Inhibition of macroautophagy by bafilomycin A1 lowers proliferation and induces apoptosis in colon cancer cells

doi:10.1016/j.bbrc.2009.03.051

Biochemical and Biophysical Research Communications

Volume 382, Issue 2, 1 May 2009, Pages 451-456

https://doi.org/10.1016/j.bbrc.2009.03.051 Get rights and content

Abstract

Macroautophagy is a process by which cytoplasmic content and organelles are sequestered by double-membrane bound vesicles and subsequently delivered to lysosomes for degradation. Macroautophagy serves as a major intracellular pathway for protein degradation and as a pro-survival mechanism in time of stress by generating nutrients. In the present study, bafilomycin A₁, a vacuolar type H⁺-ATPase inhibitor, suppresses macroautophagy by preventing acidification of lysosomes in colon cancer cells. Diminished macroautophagy was evidenced by the accumulation of undegraded LC3 protein. Suppression of macroautophagy by bafilomycin A₁ induced G₀/G₁ cell cycle arrest and apoptosis which were accompanied by the down-regulation of cyclin D₁ and cyclin E, the up-regulation of p21^Cip1 as well as cleavages of caspases-3, -7, -8, and -9 and PARP. Further investigation revealed that bafilomycin A₁ increased the phosphorylation of ERK, JNK, and p38. In this regard, p38 inhibitor partially reversed the anti-proliferative effect of bafilomycin A₁. To conclude, inhibition of macroautophagy by bafilomycin A₁ lowers G₁–S transition and induces apoptosis in colon cancer cells. Our results not only indicate that inhibitors of macroautophagy may be used therapeutically to inhibit cancer growth, but also delineate the relationship between macroautophagy and apoptosis.

Introduction

Macroautophagy is an evolutionarily conserved catabolic process by which the cell degrades its cellular content through the lysosomal system. Macroautophagy is initiated by the formation of a double-membrane bound vacuole, the autophagosome, which nonselectively sequesters cytosolic proteins and organelles such as mitochondria, endoplasmic reticulum, and ribosomes [1]. In the late stage of macroautophagy, autophagosomes fuse with acidic lysosomes to produce autolysosomes where the engulfed content is digested by lysosomal hydrolases. The resulting degraded components are then returned to the cytoplasm by permeases in the lysosomal membrane for reuse [1]. While overactivation of this cellular process can lead to cell death [2], macroautophagy serves as a crucial pro-survival mechanism in time of stress by generating nutrients [3]. For instances, protein degradation and macroautophagy are enhanced in livers of mice undergoing starvation [4]. Dysregulation of macroautophagy has been implicated in many human diseases, such as degenerative neuronal diseases and cancers [5], [6], [7]. In carcinogenesis, the role of macroautophagy is oxymoronic. As a tumor suppressing mechanism, macroautophagy serves as an alternative to apoptosis to eliminate transformed cells [1], [8]. Moreover, genes that are involved in the execution of macroautophagy are important tumor suppressors [1], [8]. Nevertheless, it has also been reported that macroautophagy may facilitate tumor growth and survival during nutrient starvation and may contribute to tumor dormancy [9]. In colon cancer, increased expression of beclin-1 and LC3, two biochemical markers of macroautophagy, have been documented [10], [11]. The exact role of macroautophagy in colon carcinogenesis, however, is unknown.

Bafilomycin A₁, a macrolide antibiotic isolated from Streptomycesgriseus, is a potent inhibitor of vacuolar type H⁺-ATPase which functions to acidify intracellular compartments including lysosomes and to transport protons across the plasma membrane [12], [13]. In relation to macroautophagy, vacuolar type H⁺-ATPase is involved in the maturation of autolysosomes and its inhibitors including bafilomycin A₁ have been used as inhibitors of macroautophagy [14]. Macroautophagy plays a crucial part in the protein degradation system [15], [16]. In this connection, inhibition of protein degradation, for example, by targeting the proteasome has been shown to lower cell proliferation or induce apoptosis in various types of epithelial and hematological malignancies [17], [18], [19]. Whether inhibition of macroautophagy can achieve similar effects on cancer growth, however, is unknown. In the present study, the effects of macroautophagy inhibitor bafilomycin A₁ on proliferation and apoptosis of colon cancer cells were investigated.

Section snippets

Methods

Reagents. All primary antibodies were purchased from Cell Signaling Technology (Beverley, MA, USA) unless otherwise specified. Acridine orange and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen (Invitrogen, Carlsbad, CA). All other chemicals and reagents were purchased from Sigma (St. Louis, MO, USA) unless otherwise specified.

Cell culture and proliferation assay. The human colon adenocarinoma cell lines HT-29, HCT-116, SW1116, and normal colon fibroblasts CCD-18Co were

Bafilomycin A₁ inhibited the acidification of vesicular organelles and induced accumulation of LC3-II

To determine the effect of bafilomycin A₁ on macroautophagy of colon cancer cells, the formation of acidic vesicular organelles was determined by acridine orange staining. This lysosomotropic agent emitted bright red fluorescence in acidic vesicles including lysosomes but fluoresced green in cytoplasm and nucleus [20]. Vital staining of HT-29 cells with acridine orange revealed that red fluorescence was reduced in bafilomycin A₁-treated cells (Fig. 1A), indicating the acidification of vesicular

Discussion

Here we show that inhibition of the macroautophagy by vacuolar type H⁺-ATPase inhibitor bafilomycin A₁ lowers G₁–S transition and induces apoptosis in colon cancer cells. The inhibition of macroautophagy is evidenced by the reduced formation of acidic vesicular organelles and the accumulation of undigested LC3-II protein. These data suggest that bafilomycin A₁ may inhibit the function of lysosomal hydrolases by preventing acidification of lysosomes. In addition, bafilomycin A₁ when used at high

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¹: These authors contributed equally to this work.

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Inhibition of macroautophagy by bafilomycin A1 lowers proliferation and induces apoptosis in colon cancer cells

Abstract

Introduction

Section snippets

Methods

Bafilomycin A1 inhibited the acidification of vesicular organelles and induced accumulation of LC3-II

Discussion

Int. J. Biochem. Cell Biol.

Cell

Curr. Opin. Cell Biol.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Am. J. Pathol.

Autophagy fights disease through cellular self-digestion

Nature

Does autophagy have a license to kill mammalian cells?

Cell Death Differ.

Quantitative correlation between proteolysis and macro- and microautophagy in mouse hepatocytes during starvation and refeeding

Proc. Natl. Acad. Sci. USA

The autophagy-lysosomal degradation pathway: role in neurodegenerative disease and therapy

Front. Biosci.

Control of autophagy by oncogenes and tumor suppressor genes

Cell Death Differ.

Autophagy and tumor suppression: recent advances in understanding the link between autophagic cell death pathways and tumor development

Adv. Exp. Med. Biol.

Autophagy-induced tumor dormancy in ovarian cancer

J. Clin. Invest.

Expression of beclin-1, an autophagy-related protein, in gastric and colorectal cancers

APMIS

LC3, an autophagosome marker, is highly expressed in gastrointestinal cancers

Int. J. Oncol.

Chemistry and structure activity relationships of bafilomycin A1, a potent and selective inhibitor of the vacuolar H+-ATPase

Curr. Med. Chem.

Inhibition of macroautophagy by bafilomycin A₁ lowers proliferation and induces apoptosis in colon cancer cells

Bafilomycin A₁ inhibited the acidification of vesicular organelles and induced accumulation of LC3-II

Chemistry and structure activity relationships of bafilomycin A₁, a potent and selective inhibitor of the vacuolar H⁺-ATPase