Abstract
Increasing the level of reactive oxygen species (ROS) in cancer cells has been suggested as a viable approach to cancer therapy. Our previous study has demonstrated that mitochondria-targeted flavone-naphthalimide-polyamine conjugate 6c elevates the level of ROS in cancer cells. However, the detailed role of ROS in 6c-treated cancer cells is not clearly stated. The biological effects and in-depth mechanisms of 6c in cancer cells need to be further investigated. In this study, we confirmed that mitochondria are the main source of 6c-induced ROS, as demonstrated by an increase in 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSox fluorescence. Compound 6c-induced mitochondrial ROS caused mitochondrial dysfunction and lysosomal destabilization confirmed by absolute quantitation (iTRAQ)-based comparative proteomics. Compound 6c-induced metabolic pathway dysfunction and lysosomal destabilization was attenuated by N-acetyl-L-cysteine (NAC). iTRAQ-based comparative proteomics showed that ROS regulated the expression of 6c-mediated proteins, and treatment with 6c promoted the formation of autophagosomes depending on ROS. Compound 6c-induced DNA damage was characterized by comet assay, p53 phosphorylation, and γH2A.X, which was diminished by pretreatment with NAC. Compound 6c-induced cell death was partially reversed by 3-methyladenine (3-MA), bafilomycin (BAF) A1, and NAC, respectively. Taken together, the data obtained in our study highlighted the involvement of mitochondrial ROS in 6c-induced autophagic cell death, mitochondrial and lysosomal dysfunction, and DNA damage.
Highlights
At present, cancer is one of the most life-threatening diseases, and cell death induced by anticancer agents in targeted cancer cells is a primary therapeutic aim of most cancer therapies [1]
To visualize the effect of 85 derivatives of naphthalimides (10 μM), which are known to interact with DNA, obtained from our compound library on cancer cells, HepG2 cells were stained with acridine orange (AO), a nucleic acid-selective fluorescent dye (~488 nm) which emits green fluorescence when in monomer form
We displayed that 6c induced mitochondrial reactive oxygen species (ROS) generation, which in turn caused mitochondrial and lysosomal dysfunction. 6c induced the formation of autophagosomes, resulting in cell death. 6c stimulated DNA damage
Summary
Cancer is one of the most life-threatening diseases, and cell death induced by anticancer agents in targeted cancer cells is a primary therapeutic aim of most cancer therapies [1]. Three different types of cell death, including apoptosis, necrosis, and autophagic cell death (ACD), are relatively well-characterized [2]. Autophagy is a cellular stress response and a highly regulated catabolic process that involves the formation of a double-membrane bound structure termed the autophagosome, which fuses with lysosomes for the bulk degradation of long-lived proteins and recycling of damaged organelles [3]. ACD, known as type II programmed cell death, is first accompanied by large-scale autophagic vacuolization in the cytoplasm and a vacuolated cellular appearance [6]. Some anticancer drugs induce cell death by triggering autophagy, indicating that ACD may be an important factor for suppressing tumors during cancer therapy [7]
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