Epigenetic activation of METTL14 promotes docetaxel resistance in prostate cancer by promoting pri-microRNA-129 maturation.

The development of resistance to Docetaxel (DTX) compromises its therapeutic efficacy and worsens the prognosis of prostate cancer (PCa), while the underlying regulatory mechanism remains poorly understood. In this study, METTL14 was found to be upregulated in DTX-resistant PCa cells and PCa tissues exhibiting progressive disease during DTX therapy. Furthermore, overexpression of METTL14 promoted the development of resistance to DTX in both in vitro and in vivo. Interestingly, it was observed that the hypermethylation of the E2F1 targeting site within DTX-resistant PCa cells hindered the binding ability of E2F1 to the promoter region of METTL14, thereby augmenting its transcriptional activity. Consequently, this elevated expression level of METTL14 facilitated m6A-dependent processing of pri-miR-129 and subsequently led to an increase in miR-129-5p expression. Our study highlights the crucial role of the E2F1-METTL14-miR-129-5p axis in modulating DTX resistance in PCa, underscoring METTL14 as a promising therapeutic target for DTX-resistant PCa patients.

Corrigendum: The Bcl-2-associated athanogene gene family in tobacco (Nicotiana tabacum) and the function of NtBAG5 in leaf senescence.

[This corrects the article DOI: 10.3389/fpls.2023.1108588.].

Silencing LINC00491 inhibits prostate cancer development through the miR-384/TRIM44 axis.

Accumulating evidence has demonstrated the key role of long noncoding (lnc)RNAs in tumorigenesis. Prostate cancer (PCa) is a cancer with high mortality that requires further exploration of the underlying molecular mechanisms. In the present study, we aimed to discover novel potential biomarkers for diagnosing PCa and targeting treatment. Overexpression of the lncRNA, LINC00491, was verified in PCa tumor tissues and cell lines using the real-time polymerase chain reaction. Cell proliferation and invasion were then analyzed via the Cell Counting Kit-8, colony formation, and transwell assays in vitro, and tumor growth in vivo. The interaction of miR-384 with LINC00491, as well as TRIM44, was investigated via bioinformatics analyses, subcellular fractionation, luciferase reporter gene assays, radioimmunoprecipitation, pull-down, and western blot analyses. LINC00491 was overexpressed in PCa tissues and cell lines. LINC00491 knockdown resulted in impaired cell proliferation and invasion in vitro and decreased tumor growth in vivo. Moreover, LINC00491 acted as a sponge for miR-384 and its downstream target, TRIM44. Additionally, miR-384 expression was downregulated in PCa tissues and cell lines, and its expression was negatively correlated with LINC00491. A miR-384 inhibitor restored the inhibitory effects of LINC00491 silencing on PCa cell proliferation and invasion. LINC00491 is a tumor promoter in PCa via enhancing TRIM44 expression by sponging miR-384 to facilitate the development of PCa. LINC00491 plays a significant role in PCa and could serve as both a biomarker for early diagnosis and a novel treatment target.

The Bcl-2-associated athanogene gene family in tobacco (Nicotiana tabacum) and the function of NtBAG5 in leaf senescence.

Leaf senescence in tobacco is closely related to leaf maturation and secondary metabolites. Bcl-2-associated athanogene (BAG) family members are highly conserved proteins and play key roles in senescence, growth and development, and resistance to biotic and abiotic stresses. Herein, the BAG family of tobacco was identified and characterized. In total, 19 tobacco BAG protein candidate genes were identified and divided into two classes, class I comprising NtBAG1a-e, NtBAG3a-b, and NtBAG4a-c and class II including NtBAG5a-e, NtBAG6a-b, and NtBAG7. Genes in the same subfamily or branch of the phylogenetic tree exhibited similarities in gene structure and the cis-element on promoters. RNA-seq and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that the expression of NtBAG5c-f and NtBAG6a-b was upregulated in senescent leaves, implying that they play a role in regulating leaf senescence. NtBAG5c was localized in the nucleus and cell wall as a homology of leaf senescence related gene AtBAG5. Further, the interaction of NtBAG5c with heat-shock protein 70 (HSP70) and sHSP20 was demonstrated using yeast two-hybrid experiment. Virus-induced gene silencing indicated that NtBAG5c reduced the lignin content and increased superoxide dismutase (SOD) activity and hydrogen peroxide (H2O2) accumulation. In NtBAG5c-silenced plants, the expression of multiple senescence-related genes cysteine proteinase (NtCP1), SENESCENCE 4 (SEN4) and SENESCENCE-ASSOCIATED GENE 12 (SAG12) was downregulated. In conclusion, tobacco BAG protein candidate genes were identified and characterized for the first time.

