Celastrol elicits antitumor effects by inhibiting the STAT3 pathway through ROS accumulation in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common lung cancer with high mortality across the world, but it is challenging to develop an effective therapy for NSCLC. Celastrol is a natural bioactive compound, which has been found to possess potential antitumor activity. However, the underlying molecular mechanisms of celastrol activity in NSCLC remain elusive. METHODS: Cellular function assays were performed to study the suppressive role of celastrol in human NSCLC cells (H460, PC-9, and H520) and human bronchial epithelial cells BEAS-2B. Cell apoptosis levels were analyzed by flow cytometry, Hoechst 33342, caspase-3 activity analysis, and western blot analysis. Intracellular reactive oxygen species (ROS) were analyzed by flow cytometry and fluorescence microscope. Expression levels of endoplasmic reticulum (ER) stress-related proteins and phosphorylated signal transducer and activator of transcription 3 (P-STAT3) were identified via western blot analysis. A heterograft model in nude mice was employed to evaluate the effect of celastrol in vivo. RESULTS: Celastrol suppressed the growth, proliferation, and metastasis of NSCLC cells. Celastrol significantly increased the level of intracellular ROS; thus, triggering the activation of the ER stress pathway and inhibition of the P-STAT3 pathway, and eventually leading to cell apoptosis, and the effects were reversed by the pre-treatment with N-Acetyl-L-cysteine (NAC). Celastrol also suppressed tumor growth in vivo. CONCLUSION: The outcomes revealed that celastrol plays a potent suppressive role in NSCLC in vitro and in vivo. Celastrol induces apoptosis via causing mitochondrial ROS accumulation to suppress the STAT3 pathway. Celastrol may have potential application prospects in the therapy of NSCLC.

Single-Cell RNA-Sequencing Reveals the Active Involvement of Macrophage Polarizations in Pulmonary Hypertension.

Background: Sustained hypoxia can trigger a progressive rise in pulmonary artery pressure and cause serious pulmonary diseases. Macrophages play important roles along the progression of pulmonary hypertension. However, the state of macrophage polarization during the early stage of pulmonary hypertension is unclear. Methods: Unlike traditional sequencing method, single-cell sequencing can accurately distinguish among cell types and better understand cell-to-cell relationships. In this study, we investigated the polarization of macrophages in pulmonary hypertension via single-cell RNA-sequencing in a mice hypoxia model, which was then validated in patients with pulmonary hypertension. Results: We identified that the intermittent exposure to hypoxic conditions could lead to the production of more M2-type macrophages than M1-type macrophages in a mouse model. Further validation analysis was performed by analyzing lung tissue of patients with pulmonary hypertension, revealing that the number of disease-associated M2 macrophages was substantially increased. Conclusions: In this study, the active anti-inflammatory response of macrophage involved in pulmonary hypertension has been identified, suggesting that intervention against the polarization of macrophages to the M2 type may be a potential way to reduce chronic pulmonary inflammation, pulmonary vascular remodeling, and artery pressure. Thus, investigation of macrophage polarization associated with hypoxia could help us better understand disease mechanism and craft effective prevention strategies and approaches.

Dihydroartemisinin Attenuates Hypoxia-Induced Pulmonary Hypertension Through the ELAVL2/miR-503/PI3K/AKT Axis.

ABSTRACT: Dihydroartemisinin (DHA) is an active form of artemisinin extracted from the traditional Chinese medicine Artemisia annua , which is used to treat malaria. Previous studies have shown that DHA has a therapeutic effect on pulmonary hypertension (PH), but its specific mechanism has not been fully elucidated. In this study, a hypoxia-induced PH mouse model was established and DHA was administered as a therapeutic intervention. We measured hemodynamics and right ventricular hypertrophy and observed hematoxylin and eosin staining of lung tissue sections, proving the therapeutic effect of DHA on PH. Furthermore, cell counting kit-8 and 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay kit were performed to examine cell proliferation of pulmonary artery smooth muscle cells cultured in hypoxia or in normoxia. Transwell migration chamber assay was performed to examine cell migration of the same cell model. Consistent with the therapeutic effect in vivo, DHA inhibited hypoxia-induced cell proliferation and migration. Through high-throughput sequencing of mouse lung tissue, we screened embryonic lethal abnormal vision-like 2 (ELAVL2) as a key RNA binding protein in PH. Mechanistically, DHA inhibited the proliferation and migration of pulmonary artery smooth muscle cells by promoting the expression of ELAVL2 and regulating the miR-503/PI3K/AKT pathway. The binding relationship between ELAVL2 and pre-miR-503 was verified by RNA binding protein immunoprecipitation assay. In conclusion, we first propose that DHA alleviates PH through the ELAVL2/miR-503/PI3K/AKT pathway, which may provide a basis for new therapeutic strategies of PH.

