Growth Arrest And DNA Damage-inducible Gene 153 Research Articles

#245 Deficiency of GADD45α expression mediated tubulointerstitial injury in diabetic nephropathy

Abstract Background and Aims The tubulointerstitial injury plays crucial roles in the development of diabetic nephropathy (DN). Recently, some studies have reported that disorder of mitochondrial fatty acid β-oxidation in proximal tubules is involved in tubulointerstitial injury of DN. The growth arrest and DNA damage-inducible gene 45 (GADD45) family proteins, especially, GADD45α, plays a crucial role in various cellular processes, such as mitochondrial biogenesis and lipid metabolism. Recent studies have demonstrated that GADD45α facilitates gene activation by inducing DNA demethylation. Therefore, this study aimed to investigate the role of GADD45α in tubulointerstitial injury of DN and its underlying mechanisms. Methods Bioinformatics and machine learning techniques were employed to identify differentially expressed genes between healthy individuals and DN patients. Experiments were conducted by using high glucose-treated HK-2 cells and streptozotocin (STZ)-induced mice. Lentivirus carrying the GADD45α gene was used to induce GADD45α overexpression in HK-2 cells. Adeno-Associated Virus vectors carrying LoxP-flanked GADD45α were intravenously injected into γGT-Cre transgenic mice expressing Cre recombinase in renal proximal tubular cells, specifically leading to GADD45α overexpression in proximal tubules. Results Through bioinformatics and machine learning techniques, GADD45α was identified and found to exhibit a strong association with tubulointerstitial injury in DN. Further analysis revealed that the expression of GADD45α was decreased in the kidneys of DN patients, diabetic mice, and high glucose-treated HK-2 cells. Notably, GADD45α overexpression ameliorated kidney damage and mitochondrial injury, leading to improved fatty acid β-oxidation, restored ATP production, and reduced ROS production. Transcriptomics analyses further demonstrated that GADD45α overexpression enhanced the expression of genes related to mitochondrial biogenesis, dynamics, and electron transport chain, with six transmembrane epithelial antigen of prostate 4 (STEAP4) being a key protein in this process. Knocking down STEAP4 using siRNA in HK-2 cells exacerbated the damage induced by high glucose. In addition, our further results indicated that high glucose increased methylation of the STEAP4 promoter, while GADD45α overexpression decreased methylation and increased STEAP4 expression. Conclusions Deficiency of GADD45α expression mediated tubulointerstitial injury in DN, which was mainly through the decreased STEAP4 expression by demethylating CpG motifs in the STEAP4 promoter region, thereby resulting in reduced fatty acid oxidation, energy synthesis, and increased ROS production, and finally accelerated tubular injuries. These findings suggested that targeting the GADD45α-STEAP4 pathway could be a potential therapeutic strategy for DN.

Chloroquine attenuates chronic hypoxia-induced testicular damage via suppressing endoplasmic reticulum stress and apoptosis in experimental rat model.

Chronic hypoxia negatively affects male fertility by causing pathological changes in male reproductive system. However, underlying mechanisms of this damage are unknown. Chloroquine (CLQ) is an anti-inflammatory agent that is widely used in the treatment of inflammation-related diseases such as malaria and rheumatoid arthritis. This study aimed to investigate the therapeutic effects of CLQ in the hypoxia-induced testicular damage via assessment of hypoxic response, endoplasmic reticulum stress and apoptosis. For this purpose, 32 Wistar albino rats were divided into 4 groups as control (given 20%-21% O2 , no treatment), CLQ (given 50 mg/kg and 20%-21% O2 for 28 days), hypoxia (HX) (given 10% O2 for 28 days) and HX + CLQ (given 50 mg/kg and 10% O2 for 28 days). After the experiment, blood samples and testicular tissues were taken. Histopathological evaluation was performed on testicular tissues and hypoxia-inducible factor 1-α (HIF1-α), heat shock proteins (HSPs) HSP70, HSP90 and growth arrest and DNA damage-inducible gene 153 (GADD153) expression levels were detected via immunohistochemistry. Moreover, apoptotic cells were detected via terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining and serum testosterone levels were determined by enzyme-linked immunosorbent assay (ELISA) assay. Histopathological changes, apoptotic cell numbers and HIF1-α, HSP70, HSP90 and GADD153 expressions significantly increased in HX group (P < .05). Moreover, serum testosterone levels decreased in this group (P > .05). However, CLQ exerted a strong ameliorative effect on all parameters in HX + CLQ group. According to our results, we suggested that CLQ can be considered as an alternative protective agent for eliminating the negative effects of hypoxic conditions on male fertility.