The alteration of the expression level of neuropathy target esterase in human neuroblastoma SK-N-SH cells disrupts cellular phospholipids homeostasis.

Neuropathy target esterase (NTE) has been proven to act as a lysophospholipase (LysoPLA) and phospholipase B (PLB) in mammalian cells. In this study, we took human neuroblastoma SK-N-SH cells as the research object and explored the effect of NTE on phospholipid homeostasis. The results showed that phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) levels significantly increased (> 40%), while glycerophosphocholine (GPC) decreased (below 60%) after NTE gene was knockdown in the cells (NTE < 30% of control), which were prepared by gene silencing with dsRNA-NTE. However, in the NTE-overexpressed cells (NTE > 50% of control), which were prepared by expressing recombinant catalytic domain of NTE, LPC remarkably decreased (below 80%) and GPC enhanced (> 40%). Mipafox, a neuropathic organophosphorus compound (OP), significantly inhibited NTE-LysoPLA and NTE-PLB activities (> 95-99% inhibition at 50 µM), which was accompanied with a decreased GPC level (below 40%) although no change of the PC and LPC levels was observed; while paraoxon, a non-neuropathic OP, suppresses neither the activities of NTE-phospholipases nor the levels of PC, LPC, and GPC. Thus, we concluded that both the stable up- or down-regulated expression of NTE gene and the loss of NTE-LysoPLA/PLB activities disrupts phospholipid homeostasis in the cells although the inhibition of NTE activity only decreased GPC content without altering PC and LPC levels.

The Patatin-Like Phospholipase Domain Containing Protein 7 Regulates Macrophage Classical Activation through SIRT1/NF-κB and p38 MAPK Pathways.

Lysophosphatidylcholine (LPC) is a bioactive lipid that modulates macrophage polarization during immune responses, inflammation, and tissue remodeling. Patatin-like phospholipase domain containing protein 7 (PNPLA7) is a lysophospholipase with a preference for LPC. However, the role of PNPLA7 in macrophage polarization as an LPC hydrolase has not been explored. In the current study, we found that PNPLA7 is highly expressed in naïve macrophages and downregulated upon lipopolysaccharide (LPS)-induced polarization towards the classically activated (M1) phenotype. Consistently, overexpression of PNPLA7 suppressed the expression of proinflammatory M1 marker genes, including interleukin 1ß (IL-1ß), IL-6, inducible nitric oxide synthase (iNOS), and tumor necrosis factor α (TNF-α), whereas knockdown of PNPLA7 augmented the inflammatory gene expression in LPS-challenged macrophages. PNPLA7 overexpression and knockdown increased and decreased Sirtuin1 (SIRT1) mRNA and protein levels, respectively, and affected the acetylation of the nuclear factor-kappa B (NF-κB) p65 subunit, a key transcription factor in M1 polarization. In addition, the levels of phosphorylated p38 mitogen-activated protein kinase (MAPK) were suppressed and enhanced by PNPLA7 overexpression and knockdown, respectively. Taken together, these findings suggest that PNPLA7 suppresses M1 polarization of LPS-challenged macrophages by modulating SIRT1/NF-κB- and p38 MAPK-dependent pathways.

EXTL3 could serve as a potential biomarker of prognosis and immunotherapy for prostate cancer and its potential mechanisms.