Acetylshikonin exerts anti-tumor effects on non-small cell lung cancer through dual inhibition of STAT3 and EGFR.

BACKGROUND: Lung cancer is one of the most common types of malignant tumor. It has one of the highest morbidity and mortality rates worldwide, and approximately 85% of cases are non-small cell lung cancer (NSCLC). Clinically, several EGFR inhibitors have been used to treat NSCLC, but resistance can develop. Studies have shown that cross talk between signal transducer and activator of transcription 3 (STAT3) and epidermal growth factor receptor (EGFR) can mediate drug resistance. Acetylshikonin has obvious antitumor effects, but the mechanism of action is still unclear. PURPOSE: To analyze the antitumor activity of acetylshikonin in lung cancer and clarify its molecular mechanism. METHODS: Methyl thiazolyl tetrazolium (MTT), colony formation and 5-ethynyl-2'-deoxyuridine (EDU) assays were performed to examine the effects of acetylshikonin in inhibiting the proliferation of NSCLC cells (PC-9, H1975 and A549). Scratch wound and transwell assays were used to evaluate the migration and invasion of NSCLC cells. Flow cytometry was employed to determine whether acetylshikonin could induce apoptosis. Proteome sequencing was used to identify the targets of acetylshikonin. Immunofluorescence staining and western blotting were utilized to verify the inhibition of STAT3 and EGFR phosphorylation. A xenotransplantation model was established to evaluate the efficacy of acetylshikonin in nude mice. RESULTS: Our data demonstrated that acetylshikonin significantly decreased the survival rate of human NSCLC cells, increased the apoptotic rate and inhibited cell migration dose-dependently. Immunofluorescence staining and western blotting analyses revealed that acetylshikonin inhibited EGFR and STAT3 pathways. Acetylshikonin also inhibited tumor growth in a xenograft model better than inhibitors of EGFR and STAT3. CONCLUSION: Acetylshikonin has anti-cancer effects on NSCLC cells by inhibiting EGFR and STAT3, indicating that acetylshikonin may be a new antitumor drug to treat NSCLC.

AT7519 against lung cancer via the IL6/STAT3 signaling pathway.

Lung cancer is a part of the commonest malignancies with the highest mortality rate in cancer-related deaths worldwide. Signal transducer and activator of transcription 3 (STAT3) and cyclin-dependent kinases (CDKs) are promising prognostic marker and therapeutic target in cancers. Our previous study has demonstrated the closely relationship between CDK9 and STAT3 in lung cancer. The inhibition of cell viability and migration in vitro by AT7519 were evaluated using methyl thiazolyl tetrazolium (MTT) assay, clonogenic assay and scratch wound model. The cell cycle analysis was evaluated using flow cytometry analysis and western blotting analysis. The apoptotic-induced efficiency was assessed by flow cytometry analysis, hoechst 33342 staining, caspase-3 activity analysis and western blotting analysis. The roles of STAT3 in AT7519 treatment for lung cancer were assessed by docking model and western blotting analysis. The patient-derived xenograft (PDX) models were used to investigate the effect of AT7519 in vivo. In this study, we found that AT7519, a CDK inhibitor, reduced the viability of lung cancer cells in vitro and strongly suppressed tumor growth in PDX model. AT7519 blocked cell cycle progression and induced apoptosis by inhibiting IL-6/STAT3 pathway. Taken together, AT519 exhibits great anti-tumor effects in lung cancer, and the mechanism was related closely to IL-6/STAT3 signaling pathway, which suggests the important roles of STAT3 in CDKs inhibitors. AT7519 might be a novel potential therapeutic agent based on this rationale.