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. 2020 Jul 28:10:1217.
doi: 10.3389/fonc.2020.01217. eCollection 2020.

Mefloquine Inhibits Esophageal Squamous Cell Carcinoma Tumor Growth by Inducing Mitochondrial Autophagy

Yifei Xie  1 , Jing Zhang  1 , Bingbing Lu  1 , Zhuo Bao  1 , Jimin Zhao  1   2 , Xianyu Lu  1 , Yaxing Wei  1 , Ke Yao  3 , Yanan Jiang  1 , Qiang Yuan  1 , Xiaofan Zhang  1 , Bo Li  1 , Xinhuan Chen  1   2 , Zigang Dong  1   3   4   5 , Kangdong Liu  1   2   3   4   5
Affiliations

Mefloquine Inhibits Esophageal Squamous Cell Carcinoma Tumor Growth by Inducing Mitochondrial Autophagy

Yifei Xie et al. Front Oncol. .

Abstract

Esophageal squamous cell carcinoma (ESCC) has a worldwide impact on human health, due to its high incidence and mortality. Therefore, identifying compounds to increase patients' survival rate is urgently needed. Mefloquine (MQ) is an FDA-approved anti-malarial drug, which has been reported to inhibit cellular proliferation in several cancers. However, the anti-tumor activities of the drug have not yet been completely defined. In this study, mass spectrometry was employed to profile proteome changes in ESCC cells after MQ treatment. Sub-cellular localization and gene ontology term enrichment analysis suggested that MQ treatment mainly affect mitochondria. The KEGG pathway enrichment map of down-regulated pathways and Venn diagram indicated that all of the top five down regulated signaling pathways contain four key mitochondrial proteins (succinate dehydrogenase complex subunit C (SDHC), succinate dehydrogenase complex subunit D, mitochondrially encoded cytochrome c oxidase III and NADH: ubiquinone oxidoreductase subunit V3). Meanwhile, mitochondrial autophagy was observed in MQ-treated KYSE150 cells. More importantly, patient-derived xenograft mouse models of ESCC with SDHC high expression were more sensitive to MQ treatment than low SDHC-expressing xenografts. Taken together, mefloquine inhibits ESCC tumor growth by inducing mitochondrial autophagy and SDHC plays a vital role in MQ-induced anti-tumor effect on ESCC.

Keywords: autophagy; esophageal squamous cell carcinoma (ESCC); mefloquine (MQ); mitochondria; succinate dehydrogenase complex subunit C (SDHC).

