Stable over‐expression of the 2‐oxoglutarate carrier enhances neuronal cell resistance to oxidative stress via Bcl‐2‐dependent mitochondrial GSH transport

Mitochondrial glutathione (GSH) is a key endogenous antioxidant and its maintenance is critical for cell survival. Here, we generated stable NSC34 motor neuron‐like cell lines over‐expressing the mitochondrial GSH transporter, the 2‐oxoglutarate carrier (OGC), to further elucidate the importance of mitochondrial GSH transport in determining neuronal resistance to oxidative stress. Two stable OGC cell lines displayed specific increases in mitochondrial GSH content and resistance to oxidative and nitrosative stressors, but not staurosporine. Inhibition of transport through OGC reduced levels of mitochondrial GSH and resensitized the stable cell lines to oxidative stress. The stable OGC cell lines displayed significant up‐regulation of the anti‐apoptotic protein, B cell lymphoma 2 (Bcl‐2). This result was reproduced in parental NSC34 cells by chronic treatment with GSH monoethylester, which specifically increased mitochondrial GSH levels. Knockdown of Bcl‐2 expression decreased mitochondrial GSH and resensitized the stable OGC cells to oxidative stress. Finally, endogenous OGC was co‐immunoprecipitated with Bcl‐2 from rat brain lysates in a GSH‐dependent manner. These data are the first to show that increased mitochondrial GSH transport is sufficient to enhance neuronal resistance to oxidative stress. Moreover, sustained and specific enhancement of mitochondrial GSH leads to increased Bcl‐2 expression, a required mechanism for the maintenance of increased mitochondrial GSH levels.

     

DJ‐1 inhibits autophagy activity of prostate cancer cells by repressing JNK–Bcl2–Beclin1 signaling

The regulation of DJ‐1 on AR signaling plays an important role in the pathogenesis of prostate cancer (PCa). DJ‐1 could alter autophagy and regulate Beclin1‐involved autophagy response through JNK‐dependent pathway. JNK is known to mediate autophagy through Bcl2–Beclin1 complex. Therefore, this study aimed to investigate the significance of autophagy in DJ‐1‐modulated PCa cells. The current studies showed that DJ‐1 overexpression in LNCaP decreased LC3 transformation and autophagosome formation. However, DJ‐1 knockdown exerted the opposite effect. Moreover, DJ‐1 silencing inhibited survival and promoted death in LNCaP, which was recovered by autophagy inhibition with 3‐MA. In addition, DJ‐1 overexpression inhibited the phosphorylation of JNK and Bcl2, and the dissociation of Beclin1 and Bcl2; while the effect of silencing DJ‐1 was completely opposite. More important, JNK activated by anisimycin inhibited the proliferation and promoted death of DJ‐1‐overexpressed LNCaP while increasing LC3 transformation and LC3‐puncta formation, but these results were reversed by the decrease of Beclin1 (by spautin‐1). In contrast, when DJ‐1 was silenced, the death of LNCaP, LC3 transformation, and LC3‐puncta formation were inhibited by JNK inhibitor SP600125, which promoted cell proliferation. However, Bcl2 inhibition (by ABT737) reversed all the effects of SP600125. Our results suggested that DJ‐1 in PCa cells could promote the growth of PCa through autophagy inhibition, and JNK–Bcl2–Beclin1 signaling played an important role in it. The study provided new insights into the role of DJ‐1 in the development of PCa.     

CREB‐mediated Bcl‐2 expression contributes to RCAN1 protection from hydrogen peroxide‐induced neuronal death

Regulator of calcineurin 1 (RCAN1) is located on the Down syndrome critical region (DSCR) locus in human chromosome 21. In this study, we investigated the functional role of RCAN1 in the reactive oxygen species (ROS)‐mediated neuronal death signaling. We found that RCAN1 was able to protect the cells from H₂O₂‐induced cytotoxicity. The expression of RCAN1 caused an inhibition of the H₂O₂‐induced activation of mitogen‐activated protein kinases (MAPKs) and AP‐1. In contrast, RCAN1 significantly enhanced the activity of cAMP response element‐binding protein (CREB). Furthermore, RCAN1 induced the expression of the CREB target gene, Bcl‐2. Consistently, knockdown of endogenous RCAN1 using shRNA down regulated the phosphorylation of CREB and the expression of Bcl‐2, which protects the cells from H₂O₂‐induced cytotoxicity. Our data provide a new mechanism for the cytoprotective function of RCAN1 in response to oxidant‐induced apoptosis. J. Cell. Biochem. 114: 1115–1123, 2013. © 2012 Wiley Periodicals, Inc.     

