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. 2016 Jun 21;7(25):38078-38090.
doi: 10.18632/oncotarget.9348.

Derivatives of 6-cinnamamido-quinoline-4-carboxamide impair lysosome function and induce apoptosis

Hsiao-Hui Kuo  1 , Rajesh Kakadiya  2 , Yi-Chen Wu  1 , Tsann-Long Su  2 , Te-Chang Lee  2 , Yi-Wen Lin  2 , Ling-Huei Yih  1
Affiliations

Derivatives of 6-cinnamamido-quinoline-4-carboxamide impair lysosome function and induce apoptosis

Hsiao-Hui Kuo et al. Oncotarget. .

Abstract

Autophagy is a lysosomal degradative process that protects cancer cells from multiple types of stress. In this study, we synthesized a series of derivatives of 6-cinnamamido-quinoline-4-carboxamide (CiQ), and investigated their effects on the proliferation and autophagy of cancer cells in vitro. These derivatives effectively inhibited the proliferation of a broad spectrum of cancer cell lines. Further study revealed that CiQ derivatives may induce autophagy and result in disruption of autophagy propagation. Consequently, these derivatives triggered massive apoptosis, as evidenced by caspase-9 activation and PARP cleavage. Blockage of autophagy by depletion of autophagy related gene ATG5 or BECN1 considerably alleviated CiQ-induced cell death, indicating that autophagy may mediate CiQ-induced cell death. Furthermore, treatment with CiQ derivatives increased lysosome membrane permeability (LMP) and enhanced accumulation of ubiquitinated proteins, which collectively indicate impaired lysosome function. In addition, treatment of cells with CiQ derivatives activated extracellular signal-regulated kinase (ERK); abrogation of ERK activation, either by treating cells with U0126, an inhibitor of mitogen-activated protein/ERK kinase 1 (MEK1), or by ectopically overexpressing a dominant-negative MEK1, significantly reduced CiQ derivative-induced LMP, LC3 and p62 accumulation, and cytotoxicity. These results indicate that CiQ derivatives activate ERK and disrupt lysosome function, thereby altering autophagic flux and resulting in apoptotic cell death.