The molecular mechanism of GADD153 in apoptosis of keloid fibroblasts exposed to botulinum toxin type A

Apoptosis plays a key role in keloids. Growth arrest and DNA damage‐inducible gene 153 (GADD153) is regulated by apoptosis. Botulinum toxin type A (BTXA) can induce apoptosis in keloid fibroblasts. This research aimed to explore the hypothesis that GADD153 mediates apoptosis in keloid fibroblasts exposed to BTXA. BTXA significantly induced GADD153 protein and mRNA expression in keloid fibroblasts. Treatment with c‐Jun N‐terminal kinase (JNK) inhibitor SP600125, JNK small interfering RNA (siRNA) and tumour necrosis factor‐alpha (TNF‐α) antibodies reversed the BTXA‐induced GADD153 expression. BTXA enhanced the transcriptional activity of GADD153, whereas the GADD153 mutant plasmid, JNK siRNA and anti‐TNF‐α antibody treatment abolished the BTXA‐induced transcriptional activity of GADD153. The addition of TNF‐α to keloid fibroblasts markedly increased GADD153 protein expression. The addition of GADD153 siRNA, SP600125 and anti‐TNF‐α antibodies reversed cell death and caspase 3 and 9 activity induced by BTXA.

Urtica dioica L. inhibits proliferation and enhances cisplatin cytotoxicity in NSCLC cells via Endoplasmic Reticulum-stress mediated apoptosis

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the ineffectiveness of the current therapies seriously limits the survival rate of NSCLC patients. In the search for new antitumor agents, nature has played a pivotal role providing a variety of molecules, which are likely to exert selective anti-tumour properties. Herein, we investigated the antiproliferative potential of Urtica dioica L. extract (UD) against NSCLC cell models with low sensitivity to cisplatin, a cytotoxic agent largely employed to cure NSCLCs. UD inhibited cell proliferation in the selected cells, while no toxic effects were observed in normal lung cells. Furthermore, the co-treatment of UD and cisplatin notably sensitised NSCLC cells to cisplatin. Mechanistically, we discovered that UD-promoted endoplasmic reticulum (ER) stress via activation of the growth arrest and DNA damage-inducible gene 153 (GADD153) triggering apoptosis. We also performed an extensive NMR analysis of UD, identifying rutin and oxylipins as the main secondary metabolites present in the mixture. Additionally, we discovered that an oxylipins’ enriched fraction contributes to the antiproliferative activity of the plant extract. In the future, this study may provide new chemical scaffolds for the design of anti-cancer agents that target NSCLCs with low sensitivity to cisplatinum.

Anti-tumor effects and mechanism of GA-13315, a novel gibberellin derivative, in human lung adenocarcinoma: an in vitro and in vivo study

ObjectiveTo investigate the anti-tumor effects and the mechanism of the compound 13-chlorine-3, 15-dioxy-gibberellic acid methyl ester (GA-13315) in lung adenocarcinoma in vitro and in vivo.MethodsThe antiproliferative effect of GA-13313 on the A549 cell line was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay. A xenograft model of A549 was established to evaluate the anti-tumor effect and histopathological examination was performed to assess the toxicity of GA-13315. Apoptosis was detected by TUNEL staining in tissues and flow cytometry in cells; activation of caspase-3, caspase-8 and caspase-9 was evaluated by immunohistochemical analysis; protein levels of Bcl-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), caspase-4, activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and growth arrest and DNA damage-inducible gene 153 (GADD153) were determined by western blotting. Mitochondrial membrane potential (MMP) was measured by the JC-1 fluorescence probe.ResultsOur results showed that GA-13315 exhibited potent, dose- and time-dependent anti-proliferative activity, and the IC50 values were 37.43 ± 2.73, 28.08 ± 7.76 and 19.29 ± 7.61 μM at 24, 48, and 72 h, respectively. The xenograft experiment revealed that tumor weight and volume were significantly decreased after GA-13315 3 mg/kg and 9 mg/kg (P < 0.05) treatment, and GA-13315 had low toxicity in bone marrow, kidney and colon tissues. GA-13315 triggered remarkable apoptosis in A549 cells at the concentration of 25.6 μM and 32 μM (P < 0.05) and activated caspase-3, − 8 and − 9. Moreover, GA-13315 induced apoptosis through the mitochondrial apoptosis pathway by elevating the Bax/Bcl-2 ratio, releasing cytochrome c and activating caspase-9 in A549 cells. In the endoplasmic reticulum apoptosis pathway, the levels of caspase-4, ATF4, GRP78 and GADD153 were markedly upregulated.ConclusionsThis study suggests that GA-13315 can be considered as a promising chemotherapeutic agent with anticancer activity in treatment of lung cancer in future.

