Identification of compound CA-5f as a novel late-stage autophagy inhibitor with potent anti-tumor effect against non-small cell lung cancer

Abstract

Currently, particular focus is placed on the implication of autophagy in a variety of human diseases, including cancer. Discovery of small-molecule modulators of autophagy as well as their potential use as anti-cancer therapeutic agents would be of great significance. To this end, a series of curcumin analogs previously synthesized in our laboratory were screened. Among these compounds, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one (CA-5f) was identified as a potent late-stage macroautophagy/autophagy inhibitor via inhibiting autophagosome-lysosome fusion. We found that CA-5f neither impaired the hydrolytic function nor the quantity of lysosomes. Use of an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic screen in combination with bioinformatics analysis suggested that treatment of human umbilical vein endothelial cells (HUVECs) with CA-5f for 1 h suppressed the levels of cytoskeletal proteins and membrane traffic proteins. Subsequent studies showed that CA-5f exhibited strong cytotoxicity against A549 non-small cell lung cancer (NSCLC) cells, but low cytotoxicity to normal human umbilical vein endothelial cells (HUVECs), by increasing mitochondrial-derived reactive oxygen species (ROS) production. Moreover, CA-5f effectively suppressed the growth of A549 lung cancer xenograft as a single agent with an excellent tolerance in vivo. Results from western blot, immunofluorescence, and TdT-mediated dUTP nick end labeling (TUNEL) assays showed that CA-5f inhibited autophagic flux, induced apoptosis, and did not affect the level of CTSB (cathepsin B) and CTSD (cathepsin D) in vivo, which were consistent with the in vitro data. Collectively, these results demonstrated that CA-5f is a novel late-stage autophagy inhibitor with potential clinical application for NSCLC therapy. Abbreviations: 3-MA, 3-methyladenine; ANXA5, annexin A5; ATG, autophagy related; CA-5f, (3E,5E)-3-(3,4-dimethoxybenzylidene)-5-[(1H-indol-3-yl)methylene]-1-methylpiperidin-4-one; CQ, chloroquine; CTSB, cathepsin B; CTSD, cathepsin D; DMSO, dimethyl sulfoxide; DNM2, dynamin 2; EBSS, Earle's balanced salt solution; GFP, green fluorescent protein; HCQ, hydroxyl CQ; HEK293, human embryonic kidney 293; HUVEC, human umbilical vein endothelial cells; LAMP1, lysosomal associated membrane protein 1; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LDH, lactic acid dehydrogenase; LMO7, LIM domain 7; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; NAC, N-acetyl cysteine; MYO1E, myosin IE; NSCLC, non-small cell lung cancer; PARP1, poly(ADP-ribose) polymerase 1; PI, propidium iodide; RFP, red fluorescent protein; ROS, reactive oxygen species; SQSTM1, sequestosome 1; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

Keywords: Autophagy inhibitor; CA-5f; cell death; curcumin analogs; lysosome; non-small cell lung cancer.

Figure 1.

CA-5f regulates autophagy both in A549 cells and HUVECs. (a) The chemical structure of CA-5f with a molecular weight of 388.46 g/mol. (b) Microscopy photographs of A549 cells and HUVECs treated with the indicated concentrations (0–40 μM) of CA-5f for 3–24 h. Scale bar: 20 μm. (c) Western blot analysis of LC3B-II and SQSTM1 levels in A549 cells and HUVECs treated with the indicated concentrations (0–40 μM) of CA-5f for 6 h, or in the cells treated with CA-5f (20 μM) at the times indicated. GAPDH was used as a loading control. (d) qRT-PCR analysis of SQSTM1 mRNA level in A549 cells and HUVECs treated with the indicated concentrations (0–40 μM) of CA-5f for 6 h, or in the cells treated with CA-5f (20 μM) at the times indicated. (n = 3; *, p < 0.05 vs. control) (e) Fluorescence photographs of GFP-LC3B puncta in A549 cells and HUVECs treated with DMSO or CA-5f (20 μM) for 6 h. Scale bar: 10 μm. Nuclei were stained with Hoechst 33,258. Histogram shows quantification of the percentage of cells with GFP-LC3B puncta. (n = 3; *, p < 0.05; **, p < 0.01 vs. control) (f) Transmission electron micrographs of A549 cells and HUVECs treated with DMSO or CA-5f (20 μM) for 6 h. The lower pictures are the enlarged representations of the boxed regions of the upper pictures. Scale bar: 500 nm.

Figure 2.

CA-5f inhibits autophagic flux both in A549 cells and HUVECs. (a) Western blot analysis of LC3B-II levels in A549 cells and HUVECs treated with DMSO or CA-5f (20 μM) in the absence or presence of 3-MA (10 mM) or wortmannin (2 μM) for 6 h. (b) Western blot analysis of LC3B-II levels in A549 cells and HUVECs treated with DMSO or CA-5f (20 μM) in the absence or presence of bafilomycin A₁ (100 nM) or CQ (30 μM) for 6 h. (c) Western blot analysis of LC3B-II levels in A549 cells and HUVECs cultured in complete medium or EBSS in the absence or presence of CA-5f (20 μM) for 6 h. (d) Fluorescence photographs of A549 cells and HUVECs transfected with mRFP-GFP-LC3B reporter. Cells were treated with DMSO or CA-5f (20 μM) in complete medium or EBSS for 6 h. CQ (30 μM)-treated cells were used as positive controls. Nuclei were stained with Hoechst 33,258. Scale bar: 10 μm. (e) Average number of autophagosomes (yellow) and autolysosomes (red) per cell was figured up and grouped as described in (d). (n = 3; *, p < 0.05; **, p < 0.01 vs. control; ^##, p < 0.01 vs. EBSS).

