Cancer stem cells (CSCs), characterized by self-renewal and unlimited proliferation, lead to therapeutic resistance in lung cancer. In this study, we aimed to investigate the expressions of stem cell-related genes in lung adenocarcinoma (LUAD). The stemness index based on mRNA expression (mRNAsi) was utilized to analyze LUAD cases in the Cancer Genome Atlas (TCGA). First, mRNAsi was analyzed with differential expressions, survival analysis, clinical stages, and gender in LUADs. Then, the weighted gene co-expression network analysis was performed to discover modules of stemness and key genes. The interplay among the key genes was explored at the transcription and protein levels. The enrichment analysis was performed to annotate the function and pathways of the key genes. The expression levels of key genes were validated in a pan-cancer scale. The pathological stage associated gene expression level and survival probability were also validated. The Gene Expression Omnibus (GEO) database was additionally used for validation. The mRNAsi was significantly upregulated in cancer cases. In general, the mRNAsi score increases according to clinical stages and differs in gender significantly. Lower mRNAsi groups had a better overall survival in major LUADs, within five years. The distinguished modules and key genes were selected according to the correlations to the mRNAsi. Thirteen key genes (CCNB1, BUB1, BUB1B, CDC20, PLK1, TTK, CDC45, ESPL1, CCNA2, MCM6, ORC1, MCM2, and CHEK1) were enriched from the cell cycle Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, relating to cell proliferation Gene Ontology (GO) terms, as well. Eight of the thirteen genes have been reported to be associated with the CSC characteristics. However, all of them have been previously ignored in LUADs. Their expression increased according to the pathological stages of LUAD, and these genes were clearly upregulated in pan-cancers. In the GEO database, only the tumor necrosis factor receptor associated factor-interacting protein (TRAIP) from the blue module was matched with the stemness microarray data. These key genes were found to have strong correlations as a whole, and could be used as therapeutic targets in the treatment of LUAD, by inhibiting the stemness features.

We previously demonstrated that sulforaphane (SFN) inhibited autophagy leading to apoptosis in human non-small cell lung cancer (NSCLC) cells, but the underlying subcellular mechanisms were unknown. Hereby, high-performance liquid chromatography-tandem mass spectrometry uncovered that SFN regulated the production of lipoproteins, and microtubule- and autophagy-associated proteins. Further, highly expressed fatty acid synthase (FASN) contributed to cancer malignancy and poor prognosis. Results showed that SFN depolymerized microtubules, downregulated FASN, and decreased its binding to α-tubulin; SFN downregulated FASN, acetyl CoA carboxylase (ACACA), and ATP citrate lyase (ACLY) via activating proteasomes and downregulating transcriptional factor SREBP1; SFN inhibited the interactions among α-tubulin and FASN, ACACA, and ACLY; SFN decreased the amount of intracellular fatty acid (FA) and mitochondrial phospholipids; and knockdown of FASN decreased mitochondrial membrane potential (ΔΨm) and increased reactive oxygen species, mitochondrial abnormality, and apoptosis. Further, SFN downregulated mitophagy-associated proteins Bnip3 and NIX, and upregulated mitochondrial LC3 II/I. Transmission electron microscopy showed mitochondrial abnormality and accumulation of mitophagosomes in response to SFN. Combined with mitophagy inducer CCCP or autophagosome–lysosome fusion inhibitor Bafilomycin A1, we found that SFN inhibited mitophagosome–lysosome fusion leading to mitophagosome accumulation. SFN reduced the interaction between NIX and LC3 II/I, and reversed CCCP-caused FA increase. Furthermore, knockdown of α-tubulin downregulated NIX and BNIP3 production, and upregulated LC3 II/I. Besides, SFN reduced the interaction and colocalization between α-tubulin and NIX. Thus, SFN might cause apoptosis via inhibiting microtubule-mediated mitophagy. These results might give us a new insight into the mechanisms of SFN-caused apoptosis in the subcellular level.

Long treatment with paclitaxel (PTX) might increase resistance and side-effects causing a failure in cancer chemotherapy. Here we uncovered that either sulforaphane-cysteine (SFN-Cys) or sulforaphane-N-acetyl-cysteine (SFN-NAC) induced apoptosis via phosphorylated ERK1/2-mediated upregulation of 26 S proteasome and Hsp70, and downregulation of βIII-tubulin, XIAP, Tau, Stathmin1 and α-tubulin causing microtubule disruption in human PTX-resistant non-small cell lung cancer (NSCLC) cells. Knockdown of either βIII-tubulin or α-tubulin via siRNA increased cell sensitivity to PTX, indicating that these two proteins help cells increase the resistance. Tissue microarray analysis showed that overexpression of βIII-tubulin correlated to NSCLC malignant grading. Immunofluorescence staining also showed that SFN metabolites induced a nest-like microtubule protein distribution with aggregation and disruption. Co-immunoprecipitation showed that SFN metabolites reduced the interaction between βIII-tubulin and Tau, and that between α-tubulin and XIAP. The combination of PTX with SFN metabolites decreased the resistance to PTX, and doses of both PTX and SFN metabolites, and enhanced apoptosis resulting from activated Caspase-3-caused microtubule degradation. Importantly, the effective dose of SFN metabolites combined with 20 nM PTX will be low to 4 μM. Thus, we might combine SFN metabolites with PTX for preclinical trial. Normally, more than 20 μM SFN metabolites only leading to apoptosis for SFN metabolites hindered their applications. These findings will help us develop a low-resistance and high-efficiency chemotherapy via PTX/SFN metabolites combination.

