Single-cell analysis of dihydroartemisinin–induced apoptosis through reactive oxygen species–mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

Abstract

Abstract. Dihydroartemisinin ͑DHA͒, a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHAinduced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species ͑ROS͒ in the DHAinduced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

Amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of ASTC-a-1 cells

“…Based on our previous study that mitochondrial apoptosis pathway plays an important role in both DHA-and ARTS-induced apoptosis, especially DHA induced apoptosis via both Bax-and caspase-9-dependent pathway [9,10], and ARTS induced apoptosis largely via a Bakmediated and caspase-independent pathway (unpublished data), we started the study of ARTE to find some difference among their pro-apoptotic mechanism in human lung adenocarcinoma (ASTC-a-1) cells. Our results reveal that ARTE induces caspase-dependent apoptosis without Bid cleavage and cytochrome c release from mitochondria.…”

“…The cleavage of endoperoxide bridge in ARTE induces the generation of organic free radicals which disturb the natural oxidation and reduction equilibrium in cells, and leads to an increase of reactive oxygen species (ROS) that can induce caspasedependent apoptotic cell death in HeLa-60 cells [6,7] and injury in a variety of mammalian cells such as lung cancer A549 cells [8]. Our recent studies showed that DHA induced ROS-dependent apoptosis via caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells [9,10].…”

Dihydroartemisinin induces apoptosis in skin cancer cell line A-431 via ROS pathway

“…The role of ROS in the apoptosis induced by Dihydroartemisinin investigated in cancer cells [18]. Lu et.al demonstrated that Dihydroartemisinin induced apoptosis via ROS formation in ASTC-a-1 cells [18]. In this study, it has been shown that Dihydroartemisinin induced ROS formation in A-431 human skin cancer cells.…”

“…Lu et al demonstrated that Dihydroartemisinin induced apoptosis in human lung adenocarcinoma cells by upregulation of Bax protein family [17].ROS are involved at several different points in the apoptotic pathway, including loss of mitochondrial membrane integrity with the attendant release of mitochondrial apoptogenic factors, intracellular caspase activation, and DNA damage [26,27]. The role of ROS in the apoptosis induced by Dihydroartemisinin investigated in cancer cells [18]. Lu et.al demonstrated that Dihydroartemisinin induced apoptosis via ROS formation in ASTC-a-1 cells [18].…”

“…It has a defined mechanism of action, with artemisinin and its derivatives effecting heme-mediated decomposition of the endoperoxide bridge in artemisinin to produce free radicals [15]. This heme-catalyzed excess of reactive oxygen species (ROS) induces apoptosis through a mitochondrial mediated pathway or blocking cell cycle kinetics [16][17][18]. However, reactive oxygen species production is not enough to explain anticancer activities of artemisinin [19].…”