Increased radiation-induced apoptosis of SaOS2 cells via inhibition of NFκB: A role for c-Jun N-terminal kinase

Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York 14642, USA.
Journal of Cellular Biochemistry (Impact Factor: 3.26). 12/2005; 96(6):1262-73. DOI: 10.1002/jcb.20607
Source: PubMed


To elucidate the possible effect of NFkappaB on radioresistance, we used the osteosarcoma cell line Saos2, stably expressing the NFkappaB constitutive inhibitor, mIkappaB (Saos2-mIkappaB) or stably transfected with the empty vector (Saos2-EV). Ionizing radiation induced "intrinsic" apoptosis in Saos2-mIkappaB cells but not in Saos2-EV control cells, with intact NFkappaB activity. We find as expected, that this NFkappaB activity was enhanced following irradiation in the Saos2-EV control cells. On the other hand, inhibition of NFkappaB signaling in Saos2-mIkappaB cells led to the upregulation of the pro-apoptotic systems, such as Bax protein and c-Jun N-terminal Kinase (JNK)/c-Jun/AP1 signaling. Inhibition of NFkappaB resulted in decreased expression of the DNA damage protein GADD45beta, a known inhibitor of JNK. Subsequently, JNK activation of c-Jun/AP-1 proteins increased radiation-induced apoptosis in these mutants. Radiation-induced apoptosis in Saos2-mIkappaB cells was inhibited by the JNK specific inhibitor SP600125 as well as by Bcl-2 over-expression. Furthermore, release of cytochrome-c from mitochondria was increased and caspase-9 and -3 were activated following irradiation in Saos2-mIkappaB cells. Antisense inhibition of GADD45beta in Saos2-EV cells significantly enhanced apoptosis following irradiation. Our results demonstrate that radioresistance of Saos2 osteosarcoma cells is due to NFkappaB-mediated inhibition of JNK. Our study brings new insight into the mechanisms underlying radiation-induced apoptosis of osteosarcoma, and may lead to development of new therapeutic strategies against osteosarcoma.