Figure 3.

CA-5f suppresses autophagosome-lysosome fusion. (a) Fluorescence photographs of the colocalization of GFP-LC3B and LysoTracker Red in A549 cells and HUVECs cultured in full medium or EBSS in the absence or presence of CA-5f (20 μM) for 6 h. Nuclei were stained with Hoechst 33,258. Scale bar: 10 μm. (b) Immunofluorescence photographs of the colocalization of LC3B (green) and LAMP1 (red) in A549 cells and HUVECs treated with DMSO or CA-5f (20 μM) for 6 h. CQ (30 μM)-treated cells were used as positive controls. Nuclei were stained with DAPI. Scale bar: 10 μm.

Figure 4.

CA-5f does not affect the pH and hydrolytic function of lysosomes. (a) Fluorescence photographs of acridine orange (AO) staining and LysoTracker Red staining of A549 cells and HUVECs treated with DMSO, CA-5f (20 μM), or bafilomycin A₁ (100 nM) for 6 h. Scale bar: 10 μm. (b) Relative enzymatic activity of ACP (acid phosphatase) in A549 cells and HUVECs treated with CA-5f (20 μM) at the times indicated. (c) Relative enzymatic activity of CTSB and CTSD in A549 cells and HUVECs treated with CA-5f (20 μM) at the times indicated.

Figure 5.

CA-5f decreases the levels of cytoskeletal proteins and membrane traffic proteins. (a) Bar graph showed the top 10 cellular functions of the differentially expressed proteins in CA-5f-treated HUVECs. (b) Bar graph showed the top 10 biological pathways associated with the differentially-expressed proteins in CA-5f-treated HUVECs. The x axis displays -log (p-value). The y axis displays a decreased order of significance. The vertical line denotes the cutoff threshold for significance (p < 0.05). (c) Table showed the cytoskeletal proteins and membrane traffic proteins as classified in (a). * indicates the proteins exist in both categories. (d) Western blot analysis of 7 cytoskeletal proteins and membrane traffic proteins selected from (c) in HUVECs and A549 cells treated with DMSO or CA-5f (20 μM) for 1 h.

Figure 6.

The cytotoxicity of CA-5f on A549 cells and HUVECs. (a) Cell viability was monitored over 96 h in real time using an xCELLigence RTCA S16 System. Cell-sensor impedance is displayed as the cell index. The vertical dotted line indicates DMSO or CA-5f (20 μM) addition time points. (b) Flow cytometric analysis of ANXA5-FITC/PI staining in A549 cells and HUVECs treated with DMSO or CA-5f (20 μM) for 24 h. Apoptosis inducers were used as a positive control. (c) Transmission electron micrographs of A549 cells and HUVECs treated with DMSO or CA-5f (20 μM) for 24 h. Scale bar: 2 μm. (d) Western blot analysis of cleaved PARP1 in A549 cells and HUVECs treated with DMSO or the indicated concentrations (0–40 μM) of CA-5f for 24 h. GAPDH was used as a loading control. (e) Bar graph showing the relative lactate dehydrogenase (LDH) activity in A549 cells and HUVECs treated with the indicated concentrations (0–40 μM) of CA-5f for 24 h. (n = 3; *, p < 0.05; **, p < 0.01 vs. 0) (F) Western blot analysis of cleaved PARP1 in A549 cells and HUVECs treated with DMSO or CA-5f (20 μM) in the absence or presence of NAC (10 mM) for 24 h. (G) Bar graph showing the relative LDH activity in A549 cells treated with DMSO or CA-5f (20 μM) in the absence or presence of NAC (10 mM) for 24 h. (n = 3; *, p < 0.05 vs. control; #, p < 0.05 vs. CA-5f) (h) Cell viability determined by MTT assay. A549 cells and HUVECs treated with DMSO or CA-5f (20 μM) in the absence or presence of NAC (10 mM) for 24 h. (n = 3; **, p < 0.01 vs. control; #, p < 0.05 vs. CA-5f).

Figure 7.

CA-5f inhibits A549 lung cancer xenograft growth and autophagic flux in vivo. 10 nude mice with palpable tumors were randomly assigned to 2 groups: control group and CA-5f group. DMSO or CA-5f (40 mg/kg) was injected via caudal vein every 2 days for up to 30 days. (a) Mouse body weight and (b) tumor volume were recorded every 2 days for up to 30 days. (c) Tumor weight was measured at the day of sacrifice (day 30). (d) Images showed that tumors of all the 10 cases both in control and CA-5f injected-mice. (e) Western blot analysis of LC3B-II and SQSTM1 in tumor tissues. 1–5 represents tumor tissues in control mice. 6–10 represents tumor tissues in CA-5f injected-mice. Scattergram showed the densitometric analysis of LC3B-II and SQSTM1 in tumor tissues. GAPDH was used as a loading control. (f) Representative images showed the immunofluorescence staining of LC3B, SQSTM1, CSTB, CSTD, and TUNEL staining (green) in tumor sections. Panels marked with enlarged are cropped sections from the overview panels. Nuclei were stained with DAPI (red). Scale bar: 10 μm.