N-Methylparoxetine Blocked Autophagic Flux and Induced Apoptosis by Activating ROS-MAPK Pathway in Non-Small Cell Lung Cancer Cells
<p>NMP inhibited human NSCLC cell proliferation. (<b>A</b>) Molecular structure of NMP. (<b>B</b>) Inhibition rates of proliferation in NMP-treated NCI-H1299 and NCI-H1650 cells (24 h) quantified by CCK-8 viability assay. Median inhibitory concentrations (IC50) were estimated by log(inhibitor) vs. normalized response of non-linear regression analysis. (<b>C</b>) Inhibition rates of proliferation in NMP-treated NCI-H1299, NCI-H1650 and BEAS-2B cells (24 h) quantified by CCK-8 viability assay. (<b>D</b>) Colony formation assay of NCI-H1299 and NCI-H1650 cells, treated with serial concentrations of NMP for 7 days. (<b>E</b>) Flow cytometry analyses of 24 h NMP (0–60 μM) treatment of CFDA-SE-labelled NCI-H1299 and NCI-H1650 cells (<b>F</b>) Fluorescence micrographs of NMP (0–60 μM, 24 h)-treated NCI-H1299 and NCI-H1650 cells with EdU incorporation. <span class="html-italic">Green</span>, EdU-positive cells; <span class="html-italic">blue</span>, Hoechst 33342 for nuclear staining. Scale bar, 20 μm. Error bars, means ± S.D. of three independent experiments; * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01,*** <span class="html-italic">p</span> < 0.001, compared to the control group.</p> "> Figure 2
<p>NMP induced apoptosis in NSCLC cells. (<b>A</b>) Flow cytometry analyses of NMP-treated NCI-H1299, NCI-H1650, and BEAS-2B cells that were subjected to PI/Annexin V staining assay for apoptosis detection. Error bars means ± S.D. of three independent experiments; *** <span class="html-italic">p</span> < 0.001, compared to the control group. (<b>B</b>,<b>C</b>) Western blots of whole cell lysates in NCI-H1299 and NCI-H1650 cells which were treated with NMP (60 µM) or cisplatin(Cis, 35 µM) at the indicated doses for 24 h (<b>B</b>) or for the indicated time courses (<b>C</b>).</p> "> Figure 3
<p>NMP induced apoptosis through a mitochondria-dependent pathway in NSCLC cell lines. (<b>A</b>,<b>B</b>) Fluorescence micrographs of mitochondria in a vehicle or 40 µM NMP-treated NCI-H1299 and NCI-H1650 cells with MitoTracker Red CMXRos staining. The length of mitochondria was quantified with ImageJ (US National Institutes of Health, Bethesda, MD, USA). Scale bar, 5 μm. Error bars mean ± S.D. of three independent experiments; *** <span class="html-italic">p</span> < 0.001, compared to the control group. (<b>C</b>) Western blot assay for mitochondria-dependent apoptosis of different cellular fractions obtained from NMP treated NCI-H1299 cells. The intensity of bands was quantified by using Gelpro32 Analyzer (Media Cybernetics, Inc., MD, USA). One-way analysis of variance (ANOVA), ** <span class="html-italic">p</span> < 0.01,*** <span class="html-italic">p</span> < 0.001, compared to the control group. <span class="html-italic">Cyto</span>, cytosolic fractions; <span class="html-italic">Mito</span>, mitochondrial fractions; <span class="html-italic">WCL</span>, whole cell lysates.</p> "> Figure 4