Relationship between CHOP/GADD153 and unstable human carotid atherosclerotic plaque

Background and aims: The signaling protein C/EBP homologous protein (CHOP) and corresponding growth-arrest-and-DNA-damage-inducible gene 153 (GADD153) is associated with endoplasmic reticulum stress (ERS), which can lead to apoptosis. Our study aims to elucidate the role of CHOP/GADD153 in unstable atherosclerotic (AS) plaque formation isolated from confounding factors such as diabetes mellitus, primary hyperlipidemia, autoimmune deficiencies/abnormalities, essential hypertension, chronic heart failure, chronic kidney disease, and smoking.Material and methods: We collected carotid artery tissue samples from patients aged 50–80 years-old who received carotid endarterectomies (CEA) at our institution. We obtained fresh AS plaque samples during CEA and preserved the specimens immediately in the operating room with liquid nitrogen. Samples were categorized as stable or unstable AS plaques according to a six-stage histologic classification. CHOP/GADD153 expression was then examined with immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).Results: A total of 32 patients met our inclusion and exclusion criteria, with 24 (75.0%) classified as unstable lesions. The mean optical density ratio normalized to GAPDH for CHOP/GADD153 in stable and unstable groups was 0.357 ± 0.025 and 0.490 ± 0.027, respectively (p < .05). Positive immunostaining of CHOP/GADD153 was found in macrophages and smooth muscle cells of unstable AS plaques with a mean integrated optical density of 0.63 ± 0.03, compared to 0.17 ± 0.05 in the stable group (p < .05).Conclusions: In conclusion, we were able to show significant elevation of CHOP/GADD153 in unstable plaques independent of other confounding factors that induce ERS.

Down-regulation of histone deacetylase 4, −5 and −6 as a mechanism of synergistic enhancement of apoptosis in human lung cancer cells treated with the combination of a synthetic retinoid, Am80 and green tea catechin

(−)-Epigallocatechin gallate (EGCG), a green tea catechin, acts as a synergist with various anticancer drugs, including retinoids. Am80 is a synthetic retinoid with a different structure from all-trans-retinoic acid: Am80 is now clinically utilized as a new drug for relapsed and intractable acute promyelocytic leukemia patients. Our experiments showed that the combination of EGCG and Am80 synergistically induced both apoptosis in human lung cancer cell line PC-9 and up-regulated expressions of growth arrest and DNA damage-inducible gene 153 (GADD153), death receptor 5, and p21waf1 genes in the cells. To understand the mechanisms of synergistic anticancer activity of the combination, we gave special attention to the lysine acetylation of proteins. Proteomic analysis using nanoLC-ESI-MS/MS revealed that PC-9 cells treated with the combination contained 331 acetylated proteins, while nontreated cells contained 553 acetylated proteins, and 59 acetylated proteins were found in both groups. Among them, the combination increased acetylated-p53 and acetylated-α-tubulin through reduction of histone deacetylase (HDAC) activity in cytosol fraction, although the levels of acetylation in histones H3 or H4 did not change, and the combination reduced protein levels of HDAC4, −5 and −6 by 20% to 80%. Moreover, we found that a specific inhibitor of HDAC4 and −5 strongly induced p21waf1 gene expression, and that of HDAC6 induced both GADD153 and p21waf1 gene expression, which resulted in apoptosis. All results demonstrate that EGCG in combination with Am80 changes levels of acetylation in nonhistone proteins via down-regulation of HDAC4, −5 and −6 and stimulates apoptotic induction.

Atorvastatin alleviates cardiomyocyte apoptosis by suppressing TRB3 induced by acute myocardial infarction and hypoxia.

TRB3 (tribbles 3), an apoptosis-regulated gene, increases during endoplasmic reticulum stress. Hypoxia can induce inflammatory mediators and apoptosis in cardiomyocytes. However, the expression of TRB3 in cardiomyocyte apoptosis under hypoxia is not thoroughly known. We investigated the regulation mechanism of TRB3 expression and apoptosis induced by hypoxia in cardiomyocytes. An invivo model of acute myocardial infarction (AMI) was applied in adult Wistar rats to induce myocardial hypoxia. Rat neonatal cardiomyocytes were subjected to 2.5% O2 to induce hypoxia. The expression of TRB3 was evaluated in cultured rat neonatal cardiomyocytes subjected to hypoxia. Hypoxia significantly enhanced TRB3 protein and mRNA expression. Adding c-jun N-terminal kinase (JNK) inhibitor SP600125, JNK small interfering RNA (siRNA), tumor necrosis factor-α (TNF-α) antibody, and atorvastatin 30minutes before hypoxia reversed the induction of TRB3 protein. A gel-shift assay showed the DNA-binding activity of growth arrest and DNA damage-inducible gene 153 (GADD153), which increased after hypoxia. Hypoxia increased, whereas the TRB3-mut plasmid, SP600125, and TNF-α antibody abolished the hypoxia-induced TRB3 promoter activity. Hypoxia increased the secretion of TNF-α from cardiomyocytes. Exogenous administration of TNF-α recombinant protein to the cardiomyocytes without hypoxia increased TRB3 protein expression, similar to that observed after hypoxia. Hypoxia-induced cardiomyocyte apoptosis is inhibited by TRB3 siRNA, the TNF-α antibody, and atorvastatin. Atorvastatin reduced the TRB3 expression and cardiomyocyte apoptosis induced by AMI. Hypoxia induces TRB3 through TNF-α, JNK, and the GADD153 pathway. Treatment of atorvastatin inhibits the expression of TRB3 and cardiomyocyte apoptosis induced by AMI and hypoxia.