The advanced glycation end product Nϵ‐carboxymethyllysine and its precursor glyoxal increase serotonin release from Caco‐2 cells

Advanced glycation end products (AGEs), comprising a highly diverse class of Maillard reaction compounds formed in vivo and during heating processes of foods, have been described in the progression of several degenerative conditions such as Alzheimer's disease and diabetes mellitus. N^?‐Carboxymethyllysine (CML) represents a well‐characterized AGE, which is frequently encountered in a Western diet and is known to mediate its cellular effects through binding to the receptor for AGEs (RAGE). As very little is known about the impact of exogenous CML and its precursor, glyoxal, on intestinal cells, a genome‐wide screening using a customized microarray was conducted in fully differentiated Caco‐2 cells. After verification of gene regulation by qPCR, functional assays on fatty acid uptake, glucose uptake, and serotonin release were performed. While only treatment with glyoxal showed a slight impact on fatty acid uptake (P < 0.05), both compounds reduced glucose uptake significantly, leading to values of 81.3% ± 22.8% (500 μM CML, control set to 100%) and 68.3% ± 20.9% (0.3 μM glyoxal). Treatment with 500 μM CML or 0.3 μM glyoxal increased serotonin release (P < 0.05) to 236% ± 111% and 264% ± 66%, respectively. Co‐incubation with the RAGE antagonist FPS‐ZM1 reduced CML‐induced serotonin release by 34%, suggesting a RAGE‐mediated mechanism. Similarly, co‐incubation with the SGLT‐1 inhibitor phloridzin attenuated serotonin release after CML treatment by 32%, hinting at a connection between CML‐stimulated serotonin release and glucose uptake. Future studies need to elucidate whether the CML/glyoxal‐induced serotonin release in enterocytes might stimulate serotonin‐mediated intestinal motility.

     

A novel BH3 mimetic efficiently induces apoptosis in melanoma cells through direct binding to anti‐apoptotic Bcl‐2 family proteins,including phosphorylated Mcl‐1

The Bcl‐2 family modulates sensitivity to chemotherapy in many cancers, including melanoma, in which the RAS/BRAF/MEK/ERK pathway is constitutively activated. Mcl‐1, a major anti‐apoptotic protein in the Bcl‐2 family, is extensively expressed in melanoma and contributes to melanoma's well‐documented chemoresistance. Here, we provide the first evidence that Mcl‐1 phosphorylation at T163 by ERK1/2 and JNK is associated with the resistance of melanoma cell lines to the existing BH3 mimetics gossypol, S1 and ABT‐737, and a novel anti‐apoptotic mechanism of phosphorylated Mcl‐1 (pMcl‐1) is revealed. pMcl‐1 antagonized the known BH3 mimetics by sequestering pro‐apoptotic proteins that were released from Bcl‐2/Mcl‐1. Furthermore, an anthraquinone BH3 mimetic, compound 6, was identified to be the first small molecule to that induces endogenous apoptosis in melanoma cells by directly binding Bcl‐2, Mcl‐1, and pMcl‐1 and disrupting the heterodimers of these proteins. Although compound 6 induced upregulation of the pro‐apoptotic protein Noxa, its apoptotic induction was independent of Noxa. These data reveal the promising therapeutic potential of targeting pMcl‐1 to treat melanoma. Compound 6 is therefore a potent drug that targets pMcl‐1 in melanoma.     

