Critical Role of Heat Shock Protein 27 in Bufalin-Induced Apoptosis in Human Osteosarcomas: A Proteomic-Based Research

Abstract

Bufalin is the primary component of the traditional Chinese herb “Chan Su”. Evidence suggests that this compound possesses potent anti-tumor activities, although the exact molecular mechanism(s) is unknown. Our previous study showed that bufalin inhibited growth of human osteosarcoma cell lines U2OS and U2OS/MTX300 in culture. Therefore, this study aims to further clarify the in vitro and in vivo anti-osteosarcoma effects of bufalin and its molecular mechanism of action. We found bufalin inhibited both methotrexate (MTX) sensitive and resistant human osteosarcoma cell growth and induced G2/M arrest and apoptosis. Using a comparative proteomics approach, 24 differentially expressed proteins following bufalin treatment were identified. In particular, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27), decreased remarkably. The down-regulation of Hsp27 and alterations of its partner signaling molecules (the decrease in p-Akt, nuclear NF-κB p65, and co-immunoprecipitated cytochrome c/Hsp27) were validated. Hsp27 over-expression protected against bufalin-induced apoptosis, reversed the dephosphorylation of Akt and preserved the level of nuclear NF-κB p65 and co-immunoprecipitated Hsp27/cytochrome c. Moreover, bufalin inhibited MTX-resistant osteosarcoma xenograft growth, and a down-regulation of Hsp27 in vivo was observed. Taken together, bufalin exerted potent anti-osteosarcoma effects in vitro and in vivo, even in MTX resistant osteosarcoma cells. The down-regulation of Hsp27 played a critical role in bufalin-induced apoptosis in osteosarcoma cells. Bufalin may have merit to be a potential chemotherapeutic agent for osteosarcoma, particularly in MTX-resistant groups.

Degalactotigonin, a Natural Compound from Solanum nigrum L., Inhibits Growth and Metastasis of Osteosarcoma through GSK3β Inactivation–Mediated Repression of the Hedgehog/Gli1 Pathway

“…Owing to their safety, long-term use, and their ability to target multiple pathways, there is a renewed interest in understanding the molecular mechanisms underlying their activity. Our previous studies have also provided evidence of some natural agents having potential anti-osteosarcoma activity, such as dihydromyricetin, cinobufagin, and bufalin (19,39,40). However, there is no evidence at the cellular level or in animal models for such an effect of DGT on osteosarcoma progression.…”

Anticancer Effects of Bufalin on Human Hepatocellular Carcinoma HepG2 Cells: Roles of Apoptosis and Autophagy

“…It has been demonstrated that bufalin exerts cardiotonic, anesthetic, blood pressure stimulation, respiration, and antineoplastic activities [8]. Several preclinical studies indicated bufalin exerts growth inhibition, cell cycle arrest, induces differentiation and apoptosis in gynecologic cancers [9], gastric cancer [10], prostate cancer [11], liver cancer [12], leukemia [13], melanoma [14], lung cancer [15], breast cancer [16], colon cancer [17], and osteosarcoma [18]. The mechanism of bufalin-induced apoptosis has been reported to be the activation of transcription factor AP-1, Rac1, cdc2 kinase, the induction of Tiam1, suppression PI3K/Akt, JNK1/2, ERK1/2 networks, and the elevation of intracellular calcium concentrations.…”

Possible target‐related proteins and signal network of bufalin in A549 cells suggested by both iTRAQ‐based and label‐free proteomic analysis

“…As far as we know, there were only two published reports about proteomic study of BF‐treated cells, one by Xie et al. and one by our lab . But, both of these two studies were based on 2DE analysis.…”