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Sulforaphane-cysteine-induced apoptosis via phosphorylated ERK1/2-mediated maspin pathway in human non-small cell lung cancer cells.

ABSTRACT: Sulforaphane (SFN) was demonstrated to induce apoptosis in a variety of cancers via multiple mechanisms. However, owing to a short half-life in circulation, SFN was not used for clinical treatment yet. Interestingly, SFN analog, sulforaphane-cysteine (SFN-Cys) has a longer half-life in metabolism, and we previously demonstrated that SFN-Cys inhibited invasion in human prostate cancer cells. Here, we would investigate whether SFN-Cys induces apoptosis and find the underlying mechanisms in human non-small cell lung cancer (NSCLC) cells. Western blots were used to test the molecular linkages among extracellular signal-regulated kinases 1/2 (ERK1/2) and downstream signal molecules. Flow cytometry and fluorescence microscopy were used to detect cell death. Cell proliferation assay showed that SFN-Cys inhibited cell viability following a dose-dependent manner. Abnormal cell morphology was viewed after the cells were exposed to SFN-Cys. Flow cytometry showed that SFN-Cys induced cell apoptosis via a dose-dependent manner. Further, SFN-Cys triggered the activation of ERK1/2, which resulted in the upregulation of maspin, Bax, cleaved caspase-3 and downregulation of pro-caspase-3, Bcl-2, α-tubulin. Meanwhile, we demonstrated that recombinant caspase-3 cleaved α-tubulin in the lysate of cells, which were treated by SFN-Cys. These data indicated that SFN-Cys activated the ERK1/2-mediated mitochondria signaling pathway with maspin upregulation and α-tubulin downregulation leading to apoptosis. These findings will help to develop a novel therapeutic to target NSCLC cells.

SUBMITTER: Lin K

PROVIDER: S-EPMC5494314 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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Project description:Both sulforaphane-cysteine (SFN-Cys) and sulforaphane-N-acetyl-L-cysteine (SFN-NAC) inhibited cancer migration and invasion, but the underlying mechanisms were not clear. Here we uncovered via tissue microarray assay that high expression of invasion-associated Claudin-5 was correlated to malignant grades in human non-small cell lung cancer (NSCLC) cells. Further, SFN-Cys (10 µM) induced the accumulated phosphorylation of ERK1/2, leading to downregulation of Claudin-5 and upregulation of Claudin-7, and the decrease of Claudin-1 in SK-1 cells and increase of Claudin-1 in A549 cells; knockdown of Claudin-5 significantly reduced invasion, whereas knockdown of Claudin-7 increased invasion; knockdown of Claudin-1 reduced invasion in SK-1 cells, whereas it increased invasion in A549 cells, indicating that SFN-Cys regulated Claudins and inhibited invasion depending on Claudin isotypes and cell types. Furthermore, immunofluorescence staining showed that SFN-Cys triggered microtubule disruption and knockdown of α-tubulin downregulated Claudin-1, 5, and 7, and inhibited migration and invasion, indicating that microtubule disruption contributed to invasive inhibition. Co-immunoprecipitation and confocal microscopy observation showed that SFN-Cys lowered the interaction between α-tubulin and Claudin-1 or 5, or 7. Meanwhile, Western blotting and immunofluorescence staining showed that SFN-NAC (15 µM) downregulated α-tubulin resulting in microtubule disruption; knockdown of α-tubulin increased SFN-NAC-induced LC3 II accumulation in SK-1 cells. Combined with the inhibitor of autolysosome formation, Bafilomycin A1 (100 nM), SFN-NAC inhibited invasion via accumulating LC3 II and blocking formation of autolysosome. Further, SFN-NAC upregulated microtubule-stabilizing protein Tau; knockdown of Tau reduced LC3 II/LC3 I inhibiting migration and invasion. These results indicated that SFN-Cys inhibited invasion via microtubule-mediated Claudins dysfunction, but SFN-NAC inhibited invasion via microtubule-mediated inhibition of autolysosome formation in human NSCLC cells.

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Sulforaphane-cysteine-induced apoptosis via phosphorylated ERK1/2-mediated maspin pathway in human non-small cell lung cancer cells.

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