Sulforaphane (SFN) inhibited growth in many cancers, but its half-life is 2 h in circulation. However, its metabolites, sulforaphane-cysteine (SFN-Cys) and sulforaphane-N-acetyl-cysteine (SFN-NAC) had longer half-lives and decreased the cell viability in both dose- and time-dependent manners in human prostate cancer. Flow cytometry assay revealed that these two SFN metabolites induced apoptosis with the features such as vacuolization, disappeared nuclear envelope, nuclear agglutination and fragmentation via transmission electron microscopy observation. Western blot showed that the sustained phosphorylation of ERK1/2 mediated by SFN metabolites caused activation and upregulation of cleaved Caspase 3 and downregulation of α-tubulin. High expression of α-tubulin was demonstrated to be positively correlated with cancer pathological grading. Both co-immunoprecipitation and immunofluorescence staining implicated the interaction between SFN metabolite-induced phosphorylated ERK1/2 and α-tubulin, and Caspase 3 cleavage assay showed that α-tubulin might be the substrate for cleaved Caspase 3. More, the SFN metabolite-mediated reduction of α-tubulin increased the depolymerization and instability of microtubules by microtubule polymerization assay. Reversely, microtubule-associated protein Stathmin-1 phosphorylation was increased via phosphorylated ERK1/2 and total Stathmin-1 was reduced, which might promote over-stability of microtubules. Immunofluorescence staining also showed that SFN metabolites induced the ‘nest-like’ structures of microtubule distribution resulting from the disrupted and aggregated microtubules, and abnormal nuclear division, suggesting that the disturbance of spindle formation and mitosis turned up. Thus, SFN-Cys and SFN-NAC triggered the dynamic imbalance of microtubules, microtubule disruption leading to cell apoptosis. These findings provided a novel insight into the chemotherapy of human prostate cancer.

Advanced prostate cancer has highly invasive potential, which may lead to metastasis associated with poor prognosis. Sulforaphane (SFN), abundant in cruciferous vegetables, exhibited effective resistance to carcinogenesis in a variety of tumors. The aim of the present study was to investigate whether SFN inhibited invasion in human prostate cancer cells via sustained activation of ERK1/2 and downstream signaling by an invasion assay, gelatin zymography and western blot analysis. The results showed that SFN inhibited invasion and we characterized the underlying mechanisms in human DU145 prostate cancer cells. SFN (15 µM) changed cell morphology leading to short‑cell pseudopodia which may suppress tumor migration and invasion. The Transwell assay showed that SFN phosphorylated ERK1/2 in a dose- and time-dependent manner and significantly inhibited cell invasion, while the effect was reduced by the ERK1/2 blocker PD98059 (25 µM). Furthermore, these effects contributed to the upregulation of E-cadherin and the downregulation of CD44v6 and were eradicated by PD98059. Western blot analysis and gelatin zymography showed that SFN decreased the expression and activity of MMP-2. Thus, SFN inhibited invasion by activating ERK1/2 to upregulate E-cadherin and downregulate CD44v6, thereby reducing MMP-2 expression and activity. E-cadherin is an invasion inhibitor, while CD44v6 and MMP-2 are invasion promoters. Therefore, SFN is a prospective therapeutic agent that may be used to prevent invasion in prostate cancer.

Glioblastoma has highly invasive potential, which might result in poor prognosis and therapeutic failure. Hence, the key we study is to find effective therapies to repress migration and invasion. Sulforaphane (SFN) was demonstrated to inhibit cell growth in a variety of tumors. Here, we will further investigate whether SFN inhibits migration and invasion and find the possible mechanisms in human glioblastoma U87MG and U373MG cells. First, the optimal time and dose of SFN for migration and invasion study were determined via cell viability and cell morphological assay. Further, scratch assay and transwell invasion assay were employed to investigate the effect of SFN on migration and invasion. Meanwhile, Western blots were used to detect the molecular linkage among invasion related proteins phosphorylated ERK1/2, matrix metalloproteinase-2 (MMP-2) and CD44v6. Furthermore, Gelatin zymography was performed to detect the inhibition of MMP-2 activation. In addition, ERK1/2 blocker PD98059 (25 µM) was integrated to find the link between activated ERK1/2 and invasion, MMP-2 and CD44v6. The results showed that SFN (20 µM) remarkably reduced the formation of cell pseudopodia, indicating that SFN might inhibit cell motility. As expected, scratch assay and transwell invasion assay showed that SFN inhibited glioblastoma cell migration and invasion. Western blot and Gelatin zymography showed that SFN phosphorylated ERK1/2 in a sustained way, which contributed to the downregulated MMP-2 expression and activity, and the upregulated CD44v6 expression. These molecular interactions resulted in the inhibition of cell invasion. SFN inhibited migration and invasion processes. Furthermore, SFN inhibited invasion via activating ERK1/2 in a sustained way. The accumulated ERK1/2 activation downregulated MMP-2 expression and decreased its activity and upregulated CD44v6. SFN might be a potential therapeutic agent by activating ERK1/2 signaling against human glioblastoma.