Abstract 4723: Innovative cancer treatment of human lung cancer cells PC-9 with a synthetic retinoid Am80 and EGCG via inhibition of HDAC4 and HDAC5

Abstract A synthetic new retinoid Am80 (tamibarotene) is used for treatment of acute promyelocytic leukemia (APL) patients with resistance of all-trans-retinoic acid (ATRA). To potentiate efficacy of Am80, we challenged combination treatment of Am80 and EGCG on human lung cancer cell line PC-9. It is now well accepted that EGCG acts as synergist with numerous anticancer compounds, and that the average reduction of tumor volume for in vivo genograft mouse models implanted using various human cancer cell lines was 70.3% with combinations of 13 anticancer compounds and EGCG. We previously reported that combinations of NSAIDs (sulindac and celecoxib) or other retinoids (ATRA, 13-cis-RA and 9-cis-RA) and EGCG synergistically induced apoptosis of the cells along with strong expression of growth arrest and DNA-damage inducible gene 153 (GADD153) and death receptor (DR5) gene. Our objective was to investigate the mechanism of the synergistic induction for GADD153-DR5 apoptosis pathway. The proteomic analysis of lysine-acetylated proteins was conducted with a shotgun analysis using nanoLC-ESI-MS/MS in PC-9 cells. We found that the numbers of acetylated proteins were different: 551 acetylated proteins were found in non-treated cells, 331 proteins in cells treated with combination, and 59 proteins were common in both cases. Acetylation was mainly found in non-histone proteins, chaperons, transcription factors, and enzymes. The combination increased acetylated p53 (Lys382) about 3-fold compared with non-treated cells. In addition, combination significantly decreased the protein amounts of histone deacetylase (HDAC) 4 and HDAC 5 to 50% and 20%, respectively, but not HDAC6 among Class II HDACs. Class I HDACs (HDAC1, 2, and 3) were not decreased. All the results suggest that combination of Am80 and EGCG induces GADD153-DR5 apoptotic pathway, and reduces the amounts of HDAC4 and HDAC5 resulting in changing protein acetylation. Citation Format: Masami Suganuma, Yukiko Oya, Sonthaya Umsumarng, Keisuke Iida, Anchalee Rawangkhan, Ryo Sakai, Hiroyuki Kagechika, Koichi Shudo, Hirota Fujiki. Innovative cancer treatment of human lung cancer cells PC-9 with a synthetic retinoid Am80 and EGCG via inhibition of HDAC4 and HDAC5. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4723.

Endoplasmic Reticulum Stress Instigates the Rotenone Induced Oxidative Apoptotic Neuronal Death: a Study in Rat Brain.

The present study was conducted to evaluate the involvement of endoplasmic reticulum stress in rotenone-induced oxidative neuronal death in rat brain. Rotenone (6μg/3μl) was administered intranigrally, unilaterally (right side) in SD rat brain. Neuronal morphology, expression level of tyrosine hydroxylase (TH) and endoplasmic reticulum (ER) stress markers like glucose-regulated protein 78 (GRP78), growth arrest and DNA damage-inducible gene 153 (GADD153), eukaryotic translation initiation factor 2α (p-eIF2α/eIF2α) and cleaved caspase-12 were estimated in the rat brain. Levels of reactive oxygen species (ROS), reduced glutathione (GSH) and enzymatic activities of glutathione peroxidase (GPx) and glutathione reductase (GRd) were estimated to assess the rotenone induced oxidative stress. Apoptotic death of neurons was assessed by estimating the mRNA level of caspase-3. Rotenone administration caused altered neuronal morphology, decreased expression of TH, augmented ROS level, decreased level of GSH and decreased activities of GPx and GRd enzymes which were significantly attenuated with the pretreatment of ER stress inhibitor, salubrinal (1mg/kg, intraperitoneal). Significantly increased levels of GRP78, GADD, dephosphorylated eIF2α and cleaved caspase-12 was also observed after rotenone administration, which was inhibited with the pretreatment of salubrinal. Rotenone-induced increased mRNA level of caspase-3 was also attenuated by pretreatment of salubrinal. Findings suggested that salubrinal treatment significantly inhibited the rotenone-induced neurotoxicity implicating that ER stress initiates the rotenone-induced oxidative stress and neuronal death.

