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A Novel Benzofuran Derivative Moracin N Induces Autophagy and Apoptosis Through ROS Generation in Lung Cancer.

ABSTRACT: Introduction:The leaves of Morus alba L is a traditional Chinese medicine widely applied in lung diseases. Moracin N (MAN), a secondary metabolite extracted form the leaves of Morus alba L, is a potent anticancer agent. But its molecular mechanism remains unveiled. Objective:In this study, we aimed to examine the effect of MAN on human lung cancer and reveal the underlying molecular mechanism. Methods:MTT assay was conducted to measure cell viability. Annexin V-FITC/PI staining was used to detect cell apoptosis. Confocal microscope was performed to determine the formation of autophagosomes and autolysosomes. Flow cytometry was performed to quantify cell death. Western blotting was used to determine the related-signaling pathway. Results:In the present study, we demonstrated for the first time that MAN inhibitd cell proliferation and induced cell apoptosis in human non-small-cell lung carcinoma (NSCLC) cells. We found that MAN treatment dysregulated mitochondrial function and led to mitochondrial apoptosis in A549 and PC9 cells. Meanwhile, MAN enhanced autophagy flux by the increase of autophagosome formation, the fusion of autophagsomes and lysosomes and lysosomal function. Moreover, mTOR signaling pathway, a classical pathway regualting autophagy, was inhibited by MAN in a time- and dose-dependent mannner, resulting in autophagy induction. Interestingly, autophagy inhibition by CQ or Atg5 knockdown attenuated cell apoptosis by MAN, indicating that autophagy serves as cell death. Furthermore, autophagy-mediated cell death by MAN can be blocked by reactive oxygen species (ROS) scavenger NAC, indicating that ROS accumulation is the inducing factor of apoptosis and autophagy. In summary, we revealed the molecular mechanism of MAN against lung cancer through apoptosis and autophagy, suggesting that MAN might be a novel therapeutic agent for NSCLC treatment.

SUBMITTER: Gao C

PROVIDER: S-EPMC7235196 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A Novel Benzofuran Derivative Moracin N Induces Autophagy and Apoptosis Through ROS Generation in Lung Cancer.

Gao Chengcheng C Sun Xin X Wu Zhipan Z Yuan Huahua H Han Haote H Huang Hongliang H Shu Yuhan Y Xu Mengting M Gao Ruilan R Li Shouxin S Zhang Jianbin J Tian Jingkui J

Frontiers in pharmacology 20200512

<h4>Introduction</h4>The leaves of <i>Morus alba L</i> is a traditional Chinese medicine widely applied in lung diseases. Moracin N (MAN), a secondary metabolite extracted form the leaves of <i>Morus alba</i> L, is a potent anticancer agent. But its molecular mechanism remains unveiled.<h4>Objective</h4>In this study, we aimed to examine the effect of MAN on human lung cancer and reveal the underlying molecular mechanism.<h4>Methods</h4>MTT assay was conducted to measure cell viability. Annexin ...[more]

PMID: 32477104

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Project description:Clinical trials have revealed that the potential anticancer agent, 2-methoxyestradiol (2ME2) has limitations due to its low bioavailability. Subsequently, 2ME2 derivatives including (8R,13S,14S,17S)-2-ethyl-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrane-3,17-diyl bis(sulphamate) (EMBS) have shown improved efficacies in inducing apoptosis. However, no conclusive data exist to explain the mode of action exerted by these drugs. This study investigated the mode of action used by EMBS as a representative of the sulphamoylated 2ME2 derivatives. Hydrogen peroxide and superoxide production was quantified using dichlorofluorescein diacetate and hydroethidine. Cell proliferation and mitochondrial metabolism were investigated using crystal violet and Alamar Blue. Apoptosis was assessed using Annexin V-FITC while mitochondrial integrity was assessed using Mitocapture. Autophagy was visualised using LC3B II antibodies. The effects of EMBS on H2A phosphorylation and nuclei were visualised using phospho H2A antibody and 4',6-diamidino-2-phenylindole, dihydrochloride. Data showed that EMBS exposure leads to increased reactive oxygen species (ROS) production which is correlated with loss of cell proliferation, mitochondrial membrane damage, decreased metabolic activity, G2/M arrest, endoreduplication, DNA double stranded breaks, micronuclei and apoptosis induction. Treatment of EMBS-exposed cells with the ROS scavenger, N-acetyl cysteine, abrogated ROS production, cell cycle arrest and apoptosis implying an essential role for ROS production in EMBS signaling. The inhibition of c-Jun N-terminal kinase (JNK) activity also inhibited EMBS-induced apoptosis suggesting that EMBS triggers apoptosis via the JNK pathway. Lastly, evaluation of LC3IIB protein levels indicated that autophagy is not activated in EMBS-exposed cells. Our data shows that EMBS targets a pathway that leads to increased ROS production as an early event that culminates in G2/M arrest and apoptosis by means of JNK-signaling in cancer cells. This study suggests a novel oxidative stress-dependent mode of action for sulphamoylated derivatives.

Project description:BACKGROUND:Hypercholesterolemia increases the risk of tendon pain and tendon rupture. Tendon-derived stem cells (TDSCs) play a vital role in the development of tendinopathy. Our previous research found that high cholesterol inhibits tendon-related gene expression in TDSCs. Whether high cholesterol has other biological effects on TDSCs remains unknown. METHODS:TDSCs isolated from female SD rats were exposed to 10 mg/dL cholesterol for 24 h. Then, cell apoptosis was assessed using flow cytometry and fluorescence microscope. RFP-GFP-LC3 adenovirus transfection was used for measuring autophagy. Signaling transduction was measured by immunofluorescence and immunoblotting. In addition, Achilles tendons from ApoE -/- mice fed with a high-fat diet were histologically assessed using HE staining and immunohistochemistry. RESULTS:In this work, we verified that 10 mg/dL cholesterol suppressed cell proliferation and migration and induced G0/G1 phase arrest. Additionally, cholesterol induced apoptosis and autophagy simultaneously in TDSCs. Apoptosis induction was related to increased expression of cleaved caspase-3 and BAX and decreased expression of Bcl-xL. The occurrence of autophagic flux and accumulation of LC3-II demonstrated the induction of autophagy by cholesterol. Compared with the effects of cholesterol treatment alone, the autophagy inhibitor 3-methyladenine (3-MA) enhanced apoptosis, while the apoptosis inhibitor Z-VAD-FMK diminished cholesterol-induced autophagy. Moreover, cholesterol triggered reactive oxygen species (ROS) generation and activated the AKT/FOXO1 pathway, while the ROS scavenger NAC blocked cholesterol-induced activation of the AKT/FOXO1 pathway. NAC and the FOXO1 inhibitor AS1842856 rescued the apoptosis and autophagy induced by cholesterol. Finally, high cholesterol elevated the expression of cleaved caspase-3, Bax, LC3-II, and FOXO1 in vivo. CONCLUSION:The present study indicated that high cholesterol induced apoptosis and autophagy through ROS-activated AKT/FOXO1 signaling in TDSCs, providing new insights into the mechanism of hypercholesterolemia-induced tendinopathy. High cholesterol induces apoptosis and autophagy through the ROS-activated AKT/FOXO1 pathway in tendon-derived stem cells.

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A Novel Benzofuran Derivative Moracin N Induces Autophagy and Apoptosis Through ROS Generation in Lung Cancer.

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A Novel Benzofuran Derivative Moracin N Induces Autophagy and Apoptosis Through ROS Generation in Lung Cancer.

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