Keywords: ERK; anticancer; autophagy; cinnamamide quinolines; lysosome.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
A. The chemical structures of selected autophagy inhibitors and CiQ. B. The IC50 of CiQ derivatives against the indicated human cancer cell lines. Cells were treated with each drug for 72 h, and the number of viable cells was subsequently determined using the WST-8 assay. Data represent the mean±SD of the IC50 of each compound, as determined based on the dose–effect relationships of six concentrations from three to six independent experiments. ND, not determined. Numbers in brackets are measures of cross-resistance, as determined by comparison with the corresponding IC50 value of the parent cell line.
Figure 2
Figure 2. Disruption of autophagy by treatment with CiQ derivatives
A. A549 cells were treated with the indicated concentration of CiQ derivative for 8 h, and then subjected to immunoblotting to detect cellular accumulation of LC3-II and p62. B. A549 cells were treated with 1 μM 5e (BO-2222) or 5r (BO-2112) for 8 h and then subjected to immunofluorescence staining of LC3 or p62 (green). Nuclei were counterstained with DAPI (blue). C. A549 cells were treated with the indicated concentration of 5e (BO-2222) alone or together with 2 mM 3MA, 20 nM bafilomycin A1 (Baf A1), or 10 μM CQ for 14 h, and the cells were subsequently subjected to LC3-II and p62 immunoblotting. GAPDH served as a loading control. D and E. The expression of ATG5 or BECN1 was downregulated by transducing cells with virion containing the corresponding shRNA. At 72 h after transduction, cells were collected for immunoblot analysis to verify the depletion efficiency (D), or for analysis of the cytotoxicity of 5e (E). The data represent the mean ± SD of at least three independent experiments. * indicates p < 0.01 as compared to untransduced and untreated cells and # indicates p < 0.01 as compared to cells transduced with control vector and treated with CiQ, according to Student's t test.
Figure 3
Figure 3. Induction of apoptosis by treatment with CiQ derivatives
A. A549 cells were treated with the indicated CiQ derivative for 48 h and then subjected to immunoblotting to detect cleavage of PARP (cPARP) and caspase-9. B. Cell cycle distribution of cPARP-positive cells. A549 cells were treated with the indicated CiQ derivative for 48 h, and then subjected to flow cytometric analysis of PARP cleavage. Cellular DNA was counterstained with propidium iodide. In the lower panel, the green dots indicate cleaved PARP-positive cells and the numbers indicate the percentage of cleaved PARP-positive cells. C. A549 cells were treated with 5e (BO-2222) for 24 or 48 h, and PARP cleavage was subsequently analyzed by flow cytometry. The percentages of cells with cleaved PARP were determined. The data represent the mean ± SD for three independent experiments.
Figure 4
Figure 4. Induction of lysosome dysfunction by treatment with CiQ derivatives
A and B. CiQ derivative treatment reduced the accumulation of LTG. A549 cells were untreated or treated for 7 h with 1–2 μM 5e (BO-2222) (A) or with 1 μM of the indicated CiQ (B), and then loaded with LTG by incubation in media containing 100 nM LTG for 30 min. Following thorough washout of excess LTG, cellular accumulation of LTG was analyzed by flow cytometry at FL1. The FL1 spectrum in (A) indicates cellular accumulation of LTG. Cells unlabeled with LTG were used as a negative control. (B) The ratio of the mean intensity of LTG at FL1 in cells treated with test compounds to that in untreated cells. The data represent the mean ± SD for three independent experiments. * indicates p < 0.01 as compared to untreated cells. C. CiQ derivatives induced the release of FITC-dextran from lysosomes. A549 cells were incubated in media containing 1 mg/ml FITC-dextran for 14 h. After thorough washout of free FITC-dextran, the FITC-dextran-loaded cells were subsequently treated with vehicle or 2 μM 5e (BO-2222) for 3 h. The cells were monitored under an inverted fluorescence microscope. D. 5e (BO-2222)-induced LC3 punctae colocalized with LAMP1. A549 cells were untreated or treated with 2 μM 5e (BO-2222) for 7 h, and then immunostained for LC3 (green) and LAMP1 (red). Nuclei were counterstained with DAPI (blue). E and F. Treatment with CiQ derivatives induced accumulation of ubiquitinated proteins. A549 cells were treated with 2 μM of the indicated CiQ derivative for 8 h, and ubiquitinated proteins were subsequently detected by immunoblotting (E) or immunofluorescence staining (E, green). For immunofluorescence staining, cells were co-immunostained with LAMP1 (red) to locate lysosomes, and nuclei were counterstained with DAPI (blue).
Figure 5
Figure 5. CiQ derivatives disrupted lysosome function and blocked autophagy in an ERK-dependent manner
A. CiQ derivative treatment induced phosphorylation of ERK. A549 cells were treated with 2 μM of the indicated CiQ derivative for 8 h, and then subjected to immunoblot with antibodies against the indicated protein. B. U0126 reduced 5e (BO-2222)-induced accumulation of LC3-II and p62. A549 cells were pre-treated with 3 μM U0126 for 14 h and then co-treated with 1 μM 5e for another 8 h. C. U0126 ameliorated CiQ-induced LMP. A549 cells were pre-treated with 3 μM U0126 for 14 h and then co-treated with 0.5 μM of the indicated CiQ derivative for 7 h. The cells were then loaded with LTG, and LTG accumulation was analyzed. D. U0126 reduced 5e-induced LC3 punctae formation. A549 cells were pre-treated with 3 μM U0126 for 14 h and then co-treated with 1 μM 5e for 8 h. LC3 punctae were detected by immunofluorescence staining. At least 300 cells were examined under a fluorescence microscope for each experiment. E. U0126 reduced the cytotoxicity of 5e. A549 cells were treated with the indicated concentration of 5e alone or together with 3 μM U0126 for 72 h. Cell viability was determined using the WST-8 assay. The data represent the mean ± SD of at least three independent experiments. * indicates p < 0.01 as compared to untreated cells and # indicates p < 0.01 as compared to CiQ treatment alone, according to Student's t test.
Figure 6
Figure 6
A and B. Ectopic overexpression of a dominant-negative MEK1 ameliorated 5e-induced phosphorylation of ERK, accumulation of LC3-II and p62, and cytotoxicity. A549 or H460 cells were transfected with the indicated MEK expression construct, and at 24 h after transfection were treated with 2 μM 5e for 8 h or 60 h (A), or with the indicated concentration of 5e for 60 h (B). Immunoblotting was used to detect the indicated protein (A). Cell viability was determined using the WST-8 assay (B). * indicates p < 0.01 as compared to empty vector (pCMV)-transfected cells subjected to the same CiQ treatment. C. A simplified model depicting how CiQ disrupts autophagy. The three main steps in the autophagic pathway are shown. The formation of the autophagosome depends on the initiation and/or nucleation of a specific membrane structure, known as the phagophore. A portion of the cytoplasm, including organelles, is sequestered in the phagophore. The autophagosome is formed following elongation and closure of the phagophore. Finally, the outer membrane of the autophagosome fuses with the lysosome, and the internal material is degraded within the autolysosome. Autophagy can be inhibited at early stages using 3MA (a PI3K inhibitor) to block initiation, or at late stages using CQ (a lysosomotropic agent) or bafilomycin A1 (Baf A1, an ATPase inhibitor) to block the fusion of autophagosomes and lysosomes. We report here that CiQ derivatives activate ERK and impair lysosome function, which disrupts the degradative activity of autolysosomes (as indicated by the bar-headed line).
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