TRIM8 promotes ovarian cancer proliferation and migration by targeting VDAC2 for ubiquitination and degradation.

BACKGROUND: Ovarian cancer is a common gynecological tumor with high malignant potential and poor prognosis. TRIM8, is involved in the development of various tumors, but its precise regulatory role in ovarian cancer is still unknown. AIMS: The aim of this study was to explore the specific mechanism by which TRIM8 regulates ovarian cancer. MATERIALS AND METHODS: We used bioinformatics analysis to screen for high expression of TRIM8 in ovarian cancer. The expression of TRIM8 in healthy and cancerous ovarian tissues was assessed by immunofluorescence. TRIM8 was silenced or overexpressed in ovarian cancer cell lines, with cell proliferation and migration evaluated by CCK8, transwell and clonal formation assays. The effect of TRIM8 on ovarian cancer cells in vivo was assessed by subcutaneous tumor formation experiments in nude mice. The potential interacting protein VDAC2 was identified by mass spectrometry. The mechanism underlying TRIM8 regulation of VDAC2 was evaluated by co-immunoprecipitation and western blotting. RESULTS: TRIM8 was overexpressed in ovarian cancer. TRIM8 promoted the proliferation and migration of ovarian cancer cells in vitro and the growth of subcutaneous tumors in mice in vivo. TRIM8 interacted with VDAC2, weakened the stability of the protein, and promoted its polyubiquitination and subsequent degradation. Knockdown of VDAC2 increased the resistance of ovarian cancer cells to iron death, whereas overexpression of VDAC2 attenuated ovarian cancer progression induced by TRIM8 overexpression. DISCUSSION: TRIM8 promotes ovarian cancer proliferation and migration by targeting VDAC2 for ubiquitination and degradation, these finding may provide new targets for the treatment of ovarian cancer. CONCLUSION: TRIM8 degraded VDAC2 through the ubiquitination pathway, increased the resistance of ovarian cancer cells to iron death, and promoted the proliferation and migration of ovarian cancer.

In situ detection of dopamine in single living cells by molecularly imprinted polymer-functionalized nanoelectrodes.

In situ detection of dopamine (DA) at single-cell level is critical for exploring neurotransmitter-related biological processes and diseases. However, the low content of DA and a variety of distractors with similar oxidation potentials as DA in cells brought great challenges. Here, a sensitive and specific electrochemical nanosensor was proposed for in situ detection of DA in single living cells based on nanodiamond (ND) and molecularly imprinted polymer (MIP)-functionalized carbon fiber nanoelectrode (ND/MIP/CFNE). Due to its excellent electrocatalytic property, ND was modified to the surface of CFNE based on amide bonding. Compared with bare CFNE, ND-modified CFNE can enhance oxidation currents of DA by about 4-fold, improving signal-to-noise ratio and detection sensitivity. MIP was further electropolymerized on the surface of nanoelectrodes to achieve specific capture and recognition of DA, which could avoid the interference of complex matrix and analogs in cells. Taking advantage of the precise positioning capability of a single-cell analyzer and micromanipulator, ND/MIP/CFNE could be precisely inserted into different locations of single cells and monitor oxidation signal of DA. The concentration of DA in the cytoplasm of single pheochromocytoma (PC12) cell was measured to be about 0.4 µM, providing a sensitive and powerful method for single-cell detection. Furthermore, the nanoelectrodes can monitor the fluctuation of intracellular DA under drug stimulation, providing new ideas and methods for new drug development and efficacy evaluation.

ANGPTL4 regulates ovarian cancer progression by activating the ERK1/2 pathway.