BACKGROUND: Exostosin like glycosyltransferase 3 (EXTL3) had been reported to be associated with immune deficiency and play prognostic roles in various cancers. However, little is known about the associations between EXTL3 and prostate cancer (PCa). Hence, this article was designed to clarify their associations. METHODS: All original data were downloaded from The Cancer Genome Atlas (TCGA) database. Gene set enrichment analysis (GSEA) and CellMiner database was utilized, respectively, to identify EXTL3-related signaling pathways and drugs. We explored the relationships between EXTL3 expression and immunity to further evaluate the involvement of EXTL3 in response to immunotherapies. LncRNA/RBP/EXTL3 mRNA networks were also identified for its potential mechanism. RESULTS: Compared with normal prostate samples, EXTL3 was poorly expressed in PCa samples not only in mRNA expression levels, but also in protein expression levels, with worse overall survival (P < 0.05) and this gene could be an independent prognostic biomarker for PCa (both P < 0.05). EXTL3 was revealed to be markedly linked with seven signaling pathways in PCa by GSEA, including calcium, chemokine, ERBB, JAK STAT, MAPK, WNT, oxidative phosphorylation pathways. EXTL3 expression was also revealed to be significantly associated with MSI, immune cells, immune checkpoint molecules, tumor microenvironment and immune cells infiltration. We further predicted immune responses of EXTL3 gene to immunotherapies by TIDE database and the IMvigor210 cohort. A total of six LncRNA/RBP/EXTL3 mRNA networks were eventually identified for its potential mechanisms. CONCLUSIONS: EXTL3 could serve as a potential biomarker of prognosis and immunotherapy for PCa and six LncRNA/RBP/EXTL3 mRNA networks were also identified for its potential mechanisms.

Genome-wide analysis and identification of the PEBP genes of Brassica juncea var. Tumida.

BACKGROUND: Phosphatidylethanolamine-binding protein (PEBP) is widely present in animals, plants, and microorganisms. Plant PEBP genes are mainly involved in flowering transition and nutritional growth. These genes have been studied in several plants; however, to the best of our knowledge, no studies have explored them in Brassica juncea var. tumida. This study identified and characterized the entire PEBP gene family of Brassica juncea var. tumida. RESULTS: A total of 21 PEBP genes were identified from Brassica juncea var. tumida. Through phylogenetic analysis, the 21 corresponding proteins were classified into the following four clusters: TERMINAL FLOWER 1 (TFL1)-like proteins (n = 8), MOTHER OF FT AND TFL1 (MFT)-like proteins (n = 5), FLOWERING LOCUS T (FT)-like proteins (n = 6), and ybhB-like proteins (n = 2). A total of 18 genes contained four exons and had similar gene structures in each subfamily except BjMFT1, BjPYBHB1, and Arabidopsis thaliana CENTRORADIALIS homolog of Brassica juncea var. tumida (BjATC1). In the analysis of conserved motif composition, the BjPEBP genes exhibited similar characteristics, except for BjFT3, BjMFT1, BjPYBHB1, BjPYBHB2, and BjATC1. The BjPEBP promoter includes multiple cis-acting elements such as the G-box and I-box elements that respond to light, ABRE and GARE-motif elements that respond to hormones, and MBSI and CAT-box elements that are associated with plant growth and development. Analysis of RNA-Seq data revealed that the expression of a few BjPEBP genes may be associated with the development of a tumorous stem. The results of qRT-PCR showed that BjTFL1 and BjPYBHB1 were highly expressed in the flower tissue, BjFT1 and BjATC1 were mainly expressed in the root, and BjMFT4 were highly detected in the stem. The results of yeast two-hybrid screening suggested that BjFT interacts with Bj14-3-3. These results indicate that BjFT is involved in flowering regulation. CONCLUSIONS: To the best of our knowledge, this study is the first to perform a genome-wide analysis of PEBP genes family in Brassica juncea var. tumida. The findings of this study may help improve the yield and molecular breeding of Brassica juncea var. tumida.

The Catalytic Domain of Neuropathy Target Esterase Influences Lipid Droplet Biogenesis and Lipid Metabolism in Human Neuroblastoma Cells.

