Depletion of COPI in cancer cells: the role of reactive oxygen species in the induction of lipid accumulation, noncanonical lipophagy and apoptosis

Abstract

The coatomer protein complex 1 (COPI) is a multisubunit complex that coats intracellular vesicles and is involved in intracellular protein trafficking. Recently we and others found that depletion of COPI complex subunits zeta (COPZ1) and delta (ARCN1) preferentially kills tumor cells relative to normal cells. Here we delineate the specific cellular effects and sequence of events of COPI complex depletion in tumor cells. We find that this depletion leads to the inhibition of mitochondrial oxidative phosphorylation and the elevation of reactive oxygen species (ROS) production, followed by accumulation of lipid droplets (LDs) and autophagy-associated proteins LC3-II and SQSTM1/p62 and, finally, apoptosis of the tumor cells. Inactivation of ROS in COPI-depleted cells with the mitochondrial-specific quencher, mitoquinone mesylate, attenuated apoptosis and markedly decreased both the size and the number of LDs. COPI depletion caused ROS-dependent accumulation of LC3-II and SQSTM1 which colocalizes with LDs. Lack of double-membrane autophagosomes and insensitivity to Atg5 deletion suggested an accumulation of a microlipophagy complex on the surface of LDs induced by depletion of the COPI complex. Our findings suggest a sequence of cellular events triggered by COPI depletion, starting with inhibition of oxidative phosphorylation, followed by ROS activation and accumulation of LDs and apoptosis.

FIGURE 1:

Depletion of the COPI complex inhibits mitochondrial activity and activates ROS production in tumor cell lines. (A) Measurements of mitochondrial metabolic activity. PC3 cells were transfected with control (siCONT) or anti COPZ1 (siCOPZ1) siRNAs and plated in Seahorse XF24 microplates. ATP production (upper panel), and maximum respiration capacity (lower panel) were detected by the OCR, see Supplemental Figure S1 for details. The bar graph shows the mean of four independent transfections ± SE. (B) Measurement of induced mitochondrial ROS production in PC3 cells. PC3 cells were transfected with control (siCONT) or anti COPA (siCOPA) siRNAs. 72 h posttransfection ROS were detected with DCFDA, and mitochondria were stained with MitoNIR in live cells. Fluorescent images of separate and merged images are presented. (C) Summary of flow cytometry analysis of ROS production in COPI-depleted PC3, DU145, and U2OS cells and the effects of MitoQ and JNK inhibitor SP600125, labeled as JNKi (examples of flow cytometry profiles are presented in Supplemental Figure S2). Plots represent the DCFDA mean fluorescent intensity in siCONT, siCOPA, and siCOPZ1-transfected cells ± MitoQ or SP600125 treatments. Results shown are three independent experiments per data point ± SD. Scale bar = 25 μm.

FIGURE 2:

Depletion of the COPI complex induces the formation of LDs. PC3, U2OS, or DU145 cells were transfected with siCONT, siCOPA, and siCOPZ1; 72 h posttransfection, LDs and nuclei were visualized with BODIPY 493/503 (green) and DAPI (blue), respectively. (A). Fluorescent images of LDs in transfected cells. (B–D). Quantification of the effects of COPI complex depletion on the number and size of LDs in DU145 (B), PC3 (C), and U2OS (D) cells transfected with siRNAs. Plots in the left panels represent the mean of the number of the droplets per cell ± SEM. The total number of LDs was quantified from fluorescent images of triplicate experiments (∼5 fields per experiment, 50–100 cells per data point). Plots in the middle panel represent the mean of droplet areas in transfected cells ± SEM. The areas of the LDs (500–1300) per data point were obtained from three independent transfections. Plots in the right-hand panel show the distribution of droplet size in the transfected cells. Distributions are presented in a logarithmic scale. Scale bar = 25 μm.

FIGURE 3:

MitoQ and JNK inhibitor SP600125 decrease the size of LDs induced by depletion of COPI complex proteins. Cells were transfected with siCONT, siCOPA, and siCOPZ1 in triplicates. 48 h posttransfection cells were treated with MitoQ, JNK inhibitor, or vehicle control (DMSO) for an additional 24 h. (A). Representative fluorescent images of LDs (green) and nuclei (blue) visualized by BODIPY and DAPI staining, respectively, in the untransfected (control) and siRNA-transfected PC3 cells treated with JNK inhibitor, MitoQ, or vehicle (DMSO). Images of DU145 and U2OS cells are shown in Supplemental Figure S4, A and B. (B) Plots represent the mean of droplet areas in transfected cells ± SEM. (C) Plots represent the mean of the number of the droplets per cells ± SEM. Scale bar = 25 μm.

FIGURE 4:

Time course of the decrease size of LDs in COPI-depleted cells by MitoQ treatment. PC3 cells were transfected with siCOPA; 48 h posttransfection, cells were treated with MitoQ or vehicle control up to 24 h. The cells were fixed at seven time points (0, 2, 4, 8, 12, and 24 h) followed by LD staining with BODIPY. (A) BODIPY staining of MitoQ-treated or vehicle-treated COPI-depleted cells. (B) Analysis of droplet size distribution. Plots show size distribution. The droplet area is shown in log10 scale. (C) Plot represents maximums of droplet size distribution over time of MitoQ treatment. Scale bar = 10 μm.