BACKGROUND: Ovarian cancer (OC) has the highest mortality rate among all gynecological malignancies. A hypoxic microenvironment is a common feature of solid tumors, including ovarian cancer, and an important driving factor of tumor cell survival and chemo- and radiotherapy resistance. Previous research identified the hypoxia-associated gene angiopoietin-like 4 (ANGPTL4) as both a pro-angiogenic and pro-metastatic factor in tumors. Hence, this work aimed to further elucidate the contribution of ANGPTL4 to OC progression. METHODS: The expression of hypoxia-associated ANGPTL4 in human ovarian cancer was examined by bioinformatics analysis of TCGA and GEO datasets. The CIBERSORT tool was used to analyze the distribution of tumor-infiltrating immune cells in ovarian cancer cases in TCGA. The effect of ANGPTL4 silencing and overexpression on the proliferation and migration of OVCAR3 and A2780 OC cells was studied in vitro, using CCK-8, colony formation, and Transwell assays, and in vivo, through subcutaneous tumorigenesis assays in nude mice. GO enrichment analysis and WGCNA were performed to explore biological processes and genetic networks associated with ANGPTL4. The results obtained were corroborated in OC cells in vitro by western blotting. RESULTS: Screening of hypoxia-associated genes in OC-related TCGA and GEO datasets revealed a significant negative association between ANGPTL4 expression and patient survival. Based on CIBERSORT analysis, differential representation of 14 distinct tumor-infiltrating immune cell types was detected between low- and high-risk patient groups. Silencing of ANGPTL4 inhibited OVCAR3 and A2780 cell proliferation and migration in vitro and reduced the growth rate of xenografted OVCAR3 cells in vivo. Based on results from WGCNA and previous studies, western blot assays in cultured OC cells demonstrated that ANGPTL4 activates the Extracellular signal-related kinases 1 and 2 (ERK1/2) pathway and this results in upregulation of c-Myc, Cyclin D1, and MMP2 expression. Suggesting that the above mechanism mediates the pro-oncogenic actions of ANGPTL4T in OC, the pro-survival effects of ANGPTL4 were largely abolished upon inhibition of ERK1/2 signaling with PD98059. CONCLUSIONS: Our work suggests that the hypoxia-associated gene ANGPTL4 stimulates OC progression through activation of the ERK1/2 pathway. These findings may offer a new prospect for targeted therapies for the treatment of OC.

Endogenous Protease-Activatable Nanosensor Based on PNA-Peptide-DNA Engineering for AND-Gated and Dual-Model Detection of MicroRNA in Single Living Tumor Cells.

The in situ detection of low-content cancer biomarkers by an endogenous activator instead of an exogenous initiator in vitro remains a great challenge, leaving a gap in the development of a tumor-specific nanosensor with an endogenous protease-activatable manner. Herein, we proposed an endogenous protease-activatable nanosensor (PA-NS) guided by peptide nucleic acid-peptide-DNA copolymers to realize AND-gated and dual-model sensing of miRNA-21 (miR-21) by combining electrochemical detection with optical imaging in living tumor cells, without an additional introduction of an exogenous activator or nanomaterials. Moreover, the PA-NS can only be activated by "dual keys" (overexpressed miR-21 and cathepsin B protease in tumor cells) simultaneously, which enables effective improvement of the tumor-to-healthy cells ratio. The fluorescence intensity measured in single tumor cells was ∼3.5-fold higher than that in single healthy cells, and the electrochemical response decreased ∼30% in the presence of target miRNA. Furthermore, studies on regulation of the protease activity and miR-21 fluctuation under external stimulation have contributed to our understanding of the biological processes and drug screenings underlying disease development. This specific endogenous protease-mediated manner for dual-model detection of miRNA guarantees excellent tumor-selective capability, which offers new opportunities to study cell heterogeneity and provides more reliable fundamentals for the diagnosis and treatment of cancer down to the single-cell level.

Circulating tumor cell associated white blood cell cluster as a biomarker for metastasis and recurrence in hepatocellular carcinoma.

Background: Recently, an in vivo study demonstrated that circulating tumor cell-associated white blood cell (CTC-WBC) cluster possess much greater potential than single CTCs. We aim to explore the correlation between the CTC-WBC cluster and the clinicopathological characteristics of hepatocellular carcinoma (HCC) patients to seek novel biomarkers for HCC metastasis and recurrence. Methods: We retrospectively analyzed 136 HCC patients from October 2014 to March 2020 who received CTC tests using the CanPatrol CTC enrichment technique. The correlation between the clinical features and total CTCs, EMT-CTCs, and CTC-WBC cluster were analyzed by a chi-square test. The ROC curves were simulated for evaluating the diagnostic performance of CTC parameters in HCC metastasis. Patients were followed up from February 2015 to November 2021, and the relapse-free survival (RFS) was analyzed using the Kaplan-Meier curve. Results: A total of 93.4% (127/136) and 31.6% (43/136) of HCC patients had detectable CTCs and CTC-WBC clusters. Baseline CTC-WBC cluster was closely correlated with microvascular invasion, portal vein tumor thrombus, and extrahepatic metastasis in pre-treatment HCC patients (P <0.05). The simulated ROC curves presented an AUC of 0.821 for the CTC-WBC cluster (sensitivity of 90.0% and specificity of 93.7%) in discriminating metastasis from non-metastatic HCC, which was higher than that for total CTCs (0.718) and EMT-CTCs (0.716). Further follow-up analysis showed that compared to the CTC-WBC cluster negative group (<1/5 ml), patients in the CTC-WBC cluster positive group (≥1/5 ml) presented an increased relapse ratio (60.0% versus 17.9%) and shorter RFS (22.9 versus 53.8 months). Dynamic analysis of CTCs parameters showed that total CTC level, EMT-CTCs proportion, and CTC-WBC cluster were decreased after microwave ablation treatment, while CTC-WBC cluster increased on average 10 months in advance of imaging (MRI) diagnosed recurrence. Conclusion: The CTC-WBC cluster is a promising biomarker for the metastasis diagnosis and prognosis of HCC metastasis. Dynamic monitoring of the CTC-WBC cluster is an effective method for early detection and intervention of HCC recurrence and metastasis.