As an endoplasmic reticulum (ER)-anchored phospholipase, neuropathy target esterase (NTE) catalyzes the deacylation of lysophosphatidylcholine (LPC) and phosphatidylcholine (PC). The catalytic domain of NTE (NEST) exhibits comparable activity to NTE and binds to lipid droplets (LD). In the current study, the nucleotide monophosphate (cNMP)-binding domains (CBDs) were firstly demonstrated not to be essential for the ER-targeting of NTE, but to be involved in the normal ER distribution and localization to LD. NEST was associated with LD surface and influenced LD formation in human neuroblastoma cells. Overexpression of NEST enhances triacylglycerol (TG) accumulation upon oleic acid loading. Quantitative targeted lipidomic analysis shows that overexpression of NEST does not alter diacylglycerol levels but reduces free fatty acids content. NEST not only lowered levels of LPC and acyl-LPC, but not PC or alkyl-PC, but also widely altered levels of other lipid metabolites. Qualitative PCR indicates that the increase in levels of TG is due to the expression of diacylglycerol acyltransferase 1 gene by NEST overexpression. Thus, NTE may broadly regulate lipid metabolism to play roles in LD biogenesis in cells.

Identification of the circRNA-miRNA-mRNA Regulatory Network in Bladder Cancer by Bioinformatics Analysis.

In recent years, increasing evidence shows that circular RNA (circRNA) disorder is closely related to tumorigenesis and cancer progression. However, the regulatory functions of most circRNAs in bladder cancer (BCa) remain unclear. This study was aimed at exploring the molecular regulatory mechanism of circRNAs in BCa. We obtained four datasets of circRNA, microRNA (miRNA), and messenger (mRNA) expression profiles from the Gene Expression Omnibus and The Cancer Genome Atlas microarray databases and identified 434, 367, and 4799/4841 differentially expressed circRNAs, miRNAs, and mRNAs, respectively. With these differentially expressed RNAs, we established a circRNA-miRNA-mRNA targeted interaction network. A total of 18, 24, and 51 central circRNAs, miRNAs, and mRNAs were identified, respectively. Among them, the top 10 mRNAs that had high connectivity with other circRNAs and miRNAs were regarded as hub genes. We detected the expression levels of these 10 mRNAs in 16 pairs of BCa tissues and adjacent normal tissues through quantitative real-time polymerase chain reaction. The differentially expressed mRNAs and central mRNAs were enriched in the processes and pathways that are associated with the growth, differentiation, proliferation, and apoptosis of tumor cells. The outstanding genes (CDCA4, GATA6, LATS2, RHOB, ZBTB4, and ZFPM2) also interacted with numerous drugs, indicating their potency as biomarkers and drug targets. The findings of this study provide a deep understanding of the circRNA-related competitive endogenous RNA regulatory mechanism in BCa pathogenesis.

Transcriptome profiling reveals new insights into the roles of neuronal nitric oxide synthase on macrophage polarization towards classically activated phenotype.

In response to various stimuli, naïve macrophages usually polarize to M1 (classically activated) or M2 (alternatively activated) cells with distinct biological functions. Neuronal nitric oxide synthase (NOS1) is involved in M1 macrophage polarization at an early stage. Here, we show for the first time that NOS1 is dispensable for M2 macrophage polarization for the first time. Further, differentially expressed genes (DEGs) regulated by NOS1 signaling in M1-polarized macrophages stimulated with lipopolysaccharide (LPS) were characterized by transcriptome analysis of wild-type (WT) and NOS1 knockout mouse macrophages. Thousands of affected genes were detected 2 h post LPS challenge, and this wide-ranging effect became greater with a longer stimulation time (8 h post LPS). NOS1 deficiency caused dysregulated expression of hundreds of LPS-responsive genes. Most DEGs were enriched in biological processes related to transcription and regulation of the immune and inflammatory response. At 2 h post-LPS, the toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction, and NOD-like receptor signaling pathway were the major pathways affected, whereas the main pathways affected at 8 h post-LPS were Th1 and Th2 cell differentiation, FoxO, and AMPK signaling pathway. Identified DEGs were validated by real-time quantitative PCR and interacted in a complicated signaling pathway network. Collectively, our data show that NOS1 is dispensable for M2 macrophage polarization and reveal novel insights in the role of NOS1 signaling at different stages of M1 macrophage polarization through distinct TLR4 plasma membrane-localized and endosome-internalized signaling pathways.

Genome-wide identification and characterization of NAC genes in Brassica juncea var. tumida.

