Locating Method for Electrical Tree Degradation in XLPE Cable Insulation Based on Broadband Impedance Spectrum.

Electrical treeing is one of the main causes of crosslinked polyethylene (XLPE) cable failure. The current methods for locating electrical trees are mainly based on the partial discharge (PD) signal. However, PD signals are easily attenuated in the long cable and the PD test voltage may cause damage to the insulation. This work proposes an improved broadband impedance spectrum (BIS) method to locate electrical trees in XLPE cable. A mathematical model of a long cable containing local electrical tree degradation is established. The Gaussian signal is chosen as the simulated incident signal to reduce the spectral leakage. The location spectrum is obtained by multiplying the frequency domain function of the single-ended reflection coefficient and the Gaussian pulse. It has been found that the location spectrum of the local capacitance change can be characterized as a typical double-peak waveform and the spectrum of the local conductance change can be regarded as a typical single-peak waveform. Electrical tree experiments at different temperatures were carried out to initiate different types of electrical trees. A vector network analyzer (VNA) was used to test the high frequency capacitance characteristics in the treeing process. The location spectra of the 20 m long cable containing different types of electrical trees was calculated by the improved location algorithm. The results show that the location error of local electrical tree degradation is less than 3%. The capacitance of the sliced sample decreases with treeing time. The effect of the bush-pine tree on capacitance parameters is greater than that of the branch-pine tree. A typical double-peak is found in the bush-pine tree location spectrum and a single-peak is found in the branch-pine tree spectrum.

Sulforaphane downregulated fatty acid synthase and inhibited microtubule-mediated mitophagy leading to apoptosis.

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.

Sulforaphane-cysteine downregulates CDK4 /CDK6 and inhibits tubulin polymerization contributing to cell cycle arrest and apoptosis in human glioblastoma cells.

Here we demonstrated that sulforaphane-cysteine (SFN-Cys) regulated cell cycle-related protein expressions in G0/G1 and G2/M phases of U87MG cells via High Performance Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (HPLC-MS/MS) and proteomics analysis. Further, mRNA products of CDK4, CDK6 and α-tubulin were significantly higher in glioblastoma than those in normal tissues, and these results were significantly correlated to pathological grades and clinical prognosis via analyzing TCGA and CGGA databases. Furthermore, Western blot showed that SFN-Cys downregulated CDK4, CDK6 and p-Rb in a dose-dependent manner and these results were reversed by p-ERK1/2 blocker PD98059 in U87MG and U373MG cells. The reductions of CDK4, CDK6 and p-Rb were reversed by proteasome inhibitor MG132; similarly, the upregulation of 26S proteasome by SFN-Cys was reversed by PD98059. Interestingly, SFN-Cys decreased CDK4 and CDK6 by phosphorylated ERK1/2-caused proteasomal degradation resulting in decreased Rb phosphorylation contributing to cell cycle arrest in G0/G1 phase. Besides, Western blot showed that SFN-Cys downregulated α-tubulin resulting in microtubule disruption and aggregation, and cell cycle arrest in G2/M phase and apoptosis. These results might help us understand the molecular etiology of glioblastoma progression to establish brand-new anti-cancer therapies.

Sulforaphane-cysteine inhibited migration and invasion via enhancing mitophagosome fusion to lysosome in human glioblastoma cells.

Here we uncovered the involved subcellular mechanisms that sulforaphane-cysteine (SFN-Cys) inhibited invasion in human glioblastoma (GBM). SFN-Cys significantly upregulated 45 and downregulated 14 microtubule-, mitophagy-, and invasion-associated proteins in GBM cells via HPLC-MS/MS and GEO ontology analysis; SFN-Cys disrupted microtubule by ERK1/2 phosphorylation-mediated downregulation of α-tubulin and Stathmin-1 leading to the inhibition of cell migration and invasion; SFN-Cys downregulated invasion-associated Claudin-5 and S100A4, and decreased the interaction of α-tubulin to Claudin-5. Knockdown of Claudin-5 and S100A4 significantly reduced the migration and invasion. Besides, SFN-Cys lowered the expressions of α-tubulin-mediated mitophagy-associated proteins Bnip3 and Nix. Transmission electron microscopy showed more membrane-deficient mitochondria and accumulated mitophagosomes in GBM cells, and mitochondria fusion might be downregulated because that SFN-Cys downregulated mitochondrial fusion protein OPA1. SFN-Cys increased the colocalization and interplay of LC3 to lysosomal membrane-associated protein LAMP1, aggravating the fusion of mitophagosome to lysosome. Nevertheless, SFN-Cys inhibited the lysosomal proteolytic capacity causing LC3II/LC3I elevation but autophagy substrate SQSTM1/p62 was not changed, mitophagosome accumulation, and the inhibition of migration and invasion in GBM cells. These results will help us develop high-efficiency and low-toxicity anticancer drugs to inhibit migration and invasion in GBM.

