Abstract

Arenobufagin (ARE) has demonstrated potent anticancer activity in various types of tumor, but the role and mechanism of ARE for lung cancer remain unclear. Oxidative stress exists under normal conditions and is an inevitable state in the body. A variety of noxious stimuli can break the equilibrium state of oxidative stress and promote apoptosis. Here, we used a CCK-8 assay to examine cell viability. We determined oxidative stress damage by measuring levels of intracellular ROS and levels of GSH, SOD, and MDA. Annexin V-FITC/PI double staining assay, as well as the Hoechst 33258 staining, was used to detect ARE-induced apoptosis in A549 cell. Evaluation of the expression level of the specified molecule was indicated by Western blot and qRT-PCR. Loss of function experiment was carried out using NAC pretreatment. The experimental results show that ARE significantly declines in the viability of A549 cells and increases the apoptosis rate of A549 cells. As reflected in cell morphology, the A549 cells showed features of shrinkage and had incompletely packed membranes; the same phenomenon is manifested in Hoechst 33258 staining. Following ARE treatment, the ROS level in A549 cells was rising in a concentration-dependent manner, and so were MDA and GSH levels, while the SOD level was decreasing. Moreover, we found that ARE can decrease mitochondrial membrane potential (MMP), and a cascade of apoptotic processes can be triggered by decreased MMP. Importantly, we found significant changes in protein expression levels and mRNA levels of apoptosis-related proteins. Furthermore, when we used NAC to restrain oxidative stress, the expression levels of apoptosis-related proteins have also changed accordingly. Our data demonstrate that apoptosis in the non-small-cell lung cancer (NSCLC) cell line A549 is caused by oxidative stress due to ARE. Our research also shows that ARE may have the potential to become a targeted therapeutic for the treatment of NSCLC in the future.

Highlights

  • Lung cancer (LC) is one of the most common malignancies from poles to poles

  • According to its biological characteristics, lung cancer can be segmented into two types, small cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC)

  • In oxidative stress-mediated apoptosis, caspase activation and changes in Bcl-2-related proteins occur repeatedly, which has an important influence on the process of apoptosis [7]. e endogenous apoptotic pathway involves the suppression of the Bcl-2-encoding gene and binding of the proapoptotic protein Bax to the mitochondrial membrane [8]

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Summary

Introduction

Lung cancer (LC) is one of the most common malignancies from poles to poles. According to the results of the American Cancer Society in 2018, lung cancer accounts for 11.6% of total cancers, and lung cancer mortality accounts for 18.4% of total cancer deaths [1]. A variety of noxious stimuli can break the equilibrium state of oxidative stress and promote apoptosis [4]. Many diseases, such as Parkinson’s disease and cancer, are associated with oxidative stress [5]. In oxidative stress-mediated apoptosis, caspase activation and changes in Bcl-2-related proteins occur repeatedly, which has an important influence on the process of apoptosis [7]. By repressing Bax activation and its secondary mitochondrial membrane translocation, anti-Bcl-2 apoptotic proteins play a role in protecting mitochondria and prevent Bax from destruction of mitochondrial outer membrane completeness and suppression of caspase precursor activation, thereby resulting in cells apoptosis [9]

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