In Situ Measurement of ATP in Single Cells by an Amphiphilic Aptamer-Assisted Electrochemical Nano-Biosensor.

In situ and quantitative measurements of adenosine 5'-triphosphate (ATP) in single living cells are highly desired for understanding several sorts of necessary physiological and pathological processes. Due to its small size and high sensitivity, an ultra-microelectrode can be used for single-cell analysis. However, ATP is difficult to detect in single cells because it is nonelectroactive and low in content. Herein, we introduced an electrochemical nano-biosensor based on an amphiphilic aptamer-assisted carbon fiber nanoelectrode (aptCFNE) with high signal-to-noise ratio. The low current (e.g., 60 pA) and the tiny diameter of the tip (ca. 400 nm) of the nanosensor made it noninvasive to living cells. The amphiphilic aptamer has good biocompatibility and can be stably modified to the surface of functionalized electrodes. CFNE, which was modified with ferrocene-labeled aptamer, could quickly and selectively detect ATP content in the nucleus, cytoplasm, and extracellular space of single HeLa cells. The results showed that the ATP contents in the nucleus, cytoplasm, and extracellular space were 568 ± 9, 461 ± 20, and 312 ± 4 µM, respectively. The anticancer drug treatment effects on the cellular level were further recorded, which was of great significance for understanding ATP-related biological processes and drug screenings. This strategy is universally applicable to detect other targets by changing the aptamer sequence, which will greatly improve our understanding of cell heterogeneity and provide a more reliable scientific basis for exploring major diseases at the single-cell level.

CDC20 is a novel biomarker for improved clinical predictions in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC), a common tumor of the female reproductive system, ranks first in fatalities among gynecological malignancies. Most patients find tumors at late stage and have extremely poor prognoses, which necessitates improvements in early detection. This study applied bioinformatic methods to identify potential biomarkers of EOC. First, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed on differentially expressed genes (DEGs) and hub genes, and a protein-protein interaction (PPI) network was constructed. The network of hub genes was analyzed using GeneMANIA, and an analysis of biological processes was constructed with BINGO. Lastly, hub genes were analyzed for EOC-related oncology using the Oncomine and TCGA databases, and the cBioPortal online platform. Overall, cell division cycle 20 (CDC20) was identified as a key gene in EOC. Short hairpin RNA (shRNA) was used to silence CDC20 to explore its effects on EOC cell proliferation, apoptosis and SRY-related HMG-box 2 (SOX2) expression. DEGs were enriched in pathways related to cell cycle signaling, cancer, progesterone-mediated oocyte maturation, Wnt signaling and P53 signaling. Analysis revealed high expression of CDC20 in EOC tissues and a correlation with histology and tumor grade. CDC20 levels are highest in serous adenocarcinoma, when compared to ovarian clear cell carcinoma, ovarian endometrioid carcinoma and mucinous adenocarcinoma. High CDC20 expression within the tumor is associated with poor EOC prognosis. After silencing CDC20, EOC cell proliferation and migration decreased, apoptosis increased, and SOX2 expression decreased. In conclusion, CDC20 is likely a key biomarker of EOC and may act as an upstream regulator of SOX2 to mediate the SOX2 signaling in the progression of EOC. Future application of CDC20 analysis to early detection may improve prognosis, and it has the potential to be a therapeutic target.

High sensitivity and rapid detection of hepatitis B virus DNA using lateral flow biosensors based on Au@Pt nanorods in the absence of hydrogen peroxide.