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Figures

Figure 1
Figure 1
Mefloquine inhibits proliferation and anchorage- independent cell growth of KYSE150 and KYSE450 cells. (A) Cell count after treatment with varying doses of MQ (0, 1, 2.5, 5, 10 μM) in KYSE150 and KYSE450 cells after 0, 24, 48, 72 and 96 h. *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA); (B) Soft agar assay of KYSE150 and KYSE450 cells treated with different concentrations of MQ (0, 1, 2.5, 5 and 10 μM) for ~7 days. **p < 0.01, ***p < 0.001 (ANOVA); (C) Clone formation assay of KYSE150 and KYSE450 cells treated with different concentrations of MQ (0, 1, 2.5, 5 and 10 μM), stained with purple crystal after ~7 days. All data are shown as means ± S.D. The asterisks (*, **, ***) indicate a significant decrease (p < 0.05, p < 0.01, p < 0.001, respectively).
Figure 2
Figure 2
Flow graph of proteomics. (A) Schematic diagram of the quantitative proteomics analysis process. KYSE150 cells were treated with DMSO or MQ (10 μM) for 24 h, after LC-MS/MS analysis, 5,151 proteins were identified in the overlap section between DMSO and MQ (10 μM) groups. (B) Volcano diagram indicated the distribution of 5,151 proteins identified. Peptide mass error was plotted as x axis, the peptide score was plotted as y axis. (C) The distribution of peptide length of 5,151 proteins identified. Peptide length was plotted as x axis, the number of peptides was plotted as y axis.
Figure 3
Figure 3
Mefloquine remodels proteome of KYSE150 cells. MS analysis of global protein changes in KYSE150 cells after MQ (10 μM) treatment for 24 h compared with DMSO treatment. (A) Heat map representation of differentially expressed proteins between DMSO and MQ (10 μM) groups. The color key shows the log2 transformed protein amount ratios; (B) WoLF PSORT software analysis of the sub-cellular localization of differentially changed proteins; (C) Gene ontology term enrichment classification of differentially changed proteins according to cellular component and molecular function, with the top 4 related components and functions shown; (D) KEGG pathway enrichment map of down-regulated pathways (blue bars). Data was shown as -Log10 (Fisher's exact test p-value); (E) Venn diagram indicating the overlap between the 5 downregulated signaling pathways. Four overlapping proteins were listed; (F) Averaged quantitative kinase data from the full proteome, ranked according to their Log2 FC between the DMSO and MQ-treated cells, and the locations of SDHC, SDHD, MTCO3, and NDUFV3 were shown; (G) Western blot for SDHC, SDHD, MTCO3, and NDUFV3 in KYSE150 cells after DMSO or MQ (10 μM) treatment.
Figure 4
Figure 4
Mefloquine induces mitochondrial autophagy of KYSE150 cells. (A) ROS generation in KYSE150 cells after DMSO and MQ (10 μM) treatment for 24 h was measured with ROS assay kit; (B) KYSE150 cells probed for OXPHOS protein levels (complexes I, II, III, IV, and V) after DMSO or MQ (10 μM) treatment for 24 h; (C) NADH was quantified in KYSE150 cells after DMSO and MQ (10 μM) treatment for 24 h by NAD+/NADH assay kit; (D) ATP generation was tested in KYSE150 cells after DMSO and MQ (10 μM) treatment for 24 h by ATPlite assay kit; (E) Mitochondrial mass was evaluated with NAD staining in mitochondria after DMSO and MQ (10 μM) treatment for 24 h; (F) KYSE150 cells were photographed by transmission electron microscopy after DMSO or MQ (10 μM) treatment for 24 h. Three electron microscopy pictures were randomly chosen, and the number of autophagic mitochondria was counted. Black boxes indicate the normal mitochondria, and red boxes indicate the autophagosomes engulfing mitochondria. Scale = 500 nm; (G) LC3-II labeling for autophagic mitochondria after 24 h of DMSO or 10 μM MQ treatment by immunofluorescence analysis; (H) Western blot for LC3A/B in KYSE150 cells after DMSO and MQ (10 μM) treatment for 24 h; (I) Co-localization analysis of MTCO1 and LC3-II in KYSE150 cells after DMSO and MQ (10 μM) treatment for 24 h by immunofluorescence analysis.
Figure 5
Figure 5
SDHC is highly expressed in ESCC tissues and knocking SDHC down suppresses proliferation of ESCC cells. (A) ESCC tissue spots were incubated with SDHC antibody, then stained with DAB and DAB density was analyzed for ESCC and normal tissues. The asterisks (***) indicate a significant change (p < 0.001); (B) Short hairpin RNA (shSDHC) in vector plko.1 was used to transduce KYSE150 and KYSE450 cells, with knock-down efficiency tested by Western blot in KYSE150 and (C) KYSE450 cells; (D) Cell count for KYSE150 and (E) KYSE450 cells at 0, 24, 48, 72, and 96 h; (F) Colony formation for KYSE150 and KYSE450 cells after 10 days. All data are shown as means ± S.D. The asterisks (*, **, ***) indicate a significant decrease (p < 0.05, p < 0.01, p < 0.001, respectively).
Figure 6
Figure 6
Mefloquine inhibits tumor growth of ESCC PDX models. (A) SDHC protein level was detected in cases of ESCC tissues by Western blot; (B,C) Effect of MQ treatment on tumor growth. MQ significantly suppressed tumor growth of case EG59 and EG60, while not in case EG20 and EG84; (D) Immunohistochemistry analysis of SDHC with DAB staining in case EG59, EG60, EG20, and EG84 after MQ treatment. All data are shown as means ± S.D. The asterisks (*, **, ***) indicate a significant decrease (p < 0.05, p < 0.01, p < 0.001, respectively).
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