Tauroursodeoxycholic acid suppresses endoplasmic reticulum stress in the chondrocytes of patients with osteoarthritis

The main pathogenic events in osteoarthritis (OA) include loss and abnormal remodeling of cartilage extracellular matrix. The present study aimed to evaluate the protective effect of tauroursodeoxycholic acid on chondrocyte apoptosis induced by endoplasmic reticulum (ER) stress. Articular cartilage tissues were collected from 18patients who underwent total knee arthroplasty and were analyzed histologically. Subsequently, chondrocyte apoptosis was assessed by TUNEL. Quantitative polymerase chain reaction and western blot analysis were employed to evaluate gene and protein expression, respectively, of ER stress markers, including glucose‑regulated protein78 (GRP78), growth arrest and DNA‑damage‑inducible gene153 (GADD153) and caspase‑12 along with typeII collagen. Chondrocytes obtained from osteoarthritis patients at different stages were cultured in three conditions including: No treatment (CON group), tunicamycin treatment to induce ER stress (ERS group) and tauroursodeoxycholic acid treatment after 4h of tunicamycin (TDA group); and cell proliferation, apoptosis, function and ER stress level were assessed. Degradation of cartilage resulted in histological damage with more apoptotic cartilage cells observed. Of note, GRP78, GADD153 and caspase‑12 mRNA and protein expression increased gradually from gradeI to III cartilage tissue, while typeII collagen expression decreased. Tunicamycin induced ER stress, as shown by a high expression of ER stress markers, reduced cell proliferation, increased apoptosis and decreased synthesis of typeII collagen. Notably, tauroursodeoxycholic acid treatment resulted in the improvement of tunicamycin‑induced ER stress. These results indicated that ER stress is highly involved in the tunicamycin‑induced apoptosis in chondrocytes, which can be prevented by tauroursodeoxycholic acid.

Misregulation of polo-like protein kinase 1, P53 and P21WAF1 in epithelial ovarian cancer suggests poor prognosis.

Polo-like protein kinase 1 (PLK1), P53 and P21WAF1 are relevant to cell cycle checkpoints and cancer biology. Misregulation of PLK1, P53 and P21WAF1 has been detected in several types of malignant tumors. The present study aimed to clarify the role of PLK1, P53 and P21WAF1 in the prognosis of ovarian cancer. PLK1 and P53 shRNA lentiviral plasmids were transfected into SK-OV-3 cells, respectively. Cell proliferation, apoptosis and invasion were examined by MTT assay, flow cytometry and Matrigel assay, respectively. Survival time of the animals was observed in a xenograft model. Expression levels of PLK1, P53 and P21WAF1 were detected in different ovarian tissues by immunohistochemistry and western blot analysis. Their correlations to the clinicopathologic characteristics of the epithelial ovarian cancer (EOC) cases and their interrelationships were analyzed. Risk factors of prognosis for EOC were determined by logistic regression analysis. The survival time of EOC patients was measured by Kaplan-Meier analysis. After PLK1 or P53 knockdown, proliferation of the SK-OV-3 cells was inhibited, the apoptosis rate was increased, and cell invasion was suppressed in vitro, and the survival time was prolonged in the animals. Expression levels of P53, p-P53 (Ser15), P21WAF1, growth arrest and DNA damage‑inducible gene 45 (GADD45) and 14-3-3σ were upregulated in the SK-OV-3 cells after PLK1 knockdown, but downregulated after P53 knockdown. Higher expression levels of PLK1 and P53 were observed in patients with a higher FIGO stage and worse histological differentiation, but lower P21WAF1 was noted at a higher FIGO stage. Negative correlations were observed between expression of PLK1 and P53 and P53 and P21WAF1 in the EOC cases. PLK1, P53 and P21WAF1 could be used to assess the prognosis of EOC, respectively, but only PLK1 was found to be an independent prognostic factor. The overall survival time of subjects exhibiting PLK1-positive/P53-positive expression and PLK1-positive/P21WAF1-negative expression was obviously shorter than the other patient groups at the end of the follow-up. These results indicate that PLK1 is implicated in ovarian carcinogenesis and may owe its ability to inhibition of the activity of P53. In addition, misregulation of PLK1 coincident with P53 and P21WAF1 in EOC suggests poor prognosis.

