glaucocalyxin-a and Osteosarcoma

Osteosarcoma (OS) is ranked as the most common primary bone malignancy in children and adolescents worldwide, and the 5-year overall survival rate of OS is not optimistic. Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been implicated in tumor cell growth, proliferation, and anti-apoptosis in OS. Therefore, the discovery of novel molecular compounds that can effectively block STAT3 activation, is essential for the treatment of OS and improving prognosis. Here, we investigate whether Glaucocalyxin A (GLA), derived from Rabdosia japonica, exhibit the potential anticancer effects in OS. First of all, we identify that GLA potently suppressed cell proliferation, induced G2/M phase arrest and promoted substantial apoptosis in OS. Next, we conclude that GLA could induce Reactive oxygen species (ROS)-mediated oxidative stress via an imbalance of GSH and GSSG. Then, we elucidate for the first time that GLA could significantly inhibit both constitutive and IL-6-inducible activation of STAT3 (Tyr705) and JAK2, the upstream regulator of STAT3. Furthermore, we elucidate that the inhibition of STAT3 is mainly induced by ROS-mediated oxidative stress. Overall, our findings demonstrate that GLA could exhibit potent anticancer effects through effectively blocking the STAT3 signaling pathway, which was induced by ROS-mediated oxidative stress in OS in vitro and in vivo.

Topics: A549 Cells; Animals; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Disease Progression; Diterpenes, Kaurane; Hep G2 Cells; Humans; Mice, Inbred BALB C; Mice, Nude; Osteosarcoma; Oxidative Stress; Phytotherapy; Signal Transduction; STAT3 Transcription Factor; Tumor Burden; Xenograft Model Antitumor Assays

Metastatic osteosarcoma usually has an unsatisfactory response to the current standard chemotherapy and causes poor prognosis. Currently, epithelial-mesenchymal transition (EMT) is reported as a critical event in osteosarcoma metastasis. Glaucocalyxin A, a bioactive ent-kauranoid diterpenoid, exerts anti-cancer effect on osteosarcoma by inducing apoptosis in previous study. However, the effect of Glaucocalyxin A on EMT and metastasis of osteosarcoma is unclear. In this study, we investigated the potential mechanisms of Glaucocalyxin A on EMT and metastasis of osteosarcoma. We found that Glaucocalyxin A inhibited migration and invasion of MG-63 and 143B cells. Moreover, Glaucocalyxin A increased the protein and mRNA levels of E-cadherin and decreased the protein and transcription expression of N-cadherin, Vimentin. Glaucocalyxin A also inhibited the protein and mRNA levels of EMT-associated transcription factor including Snail and Slug. Furthermore, Glaucocalyxin A inhibited transforming growth factor-β1 (TGF-β1)-induced migration, invasion and EMT of low-metastatic osteosarcoma U2OS cells. Glaucocalyxin A inhibited TGF-β-induced phosphorylation of Smad 2/3 in osteosarcoma U2OS cells. Finally, we established transplanted metastatic models of highly metastatic osteosarcoma 143B cells. Glaucocalyxin A inhibited lung metastasis in vivo. Interestingly, Glaucocalyxin A increased the protein expression of E-cadherin and reduced the protein expression of N-cadherin and Vimentin. Glaucocalyxin A inhibited the protein expression of Snail and Slug in vivo. In summary, this study demonstrated that Glaucocalyxin A inhibited EMT and TGF-β1-induced EMT by inhibiting TGF-β1/Smad2/3 signaling pathway in osteosarcoma. Therefore, Glaucocalyxin A might be a promising candidate against the metastasis of human osteosarcoma.

Topics: Animals; Bone Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Diterpenes, Kaurane; Epithelial-Mesenchymal Transition; Humans; Lung Neoplasms; Mice; Mice, Nude; Osteosarcoma; Phosphorylation; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta1; Vimentin

Osteosarcoma, the most common malignant bone tumor with recurring disease or lung metastases, has become one of the leading causes of death in humans. In the current study, we made an investigation on the anticancer effect of glaucocalyxin A, a bioactive ent-kauranoid diterpenoid isolated from Rabdosia japonica var., and unraveled the underlying mechanisms. Here, we found that Glaucocalyxin A inhibited the cell viability of numerous osteosarcoma cells. Our results showed that Glaucocalyxin A exerted the pro-apoptotic effect on human osteosarcoma cells, MG-63 and HOS cells. Glaucocalyxin A induced apoptosis by mitochondrial apoptotic pathway through several steps including increasing the Bax/Bcl-2 ratio, triggering the intracellular reactive oxygen species (ROS) generation, reducing mitochondrial membrane potential (MMP), and inducing cleavage of caspase-9 and caspase-3. We demonstrated that Glaucocalyxin A induced apoptosis via inhibiting Five-zinc finger Glis 1 (GLI1) activation by overexpression and knockdown of GLI1 in vitro. We also found that Glaucocalyxin A inhibited GLI1 activation via regulating phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway. We further confirmed our findings by using PI3K activator and inhibitor to verify the inhibitory effect of Glaucocalyxin A on PI3K/Akt/GLI1 pathway. Moreover, our in vivo study revealed that glaucocalyxin A possessed a remarkable antitumor effect with no toxicity in the xenograft model inoculated with HOS tumor through the same mechanisms as in vitro. In conclusion, our results suggested that Glaucocalyxin A induced apoptosis in osteosarcoma by inhibiting nuclear translocation of GLI1 via regulating PI3K/Akt signaling pathway. Thus, Glaucocalyxin A might be a potential candidate for human osteosarcoma in the future.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Diterpenes, Kaurane; Humans; Male; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Osteosarcoma; Phosphatidylinositol 3-Kinases; Protein Transport; Proto-Oncogene Proteins c-akt; Signal Transduction; Zinc Finger Protein GLI1