Sulforaphane N-Acetyl-Cysteine Inhibited Autophagy Leading To Apoptosis Via Microtubule Disruption In Non-small Cell Lung Cancer Cells

Objective :Sulforaphane(SFN)inhibits cancer growth and invasion and induces apoptosis in variety of cancers.We previously unveiled some novel mechanisms regarding SFN-induced tumor suppression by disrupting microtubule.However,SFN has shorter half-life(approximately 2 hours)in circulation so that it has not been used clinically.SFN metabolite,sulforaphane-N-acetyl-cysteine(SFN-NAC)has longer half-life(approximately 7 hours),higher enrichment in lung and is a potential drug to inhibit human non-small cell lung cancer(NSCLC).The aim of this study is to explore the underlying anti-cancer mechanisms of SFN-NAC.Methods: The morphological images of the NSCLC cells were observed by phase contrast microscope;cell proliferation assay(MTS)was performed in NSCLC cells;transmission electron microscopy was performed to observe apoptotic bodies,autophagosomes and autophagolysosomes;the expression of p ERK,ERK,Hsp70,α-tubulin,LC3,and Stathmin-1 Western blot was detected by Western blot;the interaction between Hsp70 and α-tubulin was detected by co-immunoprecipitation;confocal laser scanning microscopy was used to observe the LC3 punctas and the co-localization of Hsp70 and α-tubulin;cell apoptosis was detected by flow cytometry.Results: SFN-NAC induced morphological alterations in NSCLC cells.cell proliferation assay showed that cell viability was inhibited by SFN-NAC in a concentration-dependent manner.More,we observed that SFN-NAC triggered autophagy-mediated apoptosis with the features: autophagosomes,autophagolysosomes,sporadic vacuoles,nuclear fragmentation,nucleic condensation and apoptotic bodies under transmission electron microscope.Western blot showed that SFN-NAC induced the phosphorylation of ERK1/2 which contributed to upregulation of Hsp70,downregulation of microtubule associated proteins,α-tubulin and Stathmin-1 leading to microtubule disruption and apoptosis;LC3 II expression was not upregulated by SFN-NAC after inhibition of the fusion of autophagosomes with lysosomes by Bafilomycin A1.Tissue microarray analysis showed that both overexpression of α-tubulin and Hsp70 was correlated to NSCLC malignancy.Immunofluorescence staining showed that SFN-NAC increased the colocalization of Hsp70 and α-tubulin,LC3 punctas,disrupted and aggregated microtubules.Further,co-immunoprecipitation showed that Hsp70 interacted with α-tubulin,and knockdown of Hsp70 lowered SFN-NAC-induced autophagy and increased apoptosis.The autophagy inhibitor 3-MA also enhanced SFN-NAC-induced apoptosis.Conclusions: These results demonstrated that SFN-NAC inhibited autophagy leading to apoptosis via Hsp70-mediated microtubule disruption.These findings will help us understand the working mechanisms of SFN-NAC and establish a novel anti-cancer therapy via the low-toxicity and high-efficiency therapeutic.Purpose:Plant-derived sulforaphane suppresses cancer,but has short half-life;its metabolite sulforaphane-N-acetyl-L-cysteine(SFN-NAC)has longer retention time in circulation.We have demonstrated that SFN-NAC inhibited cell growth and induced apoptosis in human prostate cancer.The underlying mechanism is that SFN-NAC induces microtubule disruption.The aim of this study is to investigate whether SFN-NAC inhibits non-small cell lung cancer(NSCLC)cell growth and invasion via microtubule disruption.Methods: Cell proliferation assay(MTS)was performed in NSCLC SK-1 cells;laser scanning confocal microscopy was used to observe microtubule morphology and LC3 punctate;transmission electron microscopy was performed to observe autophagosomes and autophagolysosomes;cell migration was tested by scratch wound healing assay;cell invasion was determined by Transwell invasion assay;the expression of LC3,Tau and α-tubulin was detected by Western blot.Results: We unraveled that SFN-NAC inhibited cell growth at the concentration of 20μM,and inhibited migration and invasion starting from 15μM.Also,SFN-NAC induced autophagy generating autophagosomes and autophagolysosomes.Immunofluorescence staining showed that SFN-NAC increased LC3 punctas.Western blot demonstrated that SFN-NAC upregulated LC3 II/LC3 I.More,SFN-NAC upregulated microtubule stabilizing protein Tau and knockdown of Tau reduced SFN-NAC-upregulated LC3 II/LC3 I leading to migratory and invasive inhibition.Conclusions: The downregulation of microtubule protein α-tubulin contributed to inhibition of migration and invasion.Knockdown of Tau decreased SFN-NAC-upregulated LC3 II/LC3 I inhibiting cell migration and invasion in NSCLC cells.