Effect of electro-acupuncture pretreatment on endoplasmic reticulum stress during global cerebral ischemia-reperfusion in rats

Objective To evaluate the effect of electro-acupuncture pretreatment on endoplasmic reticulum stress during global cerebral ischemia-reperfusion （I/R ） in rats .Methods One hundred and forty-four adult male Sprague-Dawley rats ,weighing 400-500 g ,were randomly divided into 3 groups （ n=48 each） using a random number table：sham operation group （group S ） , I/R group and electro-acupuncture pretreatment group （group EA） .Global cerebral I/R was induced by 4-vessel occlusion method .Bilateral vertebral arteries were permanently occluded by cauterization and bilateral carotid arteries were occluded for 5 min , followed by reperfusion .Electro-acupuncture of Dazhui and Baihui acupoints lasting for 30 min was performed once a day for 5 consecutive days starting from 5 days before ischemia in group EA .At 6 ,12 ,24 and 48 h of reperfusion ,12 rats were sacrificed in each group and hippocampi were removed for microscopic examination and for determination of apoptosis rate （by TUNEL ） and expression of growth arrest-and DNA damage-inducible gene 153 （GADD153 ） protein （by Western blot ） .Results Compared with group S ,the apoptosis rate was significantly increased and the expression of GADD153 protein was up-regulated at 6 ,12 ,24 and 48 h of reperfusion in I/R and EA groups （ P〈0.05） .Compared with group I/R ,the apoptosis rate was significantly decreased and the expression of GADD 153 protein was down-regulated at 6 ,12 ,24 and 48 h of reperfusion in group EA （ P〈 0.05） .The pathological changes were significantly attenuated in group EA as compared with group I/R .Conclusion Electro-acupuncture pretreatment can down-regulate the expression of GADD 153 protein , reduce endoplasmic reticulum stress , and decrease cell apoptosis ,thus attenuating global cerebral I/R injury in rats . Key words: Electric stimulation therapy; Reperfusion injury; Brain; Endoplasmic reticulum

Clinical significance of GADD153 expression in stage I non-small cell lung cancer

The transcription factor growth arrest and DNA damage-inducible gene 153 (GADD153), also known as CHOP, is considered to function as a proapoptotic molecule. Overexpression of GADD153 leads to cell cycle arrest and/or apoptosis. However, its clinical implications in non-small cell lung cancer (NSCLC) remain controversial. Therefore, we investigated the expression of GADD153 in stage I NSCLC using immunohistochemistry. Paraffin-embedded tissue sections from 76 patients, who were diagnosed with primary stage I NSCLC and had undergone a curative lung resection, were stained using an anti-GADD153 antibody. The intensity of GADD153 immunostaining was evaluated within the cell membrane and cytoplasm of invasive cancer components. The correlation between the intratumoral expression of GADD153 and various clinical parameters were explored. GADD153 was detected in 29 (38.2%) cases. No statistically significant difference in expression was demonstrated between stage IA and stage IB tumors (35.0 vs. 39.3%; P=0.735). The expression of GADD153 was not affected by histological subtypes or histological grades of differentiation. The intratumoral expression of GADD153 did not influence the overall survival rate (53.29 vs. 52.18 months; P=0.743) or disease-free survival rate (46.97 vs. 54.19 months; P=0.084) of stage I NSCLC patients. However, patients with GADD153 expression demonstrated an improved disease-specific survival rate (28.80 vs. 53.85 months; P=0.020). No patients with GADD153 expression demonstrated distant metastasis (P=0.029). These data suggest that GADD153 expression may be a valuable prognostic factor of early-stage NSCLC in patients who have undergone curative lung resection.

Activation of PKR/eIF2α signaling cascade is associated with dihydrotestosterone‐induced cell cycle arrest and apoptosis in human liver cells

Androgen receptor (AR) signaling plays an important role in the development and progression of several liver diseases, including hepatocellular carcinoma (HCC) and non-alcoholic fatty liver disease (NAFLD). Dihydrotestosterone (DHT) is the active metabolite of the major circulating androgen, testosterone. In this study, we investigated the effect of DHT on human liver cells. We found that DHT not only induces cell cycle arrest but also initiates apoptosis in androgen-sensitive liver cells, such as SMMC-7721 and L02. Importantly, DHT/AR induces the activation of RNA-dependent protein kinase (PKR)/eukaryotic initiation factor-2 alpha (eIF2α) cascades in androgen-sensitive liver cells. PKR/eIF2α activation-induced growth arrest and DNA damage-inducible gene 153 (GADD153) and heat shock protein 27 (Hsp27) expression contribute to cell cycle arrest in response to DHT. It is notable that DHT administration results in androgen-sensitive liver cells apoptosis, at least in part, through PKR/eIF2α/GADD153 cascades. These results suggest that the androgen/AR pathway plays a pivotal role in liver cell growth and apoptosis regulating, whose deregulation might be involved in the pathogenesis of liver diseases.