Nanozymes are a kind of nanomaterial with enzymatic activity, and have attracted wide attention in signal probe fields owing to their good catalytic activity and stability. Herein, we designed gold@platinum nanorods (Au@Pt) with enhanced oxidase-like activity as signal probes to construct lateral flow biosensors (LFBs) for the detection of hepatitis B virus DNA (HBV-DNA). The enhanced oxidase-like activity of Au@Pt nanorods can effectively catalyze the oxidation of 3,3',5,5'-tetramethylenebenzidine (TMB) to a blue substrate in the absence of hydrogen peroxide (H2O2). Based on this principle, LFBs using Au@Pt nanorods as signal probes can provide an effective signal amplification strategy and prevent biomolecules from being affected by H2O2. Under optimal conditions, LFBs have a good linear relationship between 0.1 nM and 50 nM, and the calculated detection limit was 8.5 pM. The technological strategy in the detection and quantification of HBV-DNA in this work may be helpful to achieve a rapid and accurate diagnosis of early HBV-DNA and provide new ideas for the development of point-of-care testing.

miR-181a-2-3p Stimulates Gastric Cancer Progression via Targeting MYLK.

Background: The abnormal expression of miRNAs facilitates tumorigenesis and development. miR-181a-2-3p is up-regulated in various cancers, yet its mechanism in gastric cancer (GC) remains elusive. Objective: To understand mechanism of miR-181a-2-3p stimulating GC cell progression via targeting Myosin Light Chain Kinase (MYLK) expression. Methods: Downstream genes of miRNA of interest were predicted in TargetScan and miRTarBase. qRT-PCR and western blot were applied to assess miR-181a-2-3p and MYLK expression in GC cells and normal cells. Dual-luciferase and RIP assays were completed to assess binding of miR-181a-2-3p and MYLK. Cell Counting Kit-8 (CCK-8) assay was conducted for detecting viability of AGS and SNU-1 cells, while Transwell tested migratory and invasive abilities of cells. Nude mouse transplantation tumor experiment was performed to assay tumor growth in vivo. Results: miR-181a-2-3p was notably increased in human GC cell lines, while MYLK was remarkably down-regulated. RIP and dual-luciferase assay disclosed that miR-181a-2-3p targeted MYLK and repressed MYLK. Forced miR-181a-2-3p expression fostered GC cell proliferation, invasion, migration, and fostered tumor growth in vivo. Promoting effect of miR-181a-2-3p on GC cells was reversed when miR-181a-2-3p and MYLK were simultaneously overexpressed. Conclusion: miR-181a-2-3p facilitated GC cell progression by targeting MYLK, and it may be a pivotal prognostic biomarker in investigating molecular mechanism of GC.

MiR-490-5p Restrains Progression of Gastric cancer through DTL Repression.

Gastric cancer (GC) accounts for a main cause of cancer-related deaths. This study sought for molecular mechanism of miR-490-5p/DTL axis in affecting GC progression, thus bringing new hope for treatment of GC. Expression data of differentially expressed miRNAs and mRNAs in GC tissue from TCGA database were analyzed. MiR-490-5p and DTL mRNA expression levels in GC were evaluated with qRT-PCR. Cell viability was confirmed with CCK-8 method. Cell cycle distribution and apoptosis were analyzed with flow cytometry. Cell migratory and invasive potential was proved with Transwell assay. The targeted relationship between DTL and miR-490-5p was analyzed with dual-luciferase assay. The results indicated a decreased miR-490-5p level in GC cells. MiR-490-5p upregulation hampered proliferation, migration, invasion and promote cell apoptosis. DTL was the target of and inversely associated with miR-490-5p, and it could remarkably induce the carcinogenesis of GC. MiR-490-5p mediated GC cell progression by DTL repression. In conclusion, miR-490-5p and DTL may be valuable in diagnosis and treatment for GC.

Cu2+-modified hollow carbon nanospheres: an unusual nanozyme with enhanced peroxidase-like activity.

