Cytochalasin E increased the sensitivity of human lung cancer A549 cells to bortezomib via inhibition of autophagy
Introduction
Cells respond to stress by activating various signaling pathways to recover and maintain cellular homeostasis. Cells adapt to their environment and external stresses by modifying their cellular metabolism and signaling protective mechanism. Macroautophagy, hereafter referred to as “autophagy”, is a tightly orchestrated intracellular degradation pathway that mediates cellular homeostasis and stress-induced adaptation [1]. During autophagy, cytoplasmic materials are sequestered into double-membrane vesicles, called “autophagosomes”, which are delivered to lysosomes to eliminate damaged or harmful components [2], allowing for survival against multiple stressors [3] [4]. Therefore, autophagy constitutes a major protective mechanism that can suppress apoptosis in favor of adapted cell survival.
Cancer cells adapt to stress and promote survival by activating the autophagy pathway [5]. High basal levels of autophagic activity can be found in several different cancer cell lines [6]. The activation of autophagy has been suggested to promote cell survival by removing abnormal protein aggregates and damaged organelles, or by fulfilling the energy demands of proliferating cancer cells. Increasing studies have demonstrated that autophagy protects some cancers against cell death induced by chemotherapy [7] [8]. Therefore, inhibition of autophagy may sensitize tumor cells to a wide array of drugs and therapeutic strategies, such as radiation, representing a promising method to improve the efficacy of current cancer treatments [9] [10]. In fact, chloroquine (CQ) and its derivative hydroxychloroquine (HCQ), an autophagy inhibitor, exhibit antitumor effects in vitro and in vivo, and various clinical trials have been conducted using these drugs alone or in combination with various chemotherapeutic agents and/or radiation in different types of cancer [9].
We recently reported that various cytochalasins, fungal metabolites that bind to actin filaments, inhibited autophagy. Among these cytochalasins, cytochalasin E (CE) inhibited cell growth and autophagic activity of human lung cancer A549 cells [11]. CE is an epoxide-containing cytochalasin family member, derived from Aspergillus clavatus. At lower concentrations, CE was reported to exhibit unique biphasic inhibition of bovine capillary endothelial cell proliferation and inhibit basic fibroblast growth factor (bFGF)-induced angiogenesis in the mouse cornea [12] [13]. However, the potential benefits of CE as an anticancer drug remain unknown.
In the present study, we investigated the potent inhibitory effects of CE. We demonstrated that treatment of serum-starved A549 cells with CE enhanced cell death, compared to control cells. Furthermore, combination treatment with CE and bortezomib, an anticancer drug that targets the 26S proteasome, resulted in synergistically augmented cytotoxicity. From our findings, we concluded that lung cancer cells may be effectively targeted by suppressing autophagy via CE treatment alone or combination treatment with CE and bortezomib. This supports a novel therapeutic strategy for lung cancer.
Section snippets
Cell culture and chemicals
Human lung epithelial cancer A549 cells (ATCC CCL-185) were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10% fetal calf serum (FCS; Tissue Culture Biologicals, Seal Beach, CA, USA), 100 units/mL penicillin, 100 μg/mL streptomycin, and 292 μg/mL l-glutamine (Thermo Fisher Scientific, Rockford, IL, USA) at 37 °C and 5% CO2. Cytochalasin E was purchased from Cayman Chemical (Ann Arbor, MI, USA). Earle's balanced salt solution (EBSS)
Cytochalasins E induced expression of autophagy protein markers
To examine the potential anticancer effect of cytochalasin E on A549 cells, we measured the cell proliferation of A549 cells treated with increasing concentrations of CE (0.065 μM, 0.125 μM, 0.25 μM, 0.5 μM) using WST-1 assay kit. Our results showed that CE treatment of A549 cells decreased cellular viability in a dose-dependent manner (Fig. S1A). In addition, we observed using light microscopy that CE treatment induced several morphological changes in treated A549 cells compared to control
Discussion
Recently, our laboratory investigated the inhibitory effects of cytochalasins on autophagy in human lung adenocarcinoma A549 cells, and we demonstrated the potent inhibitory effects of cytochalasin E on cell viability and autophagy [11], implicating as a potential anticancer drug. In the present study, we showed that CE inhibited autophagy in a dose- and time-dependent manner by impairing autophagosome–lysosome fusion. Additionally, the results showed that serum starvation enhanced the
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
We thank Mr. R. Harada for technical assistance. This work was supported in part by Grants-in-Aid for Challenging Exploratory Research (Grant Number 16K15381) and Grants-in-Aid for Scientific Research C (Grant Number 17K09164).
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