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

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.

Figure 1

Activation of Stat3, pStat3, Stat3-mediated antiapoptotic proteins, and Pgp1 in osteosarcoma cell lines and tissues. A) Osteoblast cell lines, drug sensitive and MDR osteosarcoma cell lines. B) Osteosarcoma tissues. Expression was assessed with total cellular protein isolated from the indicated cell lines and immunoblotted with specific antibodies as described in Materials and Methods. The blots were also probed with an anti-actin monoclonal antibody to assess relative protein levels in the sample lanes.

Figure 2

CDDO-Me inhibits growth and induces apoptosis in osteosarcoma cell lines. A) To analyze the effect of CDDO-Me, KHOS, U-2OS, KHOS_R2, U-2OS_TR, and HOB-c were exposed to varying concentrations of CDDO-Me for 7 days. Growth inhibition was assessed by MTT. B) Parental cells KHOS, U-2OS and MDR cells KHOS_R2 and U-2OS_TR were treated with CDDO-Me in a dose-dependent manner. Total cellular proteins were subjected to immunoblotting with specific antibodies to PARP and β-actin. PARP cleavage was detected in all four osteosarcoma cell lines after treatment with CDDO-Me. The anti-PARP antibodies demonstrate 118 kd full length PARP and 85 kd cleaved PARP fragment.

Figure 3

Caspase-3/7 activity was measured as a second parameter of apoptotic cell death. KHOS and KHOS_R2 showed significant increase in apoptosis when they were treated with increasing concentrations of CDDO-Me. The experiment was repeated three times in triplicate.

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.

Figure 5

Dose-dependent inhibition of Stat3 phosphorylation and down-regulated Stat3-mediated antiapoptotic proteins by CDDO-Me. Parental cells KHOS, U-2OS (A) and MDR cells KHOS_R2, U-2OS_TR (B) were treated with CDDDO-Me for 24 hours in 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.

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 KHOS_R2, U-2OS_TR (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.

Figure 7

CDDO-Me shows synergy with doxorubicin and overcomes drug resistance in MDR osteosarcoma cell lines. A) CDDO-Me inhibits cell growth in KHOS_R2 and U-2OS_TR. Cells were seeded at a density of 8,000 cells per well in a 96-well plate for 24 hours. The cells were then treated with 0.6 μM doxorubicin, 0.1 μM CDDO-Me, 0.3 μM CDDO-Me or combination of different two drugs for additional 24 hours. The cells were analyzed by MTT assay as described in Materials and Methods (B and C). CDDO-Me together with doxorubicin overcome drug resistance in both MDR KHOS_R2 and U-2OS_TR cells.