A Cu2+-modified carboxylated hollow carbon nanospheres (Cu2+-HCNSs-COOH) was designed with enhanced peroxidase-like activity for the detection of hydrogen peroxide (H2O2) and degradation of methylene blue (MB). Hollow polymer nanospheres were fabricated from aniline, pyrrole, Triton-100, and ammonium persulfate via confined interfacial copolymerization reaction, which can be pyrolyzed to create HCNSs with the hollow gap diameter of about 20 nm under high temperature. Combining the synergistic effect of coordination and electrostatic interaction, Cu2+-HCNSs-COOH was constructed by anchoring Cu2+ on the surface of HCNSs-COOH. Furthermore, Cu2+-HCNSs-COOH has higher affinity for 3,3',5,5'-tetramethylbenzidine and H2O2 of 0.20 mM and 0.88 mM, respectively. Based on the rapid response of Cu2+-HCNSs-COOH to H2O2, we constructed a colorimetric sensing platform by detecting the absorbance of the 3,3',5,5'-tetramethylbenzidine-H2O2 system at 652 nm for quantifying H2O2, which holds good linear relationship between 1 and 150 µM and has a detection limit of 0.61 µM. We also investigated the degradation of MB in the presence of Cu2+-HCNSs-COOH and H2O2, which can degrade 80.7% pollutants within 30 min. This research developed an unusual nanozyme for bioassays and water pollution treatment, which broadened the way for the rapid development of clinical diagnostics and water pollution treatment.

Prognostic value of preoperative serum bilirubin levels in ovarian cancer.

Bilirubin is a promising prognostic factor for non-liver disease-related deaths in various cancers. We investigated the association between preoperative serum bilirubin levels and oncological outcomes in patients with ovarian cancer. We retrospectively analyzed the clinical data of 282 patients with epithelial ovarian carcinoma (EOC), and grouped them according to optimal threshold values of total bilirubin (TBIL), direct bilirubin (DBIL), and indirect bilirubin (IBL) measured by receiver operating characteristic curve analysis. Univariate and multivariate Cox proportional hazards regression analyses were used to evaluate various parameters that might affect overall survival (OS) and progression-free survival (PFS) in patients with EOC. The optimal cutoff values for TBIL, DBIL, and IBIL levels were 9.65 µmol/L, 2.95 µmol/L, and 6.75 µmol/L, respectively. Increased TBIL, DBIL, and IBIL levels correlated with the serum carbohydrate antigen (CA)-125 levels, International Federation of Gynecology and Obstetrics stage, and pathological differentiation (all P<0.05). Univariate analysis revealed longer OS and PFS in patients with high TBIL (≥9.65 µmol/L) and IBIL (≥6.75 µmol/L) levels (P<0.05). Multivariate analysis showed that patients with high IBIL levels (≥6.75 µmol/L) had significantly longer OS and PFS than those with low IBIL levels (<6.75 µmol/L) [hazard ratio (HR) = 0.333, 95% confidence interval (CI): 0.123~0.904, P<0.05; HR = 1.814, 95% CI: 1.169~2.816, P<0.05]. Therefore, IBIL is a potential independent prognostic factor for OS and PFS in patients with EOC. The higher the IBL level, the better the prognosis of patients with EOC.

Iron doped graphitic carbon nitride with peroxidase like activity for colorimetric detection of sarcosine and hydrogen peroxide.

The successful synthesis is reported of Mn, Fe, Co, Ni, Cu-doped g-C3N4 nanoflakes via a simple one-step pyrolysis method, respectively. Among them, the Fe-doped g-C3N4 nanoflakes exhibited the highest peroxidase-like activity, which can be used for colorimetric detection of hydrogen peroxide (H2O2) and sarcosine (SA), within the detection ranges of 2-100 µM and 10-500 µM and detection limits of 1.8 µM and 8.6 µM, respectively. The catalytic mechanism of the Fe-doped g-C3N4 nanoflake was also explored and verified the generation of hydroxyl radical (•OH) through fluorescence method. It is believed that the Fe-doped g-C3N4 nanoflakes as enzyme mimics will greatly promote the practical applications in a variety of fields in the future including biomedical science, environmental governance, antibacterial agent, and bioimaging due to their extraordinary catalytic performance and stability. Graphical abstract.

A H2O2-free electrochemical peptide biosensor based on Au@Pt bimetallic nanorods for highly sensitive sensing of matrix metalloproteinase 2.

Inspired by the excellent catalytic activity of Au@Pt bimetallic nanorods, we construct a H2O2-free electrochemical peptide biosensor based on Au@Pt bimetallic nanorods for highly efficient and sensitive sensing of MMP-2 for the first time, not only simplifying the traditional testing steps but also avoiding the potential damage caused by H2O2 to peptides and proteins.

Metabolic classification of circulating tumor cells as a biomarker for metastasis and prognosis in breast cancer.