Transforming growth factor‐β1 modulates metalloproteinase‐2 and ‐9, nitric oxide,RhoA and α‐smooth muscle actin expression in colon adenocarcinoma cells

Colon carcinoma invasiveness is a process involving cell–cell and cell–matrix alterations, local proteolysis of the ECM (extracellular matrix) or changes in cytokine and growth factor levels. In order to evaluate the role of TGF‐β1 (transforming growth factor‐β1) and small G protein RhoA in tumour progression, the influence of TGF‐β1 treatment or RhoA‐associated kinase inhibitor on the production of NO (nitric oxide) and MMP‐2 and MMP‐9 (metalloproteinases‐2 and ‐9) was analysed in three human colon adenocarcinoma cell lines (HT29, LS180, SW948) representing different stages of tumour development. All the tested cell lines produced low amounts of MMP‐2 and MMP‐9. rhTGF‐β1 and the synthetic Rho kinase inhibitor (Y‐27632) decreased MMP‐2 secretion by colon cancer cells, especially in the most advanced stage of colon cancer. rhTGF‐β1 decreased NO secretion by cells, while Y‐27632 had no effect on it. Immunoblotting with anti‐RhoA antibodies followed by densitometry revealed that RhoA levels were slightly increased after incubation of colon carcinoma cells (SW948) with rhTGF‐β1. rhTGF‐β1 induced α‐smooth muscle actin (α‐SMA) expression, especially in high Duke's grade of colon cancer, while Y‐27632 blocked it. Summing up, in colon carcinoma cells, TGF‐β1 and RhoA protein may regulate tumour invasiveness measured as MMP, NO and α‐SMA expression or assayed using motility data and may be a good target for cancer therapy.     

Unusual sterolic mixture,and 24‐isopropylcholesterol,from the sponge Ciocalypta sp. reduce cholesterol uptake and basolateral secretion in Caco‐2 cells

An unusual sterolic mixture (82.3% of 24‐isopropylated sterols) and its major component, 24‐isopropylcholesterol, isolated from a marine sponge, Ciocalypta sp. (Halichondriidae), reduce cholesterol uptake, basolateral secretion and ACAT‐2 mRNA expression and increase the expression of ABCA1 mRNA in Caco‐2 cells. The decreases of cholesterol uptake and secretion induced by 24‐isopropylcholesterol alone were more than that of both the sterolic mixture and β‐sitosterol. These data add a new sterol, 24‐isopropylcholesterol, to sterols that may reduce intestinal cholesterol absorption. J. Cell. Biochem. 106: 659–665, 2009. © 2009 Wiley‐Liss, Inc.     

Elevated hsa‐miR‐590‐3p expression down‐regulates HMGB2 expression and contributes to the severity of IgA nephropathy

Peripheral blood mononuclear cells (PBMCs) play important roles in the pathogenesis of IgA nephropathy (IgAN). Our study aimed to provide a deep understanding of IgAN and focused on the dysregulation of hsa‐miR‐590‐3p and its target gene HMGB2 in PBMCs. Three gene expression profile datasets (GSE14795, GSE73953 and GSE25590) were downloaded from the GEO database. The DEGs (differentially expressed genes)‐miRNA network that was associated with IgAN was constructed by Cytoscape, and HMGB2 and hsa‐miR‐590‐3p were selected for further exploration. The dual‐luciferase reporter system was utilized to verify their interaction. Then, the expression levels of HMGB2 and hsa‐miR‐590‐3p in PBMCs were detected by qPCR in another cohort, and the correlation of their expression levels with the clinical pathological manifestations and serum Gd‐IgA1(galactose‐deficient IgA1) levels was also investigated. HMGB2 was identified as the target gene of hsa‐miR‐590‐3p. Furtherly, the elderly patients had higher HMGB2 expression levels than the expression levels of the younger patients. As the serum creatinine, serum BUN levels increased, the expression of HMGB2 decreased; Besides, the HMGB2 expression was positively correlated with serum complement 3(C3) levels, and it also had a negative correlation with the diastolic blood pressure, but not reach statistical significance. What is more, both hsa‐miR‐590‐3p and HMGB2 expression had a slight correlation tendency with serum Gd‐IgA1 levels in the whole population. In conclusion, HMGB2, the target gene of hsa‐miR‐590‐3p, was identified to correlate with the severity of IgAN, and this provides more clues for the pathogenesis of IgAN.     