<p>NMP induced ROS accumulation and activated MAPK pathways. (<b>A</b>,<b>B</b>) Flow cytometry analyses of intracellular ROS in NMP-treated NCI-H1299 and NCI-H1650 labeled with green DCFDA fluorescent dye. Error bars mean ± S.D. of three independent experiments; *** <span class="html-italic">p</span> < 0.001, compared to the control group. (<b>C</b>) Western blot assay for MAPK pathways in NMP-treated NCI-H1299 cells.</p> "> Figure 5
<p>ROS clearance reversed JNK/p38 activation and attenuated NMP-induced apoptosis. (<b>A</b>) CCK-8 viability assay of NCI-H1299 cells treated with different concentrations of NMP with or without NAC (2 mM) for 24 h. (<b>B</b>) Flow cytometry analyses of the treated cells with Annexin V/PI labeling. (<b>C</b>) Western blot analysis for MAPK pathways of the treated cells. Error bars, means ± S.D. of three independent experiments; ** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.001, compared to the control group.</p> "> Figure 6
<p>NMP induced the accumulation of autophagosomes in NSCLC Cells. (<b>A</b>,<b>B</b>) Western blot analyses for LC3II of dose-dependent (24 h treatment, (<b>A</b>)) or time-dependent responses (<b>B</b>) in NCI-H1299 and NCI-H1650 cells treated with NMP (60 µM) or bafilomycin A1 (Baf, 0.1 µM). (<b>C</b>) Fluorescence micrographs of LC3-stably-expressed NCI-H1299 and NCI-H1650 cells treated with vehicle, rapamycin (Rapa, 0.5 µM), bafilomycin A1 (Baf, 0.1 µM) or NMP (40 µM). Scale bar, 20 μm. Error bars mean ± S.D. of three independent experiments; *** <span class="html-italic">p</span> < 0.001, compared to the control group.</p> "> Figure 7
<p>NMP impaired NSCLC cell late-staged autophagic flux. (<b>A,B</b>) Western blot analysis for p62 of NMP- of Baf-treated NCI-H1299 and NCI-H1650 cells at the indicated doses for 24 h (<b>A</b>), or 60 µM NMP- or 0.1 µM Baf-treated cells for the indicated time courses (B). (<b>C</b>) Flow cytometry analyses of GFP-LC3 mean fluorescence intensities in NCI-H1299 cells treated with NMP (40 µM), Baf (0.1 µM) or in combination. (<b>D</b>) <span class="html-italic">Left</span>, typical fluorescence micrographs of mCherry-GFP-LC3-expressing NCI-H1299 or NCI-H1650 cells treated with vehicle, NMP (40 µM), or Baf (0.1 µM) for 24 h; or HBSS for 6 h. Scale bar, 5 μm. <span class="html-italic">Right</span>, Pearson’s correlation analyses of GFP/mCherry colocalization. Error bars, means ± S.D. of three independent experiments; ** <span class="html-italic">p</span> < 0.01, ***<span class="html-italic">p</span> < 0.001, compared to the control group.</p> "> Figure 8
<p>NMP altered lysosomal pH and inhibited lysosomal cathepsins maturation. (<b>A</b>,<b>B</b>) Typical fluorescence micrograph of vehicle-, Baf (0.1 µM)- or NMP (40 µM)-treated NCI-H1299 and NCI-H1650 cells subjected to pH-dependent fluorescent dyes acridine orange (AO) and Lysotracker Red DND-99 stainings to detect lysosomal acidification. Scale bar, 20 μm. (<b>C</b>,<b>D</b>) Western blot analysis for mature cathepsin B and D detection in treated cells in dose- (<b>C</b>) or time-dependent manners (<b>D</b>).</p> "> Figure 9
<p>NMP induced apoptosis in NSCLC cells by dual pathways. An illustration demonstrating the summary of NMP actions on apoptosis in NSCLC cells. (<b>Left</b>) NMP inhibited late-staged autophagy flux by inhibiting acidification of early lysosome. (<b>Right</b>) NMP stimulated mitochondrial fission and fragmentation, leading to ROS and cytochrome C leakage. Cytochrome C is a risk factor to promote apoptosis. Excessive ROS accumulation further damages mitochondria, thus NMP could induce an effective apoptotic effect on tumor cells by promoting mitochondrial-dependent apoptosis and inhibiting autophagy in NSCLC cells in parallel.</p> ">