BACKGROUND: Circulating tumor cells (CTCs) has been demonstrated as a promising liquid biopsy marker for breast cancer (BC). However, the intra-patient heterogeneity of CTCs remains a challenge to clinical application. We aim at profiling aggressive CTCs subpopulation in BC utilizing the distinctive metabolic reprogramming which is a hallmark of metastatic tumor cells. METHODS: Oncomine, TCGA and Kaplan-Meier plotter databases were utilized to analyze expression and survival relevance of the previously screened metastasis-promoting metabolic markers (PGK1/G6PD) in BC patients. CTCs detection and metabolic classification were performed through micro-filtration and multiple RNA in situ hybridization using CD45 and PGK1/G6PD probes. Blood samples were collected from 64 BC patients before treatment for CTCs analysis. Patient characteristics were recorded to evaluate clinical applications of CTCs metabolic subtypes, as well as morphological EMT subtypes classified by epithelial (EpCAM/CKs) and mesenchymal (Vimentin/Twist) markers. RESULTS: PGK1 and G6PD expressions were up-regulated in invasive BC tissues compared with normal mammary tissues. Increased tissue expressions of PGK1 or G6PD indicated shortened overall and relapse-free survival of BC patients (P < 0.001). Blood GM+CTCs (DAPI+CD45-PGK1/G6PD+) was detectable (range 0-54 cells/5 mL) in 61.8% of tCTCs > 0 patients. Increased GM+CTCs number and positive rate were correlated with tumor metastasis and progression (P < 0.05). The GM+CTCs ≥ 2/5 mL level presented superior AUC of ROC at 0.854 (95% CI 0.741-0.968) in the diagnosis of BC metastasis (sensitivity/specificity: 66.7%/91.3%), compared with that of tCTCs (0.779) and CTCs-EMT subtypes (E-CTCs 0.645, H-CTCs 0.727 and M-CTCs 0.697). Moreover, GM+CTCs+ group had inferior survival with decreased 2 years-PFS proportion (18.5%) than GM+CTCs- group (87.9%; P = 0.001). CONCLUSIONS: This work establishes a PGK1/G6PD-based method for CTCs metabolic classification to identify the aggressive CTCs subpopulation. Metabolically active GM+CTCs subtype is suggested a favorable biomarker of distant metastasis and prognosis in BC patients.

An aptamer-based hook-effect-recognizable three-line lateral flow biosensor for rapid detection of thrombin.

In this paper, a three-line LFB was successfully developed by adding a thrombin line to a conventional two-line LFB for the detection of thrombin in a wide range of human serum. We introduced a thrombin line between the test line and the control line. The concentration of thrombin in the sample was quantitatively related to the signal formation on the three lines of the LFB. We can make use of signal on three lines to quantitative determinate the thrombin by data processing. The detection range of thrombin concentrations measured in 10 min was 1 nM to 100 µM and the LOD was 0.85 nM. Our approach paves way for rapid and sensitive thrombin detection and a superior device for testing in a wide range of physiological concentrations, which also can be used in other hook-effect-limited aptamers or antibodies based sandwich LFBs, and has a high accuracy even within the range of the hook-effect.

Blockage of ceramide metabolism exacerbates palmitate inhibition of pro-insulin gene expression in pancreatic beta-cells.

Chronic exposure to elevated levels of fatty acids (FAs) in conjunction with chronic hyperglycemia has been reported to contribute to the progressive deterioration of beta-cell function in patients with type 2 diabetes mellitus. The long-chain saturated free fatty acid (FFA) palmitate, unlike the unsaturated FFA oleate, is known to have an inhibitory effect on proinsulin gene expression through ceramide synthesis. This study was aimed at investigating whether this effect was exacerbated by the inhibition of ceramide degradation in pancreatic beta-cells and the molecular mechanism of intracellular ceramide-induced inhibition of proinsulin gene transcription in response to exposure to palmitate. We exposed insulin-secreting (INS-1) cells treated with low levels of palmitate to the ceramidase inhibitor n-oleoylethanolamine (NOE); this led to the generation of high levels of intracellular ceramide. We observed that the effects of ceramide accumulation in INS-1 cells were similar to the effects of the inhibition of this protein on proinsulin mRNA levels that are caused by the negative regulation of insulin promoter activity. In addition, we observed that ceramide accumulation induced by NOE leads to a significant decrease in the levels of activated extracellular signal-regulated kinase (ERK); the inactivation of the ERK cascade in response to palmitate stimuli is induced by protein phosphatase 2A (PP2A) activity. Based on these findings, we suggest that the aberrant accumulation of ceramide was caused by the inhibition of ceramide metabolism, which in turn leads to the inhibition of proinsulin gene expression; the inhibition of ERK cascades by PP2A serves as an important factor in the inhibitory effects of ceramide.