and MDR cells KHOSR2, U-2OSTR (B) were treated for 24 h with CDDO-Me (1 μM), and then harvested and processed for Western blotting. For Western blot analysis, 30 μg of total cellular proteins were subjected to immunoblotting with specific antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Time-dependent inhibition of Stat3 phosphorylation and down-regulated Stat3-mediated antiapoptotic proteins by CDDO-Me. Parental cells KHOS, U-2OS (A) and MDR cells KHOSR2, U-2OSTR (B) were treated for 24 h with CDDO-Me (1 μM), and then harvested and processed for Western blotting. For Western blot analysis, 30 μg of total cellular proteins were subjected to immunoblotting with specific antibodies.
Mentions: To evaluate the time-dependent inhibition of Stat3 activation, the cell lines were treated with 1 μM of CDDO-Me for varying time periods for 24 h. In a time-dependent manner, the pStat3 level decreased as early as 4 hours after the addition of 1 μM of CDDO-Me treatment (Fig. 6). We next examined whether exposure of cell lines to CDDO-Me resulted in decreased expression of the antiapoptotic proteins Bcl-XL, survivin and MCL-1 by time-dependent inhibition (Fig. 6). Results showed incubation of CDDO-Me for 24 h caused significant down-regulation of Bcl-XL, survivin and MCL-1 expression in both drug sensitive (Fig. 6A) and MDR (Fig. 6B) cell lines in a time dependent manner. The effect of CDDO-Me on the induction of apoptosis was assessed by evaluating PARP cleavage in a time-dependent manner. PARP cleavage was detected in all cells after 12 hours of treatment with 1 μM of CDDO-Me (Fig. 6).

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