Bufotalin attenuates pulmonary fibrosis via inhibiting Akt/GSK-3ß/ß-catenin signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease with no cure. Bufotalin (BT), an active component extracted from Venenum Bufonis, has been prescribed as a treatment for chronic inflammatory diseases. However, whether BT has antifibrotic properties has never been investigated. In this study, we report on the potential therapeutic effect and mechanism of BT on IPF. BT was shown to attenuate lung injury, inflammation, and fibrosis as well as preserve pulmonary function in bleomycin (BLM)-induced pulmonary fibrosis model. We next confirmed BT's ability to inhibit TGF-ß1-induced epithelial-mesenchymal transition (EMT) and myofibroblast activation (including differentiation, proliferation, migration, and extracellular matrix production) in vitro. Furthermore, transcriptional profile analysis indicated the Wnt signaling pathway as a potential target of BT. Mechanistically, BT effectively prevented ß-catenin from translocating into the nucleus to activate transcription of profibrotic genes. This was achieved by blunting TGF-ß1-induced increases in phosphorylated Akt Ser437 (p-Akt S437) and phosphorylated glycogen synthase kinase (GSK)-3ß Ser9 (p-GSK-3ß S9), thereby reactivating GSK-3ß. Additionally, the antifibrotic effects of BT were further validated in another in vivo model of radiation-induced pulmonary fibrosis. Collectively, these data demonstrated the potent antifibrotic actions of BT through inhibition of Akt/GSK-3ß/ß-catenin axis downstream of TGF-ß1. Thus, BT could be a potential option to be further explored in IPF treatment.

MDM2-mediated Inhibitory Effect of Arsenic Trioxide on Small Cell Lung Cancer Cell Line by Degrading Mutant p53.

INTRODUCTION: Small cell lung cancer (SCLC) is featured by a high TP53 mutant rate. Our previous research found that arsenic trioxide (As2O3) could significantly inhibit the growth and metastasis of SCLC. Studies have shown that the degradation of mutant p53 mediated by murine double minute 2 (MDM2) can be induced by As2O3, which probably contributes to the inhibition of SCLC, but the detailed mechanism is still unclear. We aimed to testify that As2O3 can inhibit the growth of SCLC cells by degrading mutant p53 protein via binding to MDM2. METHODS: CCK-8 assay, cell cycle analysis, and western blot of apoptosis markers were used to evaluate the inhibitory effect of As2O3 on NCI-H446 cells (containing mutant p53) and NCI-H1299 cells (p53 null). The effects of As2O3 on p53 and its downstream proteins were identified by western blot using mut-p53-knockdown and overexpressed cell models. MDM2-knockdown cell models were constructed, and western blot, co-IP of mut-p53, and ubiquitin were carried out to explore the mediating effect of MDM2 in As2O3 induced mut-p53 degradation. RESULTS: As2O3 inhibited proliferation and induced cell cycle arrest and apoptosis of SCLC cells in a dose- and timedependent manner. After mut-p53 knockdown or overexpressed, the inhibitory effect of As2O3 was dampened or enhanced. Additionally, As2O3-induced mut-p53 ubiquitination was significantly weakened after MDM2 knockdown. CONCLUSION: As2O3 could inhibit SCLC cells by inhibiting proliferation and inducing cell cycle arrest and apoptosis. These inhibitory effects were achieved at least in part by upregulating MDM2, which, in turn, promotes ubiquitination and degradation of mut-p53.

Arsenic trioxide elicits anti-tumor activity by inhibiting polarization of M2-like tumor-associated macrophages via Notch signaling pathway in lung adenocarcinoma.

Drug-resistant advanced lung adenocarcinoma (LUAD) is an aggressive malignancy with limited treatment options. A therapeutic strategy for drug-resistant LUAD is to target the tumor associated macrophages (TAMs), because they play an important role in tumor immune escape, progression and metastasis. In this study, we conducted in vivo and in vitro investigation of the inhibitory effect of arsenic trioxide (ATO) on polarization of TAMs educated by LUAD. We found that ATO at a concentration of 4 µM disrupted the Notch-dependent positive feedback loop between LUAD and TAMs. In this loop, ATO inhibited the expression of Jagged1 and Notch1 in LUAD and suppressed M2 polarization via down-regulating Notch-dependent paracrine of CCL2 and IL1ß. As a result, the secretion of M2-derived TGF-ß1 decreased, thus inducing inhibitions of LUAD proliferation, migration, invasion, colony formation and epithelial-mesenchymal transition. In xenograft mouse models, ATO significantly inhibited tumor growth and down-regulated infiltration of M2-like TAMs in tumor tissues. In clinical LUAD biopsy samples, high Jagged1/Notch1 expression positively correlated with tumor-infiltrated M2-like TAMs, leading to poor prognosis. In conclusion, our results identified a novel tumor immunomodulating function for ATO, which can inhibit the polarization of M2-type TAMs to exert anti-tumor effects in the tumor microenvironment. Our results demonstrated the translational potential of repurposing ATO to target TAMs for lung adenocarcinoma treatment.

Arsenic trioxide inhibits the growth of cancer stem cells derived from small cell lung cancer by downregulating stem cell-maintenance factors and inducing apoptosis via the Hedgehog signaling blockade.

BACKGROUND: Small cell lung cancer (SCLC) is the most deadly and aggressive type of primary lung cancer, with the 5-year survival rate lower than 5%. The FDA has approved arsenic trioxide (As2O3) for acute promyelocytic leukemia (APL) treatment. However, its role in SCLC-derived cancer stem cells (CSCs) remains largely unknown. METHODS: CSCs were enriched from SCLC cell lines by culturing them as spheres in conditioned serum-free medium. Then, qPCR, western blot, serial passage, limiting dilution, Transwell, and tumorigenesis assay were performed to verify the cells' stem phenotypic characteristics. Anticancer efficiency of As2O3 was assessed in these cells using CCK8, colony formation, sphere formation, flow cytometry, qPCR, western blot analysis in vitro, and tumor growth curve, immunofluorescence, and TUNEL staining analyses in vivo. RESULTS: The fifth-passage SCLC spheres showed a potent self-renewal capacity, higher clonal formation efficiency (CFE), SOX2, c-Myc, NANOG, and OCT4 levels, and invasion ability, and stronger tumorigenesis capacity than the parental SCLC cells, indicating that the SCLC sphere cells displayed CSC features. As2O3 inhibited the proliferation, clonality and sphere forming ability of SCLC-derived CSCs and suppressed the tumor growth of CSCs-derived xenograft tumors. As2O3 induced apoptosis and downregulation of SOX2 and c-Myc in vitro and in xenografts. Besides, SOX2 knockdown suppressed SCLC-derived CSCs to self-renew and induced apoptosis. Mechanistically, expression of GLI1 (a key transcription factor of Hedgehog pathway) and its downstream genes increased in SCLC-derived CSCs, compared to the parental cells. As2O3 dramatically downregulated GLI1 and its downstream genes in vitro and in vivo. The GLI inhibitor (GANT-61) recapitulated and enhanced the effects of As2O3 on SCLC-derived CSCs, including growth suppression, apoptosis induction, and GLI1, SOX2 and c-Myc downregulation. CONCLUSIONS: Altogether, As2O3 effectively suppressed SCLC-derived CSCs growth by downregulating stem cell-maintenance factors and inducing apoptosis. These effects are mediated at least partly via the Hedgehog signaling blockade.