Abstract
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.
Highlights
Vegetable-derived sulforaphane (SFN) inhibits carcinogenesis and induces apoptosis in a variety of cancer cells[1,2,3,4]
We found that SFN-Cys triggered ERK1/2 phosphorylation leading to microtubule-mediated Claudins dysfunction and the inhibition of migration and invasion, and SFN-NAC inhibited migration and invasion by microtubule-mediated inhibition of autolysosome formation
SFN-Cys and SFN-NAC triggered the different signal pathways, which did not mean that these two SFN metabolites worked differently, because we did not test both pathways for each SFN metabolite; several studies in our laboratory showed that two SFN metabolites initiated the same pathways to inhibit cancer growth and induce apoptosis[46]
Summary
Vegetable-derived sulforaphane (SFN) inhibits carcinogenesis and induces apoptosis in a variety of cancer cells[1,2,3,4]. Invasion-associated proteins, Claudins (1, 5, and 7), were demonstrated to correlate to cancer migration and invasion[9,10,11]. We demonstrated that SFN-NAC (30 μM) induced apoptosis via microtubule disruption-mediated inhibition of autolysosome formation in non-small cell lung cancer (NSCLC) cells[12]. As cell proliferation and death affect cell motility, either SFN-Cys or SFN-NAC might inhibit migration and invasion via regulating either Claudins or microtubule-mediated autophagy. Microtubule proteins α-tubulin and β-tubulin, microtubule-stabilizing proteins Tau, MAP1, MAP2, MAP4, and LC3, and microtubule-destabilizing protein
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