BACKGROUND: NAC (NAM, ATAF1/2, and CUC2) transcription factors play an important role in plant growth and development. However, in tumorous stem mustard (Brassica juncea var. tumida), one of the economically important crops cultivated in southwest China and some southeast Asian countries, reports on the identification of NAC family genes are lacking. In this study, we conducted a genome-wide investigation of the NAC family genes in B. juncea var. tumida, based on its recently published genome sequence data. METHODS: The NAC genes were identified in B. juncea var. tumida using the bioinformatics approach on the whole genome level. Additionally, the expression of BjuNAC genes was analyzed under high- and low-temperature stresses by quantitative real-time PCR (qRT-PCR). RESULTS: A total of 300 BjuNAC genes were identified, of which 278 were mapped to specific chromosomes. Phylogenetic analysis of B. juncea var. tumida, Brassica rapa, Brassica nigra, rice and Arabidopsis thaliana NAC proteins revealed that all NAC genes were divided into 18 subgroups. Furthermore, gene structure analysis showed that most of the NAC genes contained two or three exons. Conserved motif analysis revealed that BjuNAC genes contain a conserved NAM domain. Additionally, qRT-PCR data indicated that thirteen BjuNAC genes with a varying degree of up-regulation during high-temperature stress. Conversely, four BjuNAC genes (BjuNAC006, BjuNAC083, BjuNAC170 and BjuNAC223) were up-regulated and two BjuNAC genes (BjuNAC074 and BjuNAC295) down-regulated under low temperature, respectively. Together, the results of this study provide a strong foundation for future investigation of the biological function of NAC genes in B. juncea var. tumida.

The anti-cancer properties of heparin and its derivatives: a review and prospect.

Heparin, including unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) and heparin derivatives, are commonly used in venous thromboembolism treatment and reportedly have beneficial effects on cancer survival. Heparin can affect the proliferation, adhesion, angiogenesis, migration and invasion of cancer cells via multiple mechanisms. The main mechanisms involve inhibition of heparanase, P-/L-selectin, angiogenesis, and interference with the CXCL12-CXCR4 axis. Here we summarize the current experimental evidence regarding the anti-cancer role of heparin and its derivatives, and conclude that there is evidence to support heparin's role in inhibiting cancer progression, making it a promising anti-cancer agent.

Genome-wide identification and characterization of TCP family genes in Brassica juncea var. tumida.

BACKGROUND: Teosinte branched1/Cycloidea/proliferating cell factors (TCPs) are plant-specific transcription factors widely involved in leaf development, flowering, shoot branching, the circadian rhythm, hormone signaling, and stress responses. However, the TCP function in Brassica juncea var. tumida, the tumorous stem mustard, has not yet been reported. This study identified and characterized the entire TCP family members in B. juncea var. tumida. METHODS: We identified 62 BjTCP genes from the B. juncea var. tumida genome and analyzed their phylogenetic relationship, gene structure, protein motifs, chromosome location, and expression profile in different tissues. RESULTS: Of the 62 BjTCP genes we identified in B. juncea var. tumida, containing 34 class I and 28 class II subfamily members, 61 were distributed on 18 chromosomes. Gene structure and conserved motif analysis showed that the same clade genes displayed a similar exon/intron gene structure and conserved motifs. Cis-acting element results showed that the same clade genes also had a similar cis-acting element; however, subtle differences implied a different regulatory pathway. The BjTCP18s members were low-expressed in Dayejie strains and the unswelling stage of Yonganxiaoye strains. Treatment with gibberellin (GA) and salicylic acid (SA) showed that GA and SA affect the expression levels of multiple TCP genes. CONCLUSION: We performed the first genome-wide analysis of the TCP gene family of B. juncea var. tumida. Our results have provided valuable information for understanding the classification and functions of TCP genes in B. juncea var. tumida.

MiR-129-5p promotes docetaxel resistance in prostate cancer by down-regulating CAMK2N1 expression.