Epigallocatechin‐3‐O‐gallate up‐regulates microRNA‐199a‐3p expression by down‐regulating the expression of cyclooxygenase‐2 in stimulated human osteoarthritis chondrocytes

Osteoarthritis (OA) is a most common form of arthritis worldwide leading to significant disability. MicroRNAs (miRNAs) are non‐coding RNAs involved in various aspects of cartilage development, homoeostasis and pathology. Several miRNAs have been identified which have shown to regulate expression of target genes relevant to OA pathogenesis such as matrix metalloproteinase (MMP)‐13, cyclooxygenase (COX)‐2, etc. Epigallocatechin‐3‐O‐gallate (EGCG), the most abundant and active polyphenol in green tea, has been reported to have anti‐arthritic effects, however, the role of EGCG in the regulation of miRNAs has not been investigated in OA. Here, we showed that EGCG inhibits COX‐2 mRNA/protein expression or prostaglandin E₂ (PGE₂) production via up‐regulating microRNA hsa‐miR‐199a‐3p expression in interleukin (IL)‐1β‐stimulated human OA chondrocytes. This negative co‐regulation of hsa‐miR‐199a‐3p and COX‐2 by EGCG was confirmed by transfection of OA chondrocytes with anti‐miR‐199a‐3p. Transfection of OA chondrocytes with anti‐miR‐199a‐3p significantly enhanced COX‐2 expression and PGE₂ production (P < 0.001), while EGCG treatment significantly inhibited anti‐miR‐199a‐3p transfection‐induced COX‐2 expression or PGE₂ production in a dose‐dependent manner. These results were further re‐validated by co‐treatment of these transfection OA chondrocytes with IL‐1β and EGCG. EGCG treatment consistently up‐regulated the IL‐1β‐decreased hsa‐miR‐199a‐3p expression (P < 0.05) and significantly inhibited the IL‐1β‐induced COX‐2 expression/PGE₂ production (P < 0.05) in OA chondrocytes transfected with anti‐hsa‐miR‐199a‐3p. Taken together, these results clearly indicate that EGCG inhibits COX‐2 expression/PGE₂ production via up‐regulation of hsa‐miR‐199a‐3p expression. These novel pharmacological actions of EGCG on IL‐1β‐stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG‐derived compounds inhibit cartilage breakdown or pain by up‐regulating the expression of microRNAs in human chondrocytes.     

Troxerutin downregulates C/EBP‐β gene expression via modulating the IFNγ‐ERK1/2 signaling pathway to ameliorate rotenone‐induced retinal neurodegeneration

Troxerutin, a natural flavonoid guards against oxidative stress and apoptosis with a high capability of passing through the blood‐brain barrier. Our aim was to investigate the role of troxerutin in experimentally induced retinal neurodegeneration by modulating the interferon‐gamma (IFNγ)‐extracellular signal‐regulated kinases 1/2 (ERK1/2)‐CCAAT enhancer‐binding protein β (C/EBP‐β) signaling pathway. Three groups of rats (10 each group) were included. Group I (control group), group II (rotenone treated group): the rats were injected subcutaneously with a single rotenone dosage of 3 mg/kg repeated every 48 hours for 60 days to trigger retinal neurodegeneration. Group III (troxerutin‐treated group): rats received troxerutin (150 mg/kg/day) by oral gavage 1 hour before rotenone administration. A real‐time polymerase chain reaction technique was applied to measure messenger RNA (mRNA) levels of retinal C/EBP‐β. Enzyme‐linked immunosorbent assay technique was utilized to assay tumor necrosis factor‐α (TNF‐α), IFNγ, and ERK1/2 levels. Finally, reactive oxygen species (ROS), as well as carbonylated protein (CP) levels, were assessed spectrophotometrically. Improved retinal neurodegeneration by downregulation of C/EBP‐β mRNA gene expression, also caused a significant reduction of TNF‐α, IFNγ, ERK1/2 as well as ROS and CP levels compared with the diseased group. These findings could hold promise for the usage of troxerutin as a protective agent against rotenone‐induced retinal neurodegeneration.