Growth Arrest DNA Damage-inducible Gene Research Articles

MicroRNA-128-1-5p attenuates myocardial ischemia/reperfusion injury by suppressing Gadd45g-mediated apoptotic signaling

Myocardial ischemia/reperfusion (I/R) injury is a clinically fatal disease, caused by restoring myocardial blood supply after a period of ischemia or hypoxia. However, the underlying mechanism remains unclear. Recently, increasing evidence reveal that microRNAs (miRs) participate in myocardial I/R injury. This study aimed to investigate whether miR-128-1-5p contributed to cardiomyocyte apoptosis induced by myocardial I/R injury. Here, we showed that the expression of miR-128-1-5p was decreased in mice following myocardial I/R injury. Down-regulation of miR-128-1-5p was also showed in H9c2 cardiomyocytes after hypoxia/reoxygenation (H/R), and in neonatal rat cardiomyocytes (NRCMs) with H2O2 treatment. Importantly, we found that overexpression of miR-128-1-5p ameliorates cardiomyocyte apoptosis both in H9c2 cardiomyocytes and NRCMs. Moreover, we also found that growth arrest DNA damage-inducible gene 45 gamma (Gadd45g) is identified as a direct target of miR-128-1-5p, which negatively regulated Gadd45g expression. Additionally, silencing of Gadd45g inhibits cardiomyocyte apoptosis in H9c2 cardiomyocytes and NRCMs. These results reveal a novel mechanism by which miR-128-1-5p regulates Gadd45g-mediated cardiomyocyte apoptosis in myocardial I/R injury.

GADD45B as a Prognostic and Predictive Biomarker in Stage II Colorectal Cancer.

GADD45B acts as a member of the growth arrest DNA damage-inducible gene family, which has demonstrated to play critical roles in DNA damage repair, cell growth, and apoptosis. This study aimed to explore the potential relationship between GADD45B expression and tumor progression and evaluate the clinical value of GADD45B in stage II colorectal cancer (CRC). The expression patterns and prognostic value of GADD45B in CRC were analyzed based on The Cancer Genomic Atlas (TCGA). GADD45B expression features of 306 patients with stage II CRC and 201 patients with liver metastasis of CRC were investigated using immunochemical staining on tissue microarrays. Afterward, survival analysis and stratification analysis were performed in stage II to explore the prognostic and predictive significance of GADD45B. Overexpressed GADD45B is associated with poorer prognosis for CRC patients both in overall survival (OS) (p < 0.001) and disease-free survival (DFS) (p = 0.001) based on the TCGA database. Analysis results according to the stage II CRC cohort and the liver metastatic CRC cohort revealed that GADD45B was gradually upregulated in normal mucosa including primary colorectal cancer (PCC). Colorectal liver metastases (CLM) tissues were arranged in order (normal tissue vs. PCC p = 0.005 and PCC vs. CLM p = 0.001). The low GADD45B group had a significantly longer five-year OS (p = 0.001) and progression-free survival (PFS) (p < 0.001) than the high GADD45B group for the stage II patients. The multivariate Cox regression analysis results proved that the expression level of GADD45B was an independent prognostic factor for stage II after radical surgery (OS: Hazard Ratio (HR) 0.479, [95% confidence interval (CI) 0.305–0.753] and PFS:HR 0.490, [95% CI 0.336–0.714]). In high GADD45B expression subgroup of stage II cohort, the patients who underwent adjuvant chemotherapy had longer PFS than those who did not (p = 0.008). High expression levels of GADD45B is an independent prognostic factor of decreased OS and PFS in stage II CRC patients. The stage II CRC patients with high GADD45B expression might benefit from adjuvant chemotherapy.

MicroRNA-206 Protects against Myocardial Ischaemia-Reperfusion Injury in Rats by Targeting Gadd45β.

MicroRNAs are widely involved in the pathogenesis of cardiovascular diseases through regulating gene expression via translational inhibition or degradation of their target mRNAs. Recent studies have indicated a critical role of microRNA-206 in myocardial ischaemia-reperfusion (I/R) injury. However, the function of miR-206 in myocardial I/R injury is currently unclear. The present study was aimed to identify the specific role of miR-206 in myocardial I/R injury and explore the underlying molecular mechanism. Our results revealed that the expression level of miR-206 was significantly decreased both in rat I/R group and H9c2 cells subjected to hypoxia/reoxygenation (H/R) compared with the corresponding control. Overexpression of miR-206 observably decreased infarct size and inhibited the cardiomyocyte apoptosis induced by I/R injury. Furthermore, bioinformatics analysis, luciferase activity and western blot assay proved that Gadd45β (growth arrest DNA damage-inducible gene 45β) was a direct target gene of miR-206. In addition, the expression of pro-apoptotic-related genes, such as p53, Bax and cleaved caspase3, was decreased in association with the down-regulation of Gadd45β. In summary, this study demonstrates that miR-206 could protect against myocardial I/R injury by targeting Gadd45β.