Abstract
:1. Introduction
2. Results
2.1. NMP Inhibited NSCLC Cells Proliferation
2.2. NMP Induced Apoptosis in NSCLC Cells
2.3. NMP Induced Apoptosis via a Mitochondria-Dependent Pathway
2.4. NMP Induced ROS Accumulation and Activated MAPK Pathways
2.5. ROS Clearance Reversed the Activation of JNK/p38 and Attenuated NMP-Induced Apoptosis
2.6. NMP Induced the Accumulation of Autophagosomes in NSCLC Cells
2.7. NMP Impeded the Late-Staged Autophagic Flux in NSCLC Cells
2.8. NMP Altered the Lysosomal pH and Inhibited Lysosomal Cathepsins Maturation
3. Discussion
4. Materials and Methods
4.1. Cell Culture and Drugs
4.2. Cell Viability Assay
4.3. Colony Formation Assay
4.4. CFDA-SE Cell Tracking Assay
4.5. 5-Ethynyl-20-Deoxyuridine (EdU) Cell Proliferation Assay
4.6. Cell Apoptosis Detection
4.7. Cellular Fractionation Western Blot Analysis
4.8. Western Blot Analysis
4.9. Plasmid Transfection Assay
4.10. AO Staining
4.11. LysoTracker Red Staining
4.12. MitoTracker Red CMXRos Staining
4.13. Intracellular ROS Measurement
4.14. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
NMP | N-Methylparoxetine |
NSCLC | Non-small cell lung cancer |
NAC | N-acetylcysteine |
ROS | Reactive oxygen species |
MAPK | mitogen-activated protein kinase |
JNK | c-Jun N-terminal kinase |
p38 | p38 MAP kinase |
ADMET | Adsorption, distribution, metabolism, excretion, and toxicity |
IC50 | Median inhibitory concentrations |
MOMP | Mitochondria outer membrane permeability |
DCFDA | 2′,7′-Dichlorofluorescein diacetate |
Baf | Bafilomycin A1 |
AO | Acridine orange |
PE | Phosphatidylethanolamine |
CQ | Chloroquine |
3-MA | 3-Methyladenine |
Rapa | Rapamycin |
CCK-8 | Cell Counting Kit-8 |
PFA | Paraformaldehyde |
EdU | 5-Ethynyl-20-deoxyuridine |
BSA | Bovine serum albumin |
SDS-PAGE | Sodium dodecyl sulfate–polyacrylamide gel electrophoresis |
ECL | Enhanced chemiluminescence |
ANOVA | Analysis of variance |
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Wang, K.; Chen, B.; Yin, T.; Zhan, Y.; Lu, Y.; Zhang, Y.; Chen, J.; Wu, W.; Zhou, S.; Mao, W.; et al. N-Methylparoxetine Blocked Autophagic Flux and Induced Apoptosis by Activating ROS-MAPK Pathway in Non-Small Cell Lung Cancer Cells. Int. J. Mol. Sci. 2019, 20, 3415. https://doi.org/10.3390/ijms20143415
Wang K, Chen B, Yin T, Zhan Y, Lu Y, Zhang Y, Chen J, Wu W, Zhou S, Mao W, et al. N-Methylparoxetine Blocked Autophagic Flux and Induced Apoptosis by Activating ROS-MAPK Pathway in Non-Small Cell Lung Cancer Cells. International Journal of Molecular Sciences. 2019; 20(14):3415. https://doi.org/10.3390/ijms20143415
Chicago/Turabian StyleWang, Kun, Bonan Chen, Ting Yin, Yujuan Zhan, Yuhua Lu, Yilin Zhang, Jiawei Chen, Weijie Wu, Shikun Zhou, Wenli Mao, and et al. 2019. "N-Methylparoxetine Blocked Autophagic Flux and Induced Apoptosis by Activating ROS-MAPK Pathway in Non-Small Cell Lung Cancer Cells" International Journal of Molecular Sciences 20, no. 14: 3415. https://doi.org/10.3390/ijms20143415