Pathways of macrophage apoptosis within the interface membrane in aseptic loosening of prostheses

Aseptic loosening is a major cause of failure of total hip arthroplasty (THA). Macrophage apoptosis in interface membrane has been proved to play an important role in the pathogenesis of aseptic loosening. The purpose of current study was to identify the apoptotic mechanism of macrophages in the interface membrane of aseptic loosening. We collected periprosthetic interface membrane from 23 patients undergoing the revision operations for aseptic loosening of hip joint prostheses. To serve as the control group, samples of capsule were collected from 18 patients undergoing the primary hip arthroplasties for osteoarthritis (OA). The ultrastructure of interface membrane was examined by transmission electron microscopy (TEM), and in situ apoptotic macrophage identification was performed by TUNEL staining. Furthermore, using immunohistochemical methods we investigated the expression of some apoptosis-related markers such as inducible nitric oxide synthase (iNOS), peroxynitrite (ONOO −), cleaved caspase-3/4/8/9, cytochrome c, glucose regulated protein 78 (GRP78), and growth arrest and DNA damage-inducible gene 153 (GADD153) in macrophages. These markers were regarded as apoptotic inducers or specific indicators of different apoptotic pathways such as death receptor pathway, mitochondrial pathway and endoplasmic reticulum (ER) stress pathway. TEM showed that a great deal of wear debris was phagocytosed by macrophages, which displayed morphological changes characteristic of apoptosis. The results of TUNEL staining demonstrated that there were more apoptotic macrophages in interface membrane. The expression levels of iNOS, ONOO −, cleaved caspase-3/4/8/9, cytochrome c, GRP78 and GADD153 in macrophages in interface membrane were significantly higher than those in the control samples ( p < 0.05). Our results suggest that death receptor pathway, mitochondria/cytochrosome c caspase-dependent pathway and ER stress pathway are involved in the process of macrophage apoptosis. A therapeutic target to modulate the apoptotic pathways in macrophages may be a strategy to prevent and treat aseptic loosening.

Is there a common upstream link for autophagic and apoptotic cell death in human high-grade gliomas?

The prognosis of patients with human high-grade gliomas (HGGs) remains dismal despite major advances in their management, due mainly to the high resistance of these infiltrative tumor cells to programmed cell death (PCD). Most therapeutic strategies for HGGs are aimed to maximize PCD type I, apoptosis or type II, autophagy. These are predominantly distinctive processes, but many studies suggest a cross-talk between the two. A better understanding of the link between PCD types I and II might allow development of more effective therapies for HGGs. In this study, we examined whether there is a common upstream signaling event responsible for both apoptotic and autophagic PCD using 3 chemotherapeutic agents in human HGG cells. Our study shows that each agent caused a significant decrease in cell viability in each of the HGG cell lines tested. The increase rate of apoptosis and autophagy varied among cell lines and chemotherapeutic agents used. Increased expression of cytidine-cytidine-adenosine-adenosine-thymidine (C)/enhancer binding protein (EBP) homologous transcription factor C/EBP homologous protein (CHOP)/growth arrest and DNA damage-inducible gene 153 (GADD153) was documented after use of either pro-autophagic or pro-apoptotic agents. The involvement of CHOP/GADD153 in both type I and type II PCD was confirmed by overexpression and gene-silencing studies. Gene silencing by small-interfering RNA-mediated CHOP/GADD153 resulted in increased cell viability, decreased upregulation of microtubule-associated protein light-chain 3' type II (LC3II) and cleaved caspase-3, and inhibition of apoptosis and autophagy. Exogenous expression of CHOP/GADD153 triggered apoptosis and autophagy in the absence of other stimuli. The clinical significance of these findings was supported by the evidence that celecoxib, a nonsteroidal anti-inflammatory drug known to induce GADD153-mediated apoptosis, strongly increases both type I and type II PCD in HGG cells when combined with another inducer of GADD153. These data suggest that CHOP/GADD153 should be investigated as a novel targetable signaling step to improve therapies for HGGs.

