Gamabufotalin (CS-6), a major bufadienolide of Chansu, has been used for cancer therapy due to its desirable metabolic stability and less adverse effect. However, the underlying mechanism of CS-6 involved in anti-tumor activity remains poorly understood. The biological functions of gamabufotalin (CS-6) were investigated by migration, colony formation and apoptosis assays in NSCLC cells. The nuclear localization and interaction between transcriptional co-activator p300 and NF-κB p50/p65 and their binding to COX-2 promoter were analyzed after treatment with CS-6. Molecular docking study was used to simulate the interaction of CS-6 with IKKβ. The in vivo anti-tumor efficacy of CS-6 was also analyzed in xenografts nude mice. Western blot was used to detect the protein expression level. Gamabufotalin (CS-6) strongly suppressed COX-2 expression by inhibiting the phosphorylation of IKKβ via targeting the ATP-binding site, thereby abrogating NF-κB binding and p300 recruitment to COX-2 promoter. In addition, CS-6 induced apoptosis by activating the cytochrome c and caspase-dependent apoptotic pathway. Moreover, CS-6 markedly down-regulated the protein levels of COX-2 and phosphorylated p65 NF-κB in tumor tissues of the xenograft mice, and inhibited tumor weight and size. Our study provides pharmacological evidence that CS-6 exhibits potential use in the treatment of COX-2-mediated diseases such as lung cancer.

Ovarian cancer (OvCa) is a leading cause of mortality from gynecologic malignancy due to its disseminated peritoneal metastasis. The tumor microenvironment dominates epithelial–mesenchymal transition (EMT) development and impacts cancer metastasis as well as mediates drug resistance. Tumour cell interaction with the collagen I matrix is critical in OvCa development. To better understand the role of the collagen matrix and the underlying mechanisms in the early stage of OvCa invasion, we developed a three-dimensional (3D) culture model in vitro by embedding OvCa cells within collagen I to recreate the architecture of a solid tumour. Our results showed that tumour spheroids formed in the 3D collagen model displayed good viability and decreased growth rates, which partly recapitulated the growth behavior of in vivo tumour cells. Collagen I enhanced the OvCa cell motility/invasion capability by up-regulating the expression of MMPs and α5β1 integrin. Moreover, highly invasive OvCa cells in collagen showed the overexpression of mesenchymal markers (N-cadherin, vimentin and fibronectin) and transcriptional factors (Snail and Slug). EMT-associated TGF-β1/Smad4 and Wnt5b/β-catenin signaling pathways were significantly up-regulated accordingly. Additionally, a remarkably enhanced drug resistance to chemotherapeutics was also detected in the 3D cultures. Collectively, the bioengineered 3D collagen models could recapitulate the in vivo tumour-like microenvironment and reflect some biological characteristics of human OvCa more accurately. The collagen I matrix promoted local invasion via EMT and enhanced the multidrug resistance in OvCa. This system might serve as a comprehensive in vitro model to better understand the manifold mechanisms of OvCa metastasis and also provide a robust tool for screening new anti-ovarian cancer therapeutics.

Autophagy predominantly promotes cell survival by recycling cell components, while it kills cells in specific contexts. Cell death related to autophagy plays important roles in multiple physiological and pathological situations including tumorigenesis, and the mechanism needs to be defined further. PRAS40 was found to be crucial in various cancers, and phosphorylation was reported to be involved in autophagy inhibition in monocytes. However, the detailed role of PRAS40 in autophagy and the relationship to tumorigenesis remain largely unknown. Herein we screened the binding partners of PRAS40, and found that PRAS40 interacted with Phosphoglycerate kinase 1 (PGK1). PGK1 phosphorylated PRAS40 at Threonine 246, which could be inhibited by blocking the interaction. Both in vitro and in vivo results revealed that PRAS40 mediated PGK1-induced cell growth. By tracing the mechanism, we found that PGK1 suppressed autophagy-mediated cell death, in which PRAS40 was crucial. Thus PGK1 phosphorylates PRAS40 to repress autophagy-mediated cell death under normoxia, promoting cellular proliferation. The binding of PGK1 to PRAS40 was transferred to Beclin1 under hypoxia, resulting in the increase of Beclin1 phosphorylation. These results suggest a novel model of tumorigenesis, in which PGK1 switches between repressing autophagy-mediated cell death via PRAS40 and inducing autophagy through Beclin1 according to the environmental oxygen level. Our study is anticipated to be able to offer novel insights in understanding PGK1/PRAS40 signaling hyperactivated cancers.