This study focuses on the effect of miR-129-5p on docetaxel-resistant (DR) prostate cancer (PCa) cells invasion, migration and apoptosis. In our study, the expression of CAMK2N1 was assessed by qRT-PCR in PCa patient tissues and cell lines including PC-3 and PC-3-DR. Cells transfected with miR-129-5p mimics, inhibitor, CAMK2N1 or negative controls (NC) were used to interrogate their effects on DR cell invasions, migrations and apoptosis during docetaxel (DTX) treatments. The apoptosis rate of the PCa cells was validated by flow cytometry. Relationships between miR-129-5p and CAMK2N1 levels were identified by qRT-PCR and dual-luciferase reporter assay. CAMK2N1 was found to be down-expressed in DR PCa tissue sample, and low levels of CAMK2N1 were correlated with high docetaxel resistance and clinical prediction of poor survival. CAMK2N1 levels were decreased in DR PCa cells treated with DXT. We further explored that up-regulation of miR-129-5p could promote DR PCa cells viability, invasion and migration but demote apoptosis. Involved molecular mechanism studies revealed that miR-129-5p reduced downstream CAMK2N1 expression to further impact on chemoresistance to docetaxel of PCa cells, indicating its vital role in PCa docetaxel resistance. Our findings revealed that miR-129-5p contributed to the resistance of PC-3-DR cells to docetaxel through suppressing CAMK2N1 expression, and thus targeting miR-129-5p may provide a novel therapeutic approach in sensitizing PCa to future docetaxel treatment.

Identification of human patatin-like phospholipase domain-containing protein 1 and a mutant in human cervical cancer HeLa cells.

Recently members of mammalian patatin-like phospholipase domain containing (PNPLA) protein family have attracted attention for their critical roles in diverse aspects of lipid metabolism and signal pathway. Until now little has been known about the characteristics of PNPLA1. Here, the full length coding cDNA sequence of human PNPLA1 (hPNPLA1) was cloned for the first time, which encoded a polypeptide with 532 amino acids containing the whole patatin domain. Tissue expression profiles analysis showed that low mRNA levels of hPNPLA1 existed in various tissues, except high expression in the digestive system, bone marrow and spleen. Subcellular distribution of hPNPLA1 tagged with green fluorescence protein mainly localized to lipid droplets. Furthermore, a nonsense mutation of PNPLA1 in human cervical cancer HeLa cells was identified. The hPNPLA1 mutant encoded a protein of 412 amino acids without the C-terminal domain and did not colocalize to lipid droplets, which suggested that the C-terminal region of hPNPLA1 affected lipid droplet binding. These results identified hPNPLA1 and a mutant in HeLa cells, and provided insights into the structure and function of PNPLA1.

The effect of focal adhesion kinase gene silencing on 5-fluorouracil chemosensitivity involves an Akt/NF-kappaB signaling pathway in colorectal carcinomas.

Multicellular resistance (MCR) is produced because multicellular spheroids (MCSs) are formed with a broad cell-cell connection when cultured in three-dimensions, which limits the clinical treatment efficacy in solid tumors. Focal adhesion kinase (FAK) plays an important role in apoptosis, survival and cell adhesion between cells and their extracellular matrix. In this study, we investigated the expressions of FAK, Akt and NF-kappaB in human colorectal cancer (CRC), and the effects of FAK gene silencing on MCSs formation and 5-fluorouracil (5-FU) chemosensitivity in colon carcinoma MCSs culture cells. In CRC samples, FAK, Akt and NF-kappaB were overexpressed. The positive expression of FAK correlated notably with lymph node metastasis and cellular differentiation. Positive expressions of Akt and NF-kappaB were significantly related to cellular differentiation and lymph node metastasis, respectively. Furthermore, positive expression of FAK correlated with that of Akt and NF-kappaB. The expression of FAK was inhibited significantly by a small hairpin RNA targeting FAK. Knockdown of FAK reversed the formation and aggregation of MCSs, significantly decreased the 50% inhibitory concentration of 5-FU, and markedly increased MCS culture cells apoptosis. These effects were associated with reduced levels of Akt and NF-kappaB. These results indicate that suppressing FAK expression potentiated 5-FU-induced cytotoxicity and contributed to its chemosensitizing effect by suppressing Akt/NF-kappaB signaling in colon carcinoma MCS culture cells. These data also imply that FAK mediates MCR of CRC through the survival signaling pathway FAK/Akt/NF-kappaB.