Garcinol inhibits cell growth in hepatocellular carcinoma Hep3B cells through induction of ROS‐dependent apoptosis

Garcinol, derived from Garcinia indica and other related species, has been found to modulate several cell signalling pathways involved in apoptosis and cancer development. Growth arrest and DNA damage‐inducible gene 153 (GADD153) is a member of the CCAAT/enhancer‐binding protein (C/EBP) family of transcription factors; it is expressed at low levels under normal conditions but strongly induced upon growth arrest, DNA damage, and endoplasmic reticulum (ER) stress. This study investigated the effect of garcinol on Hep3B cells, a human hepatocellular cancer cell line lacking functional p53, with the goal of elucidating the molecular mechanisms of p53‐independent apoptosis in hepatocellular cancer. Overall, garcinol activated not only the death receptor and the mitochondrial apoptosis pathways but also the ER stress modulator GADD153. Garcinol treatment led to the accumulation of reactive oxygen species (ROS), increased GADD153 expression, and reduced mitochondrial membrane potential. An increase in the Bax/Bcl‐2 ratio resulted in enhanced apoptosis. Our data suggest a promising therapeutic application of garcinol in p53‐independent apoptosis in cancers.

Precursor of Advanced Glycation End Products Mediates ER-Stress-Induced Caspase-3 Activation of Human Dermal Fibroblasts through NAD(P)H Oxidase 4

BackgroundThe precursor for advanced glycation end products, 3-deoxyglucosone (3DG) is highly upregulated in skin explants of diabetic cutaneous wounds, and has been shown to negatively impact dermal fibroblasts, which are crucial in wound remodeling. 3DG induces apoptosis however; the mechanisms involved in the apoptotic action of 3DG in the pathogenesis of diabetic chronic wounds are poorly understood. Therefore, we sought to delineate novel mechanisms involved with the 3DG-collagen induced apoptosis.Methodology/Principal FindingsUsing human dermal fibroblasts, we demonstrated that 3DG-modified collagen induces oxidative stress and caspase-3 activation. Oxidative stress was found to be dependent on the upregulation of NAD(P)H oxidase 4 (Nox4), a reactive oxygen species (ROS) Nox homologue, triggering endoplasmic reticulum (ER) stress, as assessed by the ER stress-induced apoptosis marker Growth Arrest and DNA Damage-inducible gene 153 (GADD153). We demonstrated that 3DG-collagen activated GADD153 via phosphorylation of p38 mitogen activated protein kinase (MAPK), and this was dependent on upstream ROS. Inhibition of ROS and/or p38 MAPK abrogated 3DG-collagen induced caspase-3 activation. Our investigations also demonstrated that 3DG-collagen-induced caspase-3 activation did not signal through the canonical receptor for advanced glycation end products (RAGE) but through integrin α1β1. To further verify the role of integrins, neutralization of integrins α1β1 prevented 3DG-collagen-induced upregulation of ROS, GADD153, and caspase-3 activation; suggesting that 3DG-collagen signaling to the fibroblast is dependent on integrins α1β1.Conclusions/SignificanceTaken together, these findings demonstrate for the first time that a RAGE independent mechanism is involved in 3DG-collagen-induced apoptosis. Moreover, the ER stress pathway through activation of Nox4 by integrins α1β1 plays a key role in 3DG-collagen-induced caspase-3 activation, which may play an important role in the pathogenesis of diabetic wounds.

Garcinol inhibits cell growth in hepatocellular carcinoma Hep3B cells through induction of ROS-dependent apoptosis

Garcinol, derived from Garcinia indica and other related species, has been found to modulate several cell signalling pathways involved in apoptosis and cancer development. Growth arrest and DNA damage-inducible gene 153 (GADD153) is a member of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors; it is expressed at low levels under normal conditions but strongly induced upon growth arrest, DNA damage, and endoplasmic reticulum (ER) stress. This study investigated the effect of garcinol on Hep3B cells, a human hepatocellular cancer cell line lacking functional p53, with the goal of elucidating the molecular mechanisms of p53-independent apoptosis in hepatocellular cancer. Overall, garcinol activated not only the death receptor and the mitochondrial apoptosis pathways but also the ER stress modulator GADD153. Garcinol treatment led to the accumulation of reactive oxygen species (ROS), increased GADD153 expression, and reduced mitochondrial membrane potential. An increase in the Bax/Bcl-2 ratio resulted in enhanced apoptosis. Caspase-8 and tBid (truncated Bid) expression also increased in a time-dependent manner. The enzymatic activities of caspase-3 and caspase-9 increased approximately 13-fold and 7.8-fold, respectively. In addition, the proteolytic cleavage of poly-(ADP-ribose)-polymerase (PARP) and DNA fragmentation factor-45 (DFF-45) increased in dose- and time-dependent manners. Our data suggest a promising therapeutic application of garcinol in p53-independent apoptosis in cancers.