Variation in the activity of distinct cytochalasins as autophagy inhibitiors in human lung A549 cells

https://doi.org/10.1016/j.bbrc.2017.10.135Get rights and content

Highlights

  • Cytochalasin E and H are novel inhibitors of autophgic flux.

  • Cytochalasin D, E, and H inhibit autophagy and are new anti-cancer drug candidates.

  • Phenochalasin A inhibits lipid droplet formation without autophagic effects.

Abstract

Autophagy is a cell survival process that represents a therapeutic target in cancer treatment. Many types of cytochalasins have been identified and some of them have been reported to interfere with the formation of the autophagosome, although only limited data are available to assess their potential effects. Therefore, in this study, we examined the effects of cytochalasins and structurally related compounds on cell survival and the regulation of autophagy in human lung A549 adenocarcinoma cells. Cytochalasin D (CD) and cytochalasin E (CE) prominently inhibited the growth of A549 cells in a dose-dependent manner. Following treatment with CE, F-actin filaments were disrupted, and the proportion of binucleated cells increased, whereas no such effects were observed with the seven other cytochalasins tested. We found that cytochalasin H (CH), CD, and especially CE could induce the up-regulation of autophagy-related protein (LC3-II) and SQSTM1/p62. Using bafilomycin A1, we demonstrated that CD, CE, and CH inhibited autophagosome turnover, resulting in a dysfunctional autophagic process. The results of this study reveal that CE is the most potent cytochalasin in terms of its ability to induce cell death and inhibit autophagy. CE may therefore be an effective therapeutic agent against lung cancer.

Introduction

Autophagy is a conserved catabolic process that allows cells to maintain cellular homeostasis under starvation and stress conditions. This process is responsible for degrading protein aggregates and damaged organelles by delivering them to autophagosomes [1], [2]. The autophagosome fuses with a lysosome for degrading the sequestered materials. Autophagy is involved in many physiological processes and disease states [3] and has recently emerged as an important determinant in cancer biology, as cancer cells often endure stresses from a variety of sources and are still able to survive and proliferate. Autophagy occurs constitutively at low levels to perform homeostatic functions such as protein and organelle turnover, whereas tumor cells can induce high levels of basal autophagy and can be constitutively dependent on autophagy for survival. In particular, autophagy is upregulated during the later stages of cancer development, allowing cancer cells to survive in the central areas of tumours, which are usually poorly vascularized and lack nutrients and oxygen [4], [5], [6]. A number of anticancer therapies, including DNA-damaging chemotherapeutic drugs, have been observed to induce the accumulation of autophagosomes in tumor cell lines. In addition, the inhibition of autophagy by pharmacologic drugs or the genetic knockdown of phylogenetically conserved autophagy-related genes, such as ATG5 and ATG7, usually enhances drug-induced cytotoxicity [7]. For example, the inhibition of autophagy via treatment with chloroquine (CQ) and siRNAs targeting ATG5 or ATG7 increases the sensitivity of lung cancer cells to gefitinib [8] and of myeloid leukemia to daunorubicin [9]. Autophagic inhibition may thus sensitize tumor cells to many cytotoxic drugs or reverse resistance to chemotherapeutic drugs, representing a promising strategy for improving the efficacy of cancer treatment [10], [11].

Cytochalasins are alkaloid mycotoxins widely present in fungi and are known to disrupt the formation of actin polymers, thereby preventing the formation of functional microfilaments. It is generally accepted that cytochalasins slow the rate of filament polymerization by inhibiting the rate of elongation. This action is caused by the high-affinity binding of cytochalasins to the barbed (plus) end of F-actin and inhibits both the dissociation and association of actin monomers (G-actin) at that end. This leads to the almost complete disorganization of the actin network and accumulation of numerous foci of short actin filaments in cells [12]. Many kinds of cytochalasins (A, B, C, D, E, H, and J) have been described, each affecting different actin-based processes. For example, cytochalasin B and D (abbreviated CB and CD, respectively) inhibit prokaryotic cell division [13] and eukaryotic cell division [14], [15] and affect the cell cycles [16] as well as antitumor activities [17], whereas cytochalasin E and H (CE, CH) are reported to inhibit angiogenesis and tumor growth [18], [19]. Phenochalasin A (PCA), which also belongs to the cytochalasan family, is structurally similar to CE, and inhibits lipid droplet formation in mouse peritoneal macrophages [20], [21]. Among the numerous cellular activities of cytochalasins, CB and CD have been shown to interfere with autophagosome formation [22]. However, it is not yet clear whether other cytochalasins also have inhibitory effects on autophagy.

Knowledge of the roles of cytochalasins in anticancer activity is essential for the design and improvement of chemotherapeutic agents. Therefore, in this study, we sought to determine the potential anti-cancer effects of the cytochalasins A, B, C, D, E, H, and J as well as PCA on human lung A549 adenocarcinoma cells by observing their effects on cell survival and autophagy. Our results demonstrate that among the cytochalasins, CE is the most effective inhibitor of autophagy and inducer of cell death in A549 cells.

Section snippets

Cell culture and chemicals

Human lung epithelial A549 cancer cells (ATCC CCL-185) were grown 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, 10368–016) at 37 °C and 5% CO2. Cytochalasins A, B, C, D, H, and J and bafilomycin A1 (BAF-A1; B1793) were purchased from Sigma-Aldrich.

Effects of cytochalasin on cell viability

As illustrated in Fig. S1A, we used commercially available cytochalasins and PCA), which is structurally similar to CE, in this study. A549 cells were treated with different concentrations of the cytochalasins for 24 h, and cellular viability was assessed by WST-8 assay. As shown in Fig. 1A, in comparison with the control group, cytochalasin C (CC), CD, CE, and CH resulted in concentration-dependent decreases in cell viability. The viability of cells was reduced to less than 50% by treatment

Discussion

Cancer cells generally exhibit higher levels of autophagy, which allows them to adapt to and survive unfavorable metabolic conditions such as hypoxia and acidity. Accordingly, it is generally accepted that inhibiting autophagy is a logical approach for cancer therapy, with autophagic inhibitors used as either standalone therapeutic agents or, more practically, as potential adjuvant therapy in combination with other established therapeutic agents. Cytochalasins are known to inhibit a number of

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

We thank Miss Y. Kojima and K. Hanada for technical assistance. This work was supported in part by a Grant-in-Aid for Challenging Exploratory Research (Grant Number 16K15381) and a Grant-in-Aid for Scientific Research C (Grant Number 17K09164).

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