Methylation mediated Gadd45\u03b2 enhanced the chemosensitivity of hepatocellular carcinoma by inhibiting the stemness of liver cancer cells

BackgroundDefects of the growth arrest DNA damage-inducible gene 45β (Gadd45β) play an important role in the progression of tumor and confer resistance to chemotherapy. However, the role of Gadd45β in the apoptosis of hepatocellular carcinoma is still not clear. Purpose of this study was to explore the effect of Gadd45β on the apoptosis of liver cancer cells, and the possible mechanism was examined.ResultIn this study, we first confirmed the decreased expression of Gadd45β in human liver cancer tissues and human liver cancer cell lines, when compared to the peri-tumor liver tissue and normal liver cells. And, it was found that Gadd45β could inhibit the stemness of liver cancer cells, enhancing the apoptosis of cancer cells induced by chemotherapy. Furthermore, the results showed that HCC tissues and cell lines showed a higher methylation status in Gadd45β promoter than that in peri-tumor tissues and normal liver cells. Methylation was then reversed by pretreatment of SMMC-7721 and Hep-3B with 5-azacytidine which is the DNA methyltransferase inhibitor. And the 5-azacytidine decreased the stemness of SMMC-7721 and Hep-3B, enhanced the sensitivity of SMMC-7721 and Hep-3B to cisplatin.ConclusionsMethylation mediated Gadd45β expression inhibited the stemness of liver cancer cells, promoting the chemotherapy-induced apoptosis. Thus Gadd45β may be the potential target for enhancing the chemosensitivity of human hepatocellular carcinoma.

Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs Target GADD45B To Protect Infected Cells from Cell Cycle Arrest and Apoptosis.

Kaposi's sarcoma-associated herpesvirus is a leading cause of cancers in individuals with AIDS. Promoting survival of infected cells is essential for maintaining viral infections. A virus needs to combat various cellular defense mechanisms designed to eradicate the viral infection. One such response can include DNA damage response factors, which can promote an arrest in cell growth and trigger cell death. We used a new approach to search for human genes repressed by small nucleic acids (microRNAs) expressed by a gammaherpesvirus (KSHV), which identified a gene called GADD45B as a target of microRNAs. Repression of GADD45B, which is expressed in response to DNA damage, benefited survival of infected cells in response to a DNA damage response. This information could be used to design new treatments for herpesvirus infections.

GADD45β induction by S-adenosylmethionine inhibits hepatocellular carcinoma cell proliferation during acute ischemia-hypoxia.

Growth arrest DNA damage-inducible gene 45β (GADD45β), which influences cell growth, apoptosis and cellular response to DNA damage, is downregulated in hepatocellular carcinoma (HCC). S-adenosylmethionine (SAMe) serves as an essential methyl donor in multiple metabolic pathways and is a polyamine and glutathione (GSH) precursor. In this study, we assessed the roles of GADD45β and SAMe in cell survival during acute ischemia-hypoxia (I/H). SAMe treatment induced growth of HL-7702 normal hepatic cells, but decreased the viability of HepG2 (p53 wild-type) and Hep3B (p53 null) HCC cells. Cells were exposed to I/H with or without SAMe pre-treatment. I/H exposure alone triggered HCC cell proliferation promoted by autophagy. SAMe pre-treatment restored GADD45β expression and activated HCC cell apoptosis and eliminated I/H-induced HCC cell proliferation. p53 loss blunted the response to SAMe and I/H exposure in Hep3B cells; thus, the inhibitory effect of SAMe on cell proliferation may be reduced in p53-null cells as compared to wild-type cells. These results indicate that GADD45β induction by SAMe inhibits HCC cell proliferation during I/H as a result of increased apoptosis, and that SAMe also protects normal hepatocytes from apoptotic cell death and promotes normal cell regeneration. SAMe should be considered a potential therapeutic agent for the management of HCC.

Multiple functions of the first EGF domain in matrilin-3: Secretion and endoplasmic reticulum stress.

Mutations in matrilin-3 are associated with common skeletal diseases, such as hand osteoarthritis (HOA), as well as rare chondrodysplasias, such as multiple epiphyseal dysplasia (MED) and spondyloepimetaphyseal dysplasia (SEMD). In the present study, we constructed the mutations R116W [at the von Willebrand factor, type A (vWFA) domain], T298M [at the first epidermal growth factor (EGF) domain] and C299S (at the first EGF domain), according to the mouse sequence, which are associated with human MED, HOA and SEMD, respectively, by overlap extension PCR and inserted them into an expression vector (pcDNA3.1/v5-His). We transfected these contructs into the COS-1 or MCT cells, and the results revealed that the HOA-related matrilin-3 mutation (T298M) leads to a high expression level of growth arrest DNA damage-inducible gene 153 (GADD153, also known as CHOP; an endoplasmic reticulum stress marker), as shown by western blot analysis and does not significantly affect protein secretion, as shown by immunofluorescence staining; however, osteochondroplasia, i.e., MED-related (R116W) and SEMD-related (C299S) mutations lead to both high levels of GADD153 expression and protein trafficking into the cytoplasm and form multiple vacuoles in cells, which in turn leads to insufficient protein secretion.