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    • "We reviewed experimentally identified miR-221 targets or upstream genes and pathways. Among them, there are several genes and pathways mediate osteosarcoma tumorigenicity such as p57 [54], [55], NF-κb [56], [57], p27 [21], [58], Notch pathway [59] and so on. Our work identified a role for PTEN in miRNA-221-induced biology and confirmed p27 but not p57 could be regulated by miR-221, these results indicate that miR-221 induces cisplatin resistance directly through at least p27 and PTEN pathway, it remains possible other factors might be partially involved. "
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    ABSTRACT: Background MicroRNAs are short regulatory RNAs that negatively modulate protein expression at a post-transcriptional and/or translational level and are deeply involved in the pathogenesis of several types of cancers. Specifically, microRNA-221 (miR-221) is overexpressed in many human cancers, wherein accumulating evidence indicates that it functions as an oncogene. However, the function of miR-221 in human osteosarcoma has not been totally elucidated. In the present study, the effects of miR-221 on osteosarcoma and the possible mechanism by which miR-221 affected the survival, apoptosis, and cisplatin resistance of osteosarcoma were investigated. Methodology/Principal Findings Real-time quantitative PCR analysis revealed miR-221 was significantly upregulated in osteosarcoma cell lines than in osteoblasts. Both human osteosarcoma cell lines SOSP-9607 and MG63 were transfected with miR-221 mimic or inhibitor to regulate miR-221 expression. The effects of miR-221 were then assessed by cell viability, cell cycle analysis, apoptosis assay, and cisplatin resistance assay. In both cells, upregulation of miR-221 induced cell survival and cisplatin resistance and reduced cell apoptosis. In addition, knockdown of miR-221 inhibited cell growth and cisplatin resistance and induced cell apoptosis. Potential target genes of miR-221 were predicted using bioinformatics. Moreover, luciferase reporter assay and western blot confirmed that PTEN was a direct target of miR-221. Furthermore, introduction of PTEN cDNA lacking 3′-UTR or PI3K inhibitor LY294002 abrogated miR-221-induced cisplatin resistance. Finally, both miR-221 and PTEN expression levels in osteosarcoma samples were examined by using real-time quantitative PCR and immunohistochemistry. High miR-221 expression level and inverse correlation between miR-221 and PTEN levels were revealed in osteosarcoma tissues. Conclusions/Significance These results for the first time demonstrate that upregulation of miR-221 induces the malignant phenotype of human osteosarcoma whereas knockdown of miR-221 reverses this phenotype, suggesting that miR-221 could be a potential target for osteosarcoma treatment.
    PLoS ONE 01/2013; 8(1):e53906. DOI:10.1371/journal.pone.0053906 · 3.23 Impact Factor
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    • "In non-pathogenic cell lines with minimal basal levels of NF-κB activity, exposure to ionizing radiation has been shown to stimulate the JNK pathway, leading to initiation of the intrinsic apoptotic program (Kuwabara et al., 2003; Dent et al., 2003). Elevated NF-κB signaling in osteosarcoma enables cells to evade radiation-induced apoptosis by inhibiting JNK pathway signaling (Eliseev et al., 2005). In addition, our data indicate that irradiation further induces NF-κB activity, subsequently escalating the resistance to radiation. "
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    ABSTRACT: Osteosarcoma is a devastating tumor of bone, primarily affecting adolescents. Osteosarcoma tumors are notoriously radioresistant. Radioresistant cancers, including osteosarcoma, typically exhibit a considerable potential for relapse and development of metastases following treatment. Relapse and metastatic potential can, in part, be due to a specific radioresistant subpopulation of cells with stem-like characteristics, cancer stem cells, which maintain the capacity to regenerate entire tumors. In the current study, we have investigated whether in vitro treatments with parthenolide, a naturally occurring small molecule that interferes with NF-κB signaling and has various other effects, will re-sensitize cancer stem cells and the entire cell population to radiotherapy in osteosarcoma. Our results indicate that parthenolide and ionizing radiation synergistically induce cell death in LM7 osteosarcoma cells. Importantly, the combination treatment results in a significant reduction in the viability of both the overall population of osteosarcoma cells and the cancer stem cell subpopulation. This effect is dependent on the ability of parthenolide to induce oxidative stress. Therefore, as a supplement to current multimodal therapy, parthenolide may sensitize osteosarcoma tumors to radiation and greatly reduce the prevalence of relapse and metastatic progression.
    Journal of Cellular Biochemistry 04/2012; 113(4):1282-91. DOI:10.1002/jcb.24002 · 3.26 Impact Factor
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    ABSTRACT: Nuclear Factor kappa B (NFkappaB) is a eukaryotic transcription factor that is constitutively active in human cancers and can be inhibited by the naturally occurring sesquiterpene lactone, parthenolide (P). The in vitro effects of P were assessed using the androgen independent cell line, CWR22Rv1, and human umbilical endothelial cells (HUVECs). The in vivo activity of P as a single agent and its ability to augment the efficacy of docetaxel and the anti-androgen, bicalutamide, were determined using the CWR22Rv1 xenograft model. Parthenolide at low micromolar concentration inhibited proliferation of CWR22Rv1 and HUVEC cells, promoted apoptosis and abrogated NFkappaB-DNA binding. Parthenolide downregulated anti-apoptotic genes under NFkappaB control, TRAF 1 and 2, and promoted sustained activation of c-jun-NH2 kinase (JNK). Parthenolide also augmented the in vivo efficacy of docetaxel and restored sensitivity to anti-androgen therapy. These studies demonstrate parthenolide's anti-tumor and anti-angiogenic activity, and its potential to augment the efficacy of chemotherapy and hormonal therapy.
    The Prostate 10/2006; 66(14):1498-511. DOI:10.1002/pros.20482 · 3.57 Impact Factor
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