Trichlorfon induces apoptosis in SH-SY5Y neuroblastoma cells via the endoplasmic reticulum?

This study investigated the role of the endoplasmic reticulum pathway in apoptosis induced by trichlorfon in SH-SY5Y human neuroblastoma cells. Flow cytometric analysis demonstrated that trichlorfon and its degradation product dichlorvos-induced apoptosis in a dose-dependent manner and Hoechst 33342 staining experiments revealed trichlorfon/dichlorvos-induced nucleus condensation. Western blot analysis indicated decreased expression of caspase-12 and increased activated caspase-12 in trichlorfon-treated cells compared to a control, suggesting that trichlorfon may induce apoptosis in SH-SY5Y partly via the endoplasmic reticulum. Intracellular Ca(2+) level ([Ca(2+)](i)) in SH-SY5Y cells increased after treatment with trichlorfon but was significantly reduced by pre-treatment with a combination of a calcium channel blocker, an inositol trisphosphate receptor inhibitor, and a ryanodine receptor inhibitor. Percent apoptosis and activated caspase-3 and caspase-12 decreased in pre-treated cells compared to those treated with trichlorfon alone. Trichlorfon-induced apoptosis was also inhibited by the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). These results suggest that endoplasmic reticulum stress, which is related to calcium, may be involved in the cytotoxicity of trichlorfon.

Knockdown of focal adhesion kinase reverses colon carcinoma multicellular resistance.

Chemotherapy resistance in solid tumors is broad and encompasses diverse unrelated drugs. Three-dimensional multicellular spheroids (MCSs) are a good model for studying in vitro drug resistance. In the current study, we investigated the role of focal adhesion kinase (FAK) in 5-fluorouracil (5-FU) chemoresistance in colon carcinoma MCS culture cells. The expression of FAK was inhibited significantly by specific small hairpin RNA targeting FAK. The suppression of FAK expression did not affect the growth of spheroid cells. However, silencing of FAK combined with 5-FU treatment significantly decreased the 50% inhibitory concentration (IC(50)) of 5-FU and markedly increased the population of apoptosis cells, which was associated with the reduction of the levels of Akt and nuclear factor-kappa B (NF-kappaB). Moreover, knockdown of FAK could inhibit tumor growth and increase the sensitivity of the tumor to 5-FU in the nude mouse xenograft. These results indicate that while not affecting cellular proliferation in the absence of 5-FU, RNA interference targeting FAK potentiated 5-FU-induced cytotoxicity in vitro and in vivo, and partially reversed multicellular resistance, which may contribute to its chemosensitizing effect through efficiently suppressing Akt/NF-kappaB activity.

Degradation of neuropathy target esterase by the macroautophagic lysosomal pathway.

AIMS: Neuropathy target esterase (NTE) was proposed as the initial target during the process of organophosphate-induced delayed neuropathy (OPIDN) in humans and some sensitive animals. NTE was recently identified as a novel phospholipase B that is anchored to the cytoplasmic side of the endoplasmic reticulum. However, little is known about the degradation of NTE. In this study, we have investigated the role of the macroautophagic-lysosomal pathway in NTE degradation in neuronal and non-neuronal cells. MAIN METHODS: Macroautophagy inhibitors and activators were used to interrupt the lysosomal pathway, and NTE protein level was followed using western blotting analysis. A fluorescent microscopy assay was used to determine the co-localization of NTE and lysosomes. KEY FINDINGS: Western blotting analysis showed that the macroautophagy inhibitors 3-methyladenine and ammonium chloride increased the levels of a heterologously expressed NTE-GFP fusion protein as well as endogenous NTE. Starvation had the opposite effect. The role of macroautophagy in NTE degradation was further supported by the co-localization of exogenous NTE with lysosomes in starved COS7 cells. Furthermore, the contribution of NTE activity and protein domains to the degradation of NTE by macroautophagy was investigated, showing that both the transmembrane and regulatory domains played a role in the degradation of NTE and that the catalytic domain, and thus NTE activity, was not involved. SIGNIFICANCE: Our findings clearly demonstrate, for the first time, that the macroautophagy/lysosome pathway plays a role in controlling NTE quantity, providing a further understanding of the function of NTE.