Sulforaphane metabolites inhibit migration and invasion via microtubule-mediated Claudins dysfunction or inhibition of autolysosome formation in human non-small cell lung cancer cells.

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.

Sulforaphane metabolites reduce resistance to paclitaxel via microtubule disruption.

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 ßIII-tubulin, XIAP, Tau, Stathmin1 and α-tubulin causing microtubule disruption in human PTX-resistant non-small cell lung cancer (NSCLC) cells. Knockdown of either ßIII-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 ßIII-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 ßIII-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-N-Acetyl-Cysteine inhibited autophagy leading to apoptosis via Hsp70-mediated microtubule disruption.

Sulforaphane-N-acetyl-cysteine (SFN-NAC) is a potential drug to inhibit human non-small cell lung cancer (NSCLC), but the underlying mechanisms are elusive. Here, we uncovered that SFN-NAC induced apoptosis via flow cytometer assay and transmission electron microscopy. Further, SFN-NAC increased LC3 II/LC3 I and the number of LC3 punctas, but Western blot showed that SFN-NAC inhibited cell autophagy in response to a co-treatment of Bafilomycin A1 and SFN-NAC. Furthermore, immunofluorescence staining and Western blot showed that SFN-NAC triggered microtubule disruption causing apoptosis via downregulating α-tubulin and phosphorylated ERK1/2-mediated Stathmin-1. Besides, SFN-NAC upregulated Hsp70 via phosphorylating ERK1/2. Confocal microscopy and immunoprecipitation assay showed that SFN-NAC promoted the colocalization and interaction of Hsp70 and α-tubulin; knockdown of Hsp70 enhanced SFN-NAC-induced microtubule disruption, lowered LC3 II/LC3 I and promoted apoptosis. Interestingly, tissue microarray analysis showed that the increased expression of either α-tubulin or Hsp70 correlated to NSCLC malignant grading, indicating that microtubule and Hsp70 are two key targets for SFN-NAC. These results will give us a new insight into SFN-NAC-induced apoptosis so that we develop more efficient therapeutics to treat NSCLC.

Sulforaphane Induced Apoptosis via Promotion of Mitochondrial Fusion and ERK1/2-Mediated 26S Proteasome Degradation of Novel Pro-survival Bim and Upregulation of Bax in Human Non-Small Cell Lung Cancer Cells.

Previous studies in our laboratory showed that sulforaphane (SFN) induced apoptosis by sustained activation of extracellular regulated protein kinases 1/2 (ERK1/2). However, the underlying mechanisms associated with SFN-induced apoptosis and downstream cascades which are modulated by ERK1/2 were not elucidated. Herein we demonstrated for the first time that alteration of mitochondrial dynamics contributed to SFN-induced apoptosis in human non-small cell lung cancer (NSCLC) cells. Reports showed that protein Bim not only induced apoptosis but also promoted proliferation under certain circumstances. We found that Bim was related to cell growth in NSCLC cells. Pro-survival Bim downregulation was shown to induce apoptosis in response to SFN. Further, Using the ERK1/2 inhibitor, PD98059, we found that SFN upregulated Bax and downregulated Bim through the ERK1/2-dependent signaling pathway. Furthermore, SFN activated ERK1/2 to increase 26S proteasome activity to degrade Bim, while the proteasome inhibitor MG132 reversed this effect. Therefore, SFN phosphorylated ERK1/2 and activated the proteasome system leading to the degradation of Bim, which contributed to apoptosis in NSCLC cells. These findings provided a novel insight into SFN-related therapeutics in cancer treatment.

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

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.