FIGURE 5:

COPI depletion does not affect the rate of lipolysis and inhibition of lipolysis results in droplets insensitive to ROS depletion. (A) Representative immunofluorescence images of LDs in PC3 cells were treated with 100 µM Atglistatin for 48 h, ± MitoQ, JNK inhibitor, or DMSO (vehicle). LDs are visualized by BODIPY (green) and nuclei by DAPI (blue) staining. (B) Plot represents the average size of the LDs in the treated cells ± SEM. (C) Plot showing the release of [³H]-NEFA into the medium from cells treated with Atglistatin or transfected with siCOPA ± MitoQ or JNK inhibitor. The bars are the average of quadruplicates ±SEM. Scale bar = 40 μm.

FIGURE 6:

Depletion of the COPI elevates expression of markers of autophagy and number of LDs colocalized with autophagy markers. (A) Western blotting analysis of the expression of LC3 and p62/SQSTM1 in COPZ1 or COPA-depleted cells. (B) Western blotting analysis of Atg5 expression in PC3, U2OS, and DU145 cell lines. (C) Representative immunofluorescent images of LDs and LC3 in the control or siRNA-transfected PC3 cells; 72 h posttransfection, the LC3 was visualized by staining with specific antibodies (red), LDs by BODIPY (green), and nuclei by DAPI (blue) staining. Images of U2OS and DU145 cells are presented in Supplemental Figure S5. Scale bar = 25 μm. (D) Quantification of LC3-positive LDs. The total number of LDs and the number of droplets positive for LC3 were determined. Plot represents the average quantification of at least 12 different fields from three independent transfections, ± SEM.

FIGURE 7:

Treatment with MitoQ and JNK inhibitor decreases accumulation and colocalization of autophagic markers with LDs in COPI-depleted cells. U2OS and PC3 cells were transfected with siCONT, siCOPA, siCOPZ1, or mock transfection. MitoQ, SP600125, or vehicle control was added 48 h posttransfection; 72 h posttransfection, cells were either fixed or lysed for IF or Western blotting analysis. (A) Western blotting determination of the level of LC3-II and SQSTM1/p62 in COPA-depleted or COPZ1-depleted PC3 and U2OS cells treated with MitoQ. (B) Western blotting determination of the level of LC3-II and SQSTM1/p62 in COPZ1-depleted PC3 and U2OS cells treated with JNK inhibitor. (C) Quantification of LC3-positive LDs. The total number of LDs and the number of droplets positive for LC3 were determined. Plot represents the average of quantification of at least 12 different fields from three independent transfections, ± SEM. (D) Representative immunofluorescent images of LDs (BODIPY, green) and LC3 (red) in the control or siCOPZ1-transfected PC3 cells, treated with MitoQ or JNK inhibitor. Images of U2OS and DU145 cells are presented in Supplemental Figure S6. Scale bar = 25 μm.

FIGURE 8:

EM analysis of LDs in COPI knockdown cells did not reveal any accumulation of double-membrane autophagosomes. PC3 cells were transfected with siCONT or siCOPA for 48 h, then treated with MitoQ or vehicle control, followed by fixation at 72 h posttransfection. The structure of the droplets was analyzed by TEM with either low (7,000× or 8,000×) or high (12,000×) magnifications. Panels B and D show parts of panels A and C, respectively, at the higher magnifications. Panels E and F show separate areas taken at different magnifications. LDs = lipid droplets. Scale bar = 0.5 μm.

FIGURE 9:

Apoptotic cell death in COPI-depleted cells is attenuated by MitoQ and a JNK inhibitor and increased by treatment with external lipids. (A) Depletion of the COPI complex induced apoptosis. U2OS, PC3, and DU145 cells were transfected either with control or COPZ1 siRNAs in triplicates. The percentage of apoptotic cells was determined by TUNEL assay. Plot shows the percentage of TUNEL positive cells ±SD. (B) MitoQ effects on JNK phosphorylation and apoptosis. DU145, U2OS, or PC3 cells were transfected with siCONT, siCOZ1 or siCOPA. 48 h posttransfection cells were treated with vehicle control or MitoQ. At 72 h cells were lysed and PARP cleavage, JNK phosphorylation, total level of JNK, COPA, and COPZ1 expression were determined by WB with the corresponding antibodies. (C). Effects of JNK inhibitor on JNK phosphorylation and apoptosis. DU145, U2OS, or PC3 cells were transfected with siCONT, or siCOPZ1. 48 h posttransfection cells were treated with vehicle control or JNK inhibitor. At 72 h cells were lysed and PARP cleavage, JNK phosphorylation, and total level of JNK, COPA, and COPZ1 expression were determined by Western blotting with the corresponding antibodies. (D). Depletion of the COPI complex sensitizes cells to lipotoxicity. U20S cells were transfected with siCONT or siCOPA; 48 h posttransfection, cells were treated with different concentrations of oleic acids for 24 h. The percentage of dead cells was analyzed using live/dead viability/cytotoxicity assay. Plot represents dependence of percentage of alive ethidium homodimer-1 negative cells. Average of triplicates, ± SE.

FIGURE 10:

Scheme of the effects of COPI depletion in eukaryotic cells.