Growth arrest DNA damage-inducible gene 45 gamma expression as a prognostic and predictive biomarker in hepatocellular carcinoma.

Growth arrest DNA damage-inducible gene 45 (GADD45) family proteins play a crucial role in regulating cellular stress responses and apoptosis. The present study explored the prognostic and predictive role of GADD45γ in hepatocellular carcinoma (HCC) treatment. GADD45γ expression in HCC cells was examined using quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. The control of GADD45γ transcription was examined using a luciferase reporter assay and chromatin immunoprecipitation. The in vivo induction of GADD45γ was performed using adenoviral transfer. The expression of GADD45γ in HCC tumor tissues from patients who had undergone curative resection was measured using qRT-PCR. Sorafenib induced expression of GADD45γ mRNA and protein, independent of its RAF kinase inhibitor activity. GADD45γ induction was more prominent in sorafenib-sensitive HCC cells (Huh-7 and HepG2, IC50 6-7 μM) than in sorafenib-resistant HCC cells (Hep3B, Huh-7R, and HepG2R, IC50 12-15 μM). Overexpression of GADD45γ reversed sorafenib resistance in vitro and in vivo, whereas GADD45γ expression knockdown by using siRNA partially abrogated the proapoptotic effects of sorafenib on sorafenib-sensitive cells. Overexpression of survivin in HCC cells abolished the antitumor enhancement between GADD45γ overexpression and sorafenib treatment, suggesting that survivin is a crucial mediator of antitumor effects of GADD45γ. GADD45γ expression decreased in tumors from patients with HCC who had undergone curative surgery, and low GADD45γ expression was an independent prognostic factor for poor survival, in addition to old age and vascular invasion. The preceding data indicate that GADD45γ suppression is a poor prognostic factor in patients with HCC and may help predict sorafenib efficacy in HCC.

Abstract 2522: Development of GADD45β (growth arrest DNA damage-inducible gene 45 beta) agonists for the treatment of hepatocellular carcinoma (HCC)

Abstract Induction of GADD45β expression, which is associated with cellular stress response and apoptosis regulation, in HCC cells was found to correlate with sensitivity of sorafenib, the standard molecular targeted therapy for advanced HCC. In this study, we use this GADD45β induction as a screening platform to identify novel agents for HCC treatment from sorafenib derivatives that lack RAF kinase inhibition activity. GADD45β expression in Huh-7 cells was measured by quantitative RT-PCR. Cell viability was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was examined by flow cytometry. Signal transduction pathways in cells were assessed by western blotting. In vivo anti-tumor efficacy was tested in mouse xenograft models using Huh7 and Huh7R cells, the latter had acquired resistance to sorafenib. Pharmacokinetic studies were done in Sprague-Dawley rat model. The compound SC-20, which had a N-(4-(quinolin-4-yloxy)phenyl)benzamide scaffold, showed greater GADD45β induction than other derivatives. The nitro group on the quinolone ring of SC-20 was considered important in GADD45β induction. SC-20 induced more prominent apoptosis and inhibition of proliferation in Huh7 cells than sorafenib, independent of MEK/ERK signaling activity in Huh7 cells. SC-20 showed more prominent GADD45β induction and better antitumor activity than sorafenib in both Huh-7 and Huh7R xenograft models and good safety. Pharmacokinetic studies indicated that SC-20 had long plasma half-life (22.5 ± 0.51 hours), low water solubility, high (&amp;gt; 99%) plasma protein binding, and inhibited CYP2C9 and 2C19. GADD45β agonists warrant further development for HCC treatment. (supported by NSC 101-2325-B-002 -039, NSC 101-2321-B-002 -014, NSC 102-2325-B-002 -038) Citation Format: Chiun Hsu, Da-Liang Ou, Kuen-Feng Chen, Zhong-Zhe Lin, Ann-Lii Cheng, Chung-Wai Shiau. Development of GADD45β (growth arrest DNA damage-inducible gene 45 beta) agonists for the treatment of hepatocellular carcinoma (HCC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2522. doi:10.1158/1538-7445.AM2014-2522

Decreased expression and aberrant methylation of Gadd45G is associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma

The growth arrest DNA damage-inducible gene (Gadd45) family, which is composed of Gadd45A, Gadd45B, and Gadd45G, is involved in DNA damage response and cell growth arrest. The present study was to detect the role of Gadd45 gene family in esophageal cancer and the relationship of Gadd45G methylation to a series of pathological parameters in a large esophageal squamous cell carcinoma (ESCC) sample, in order to elucidate more information on the role of Gadd45 gene family with regard to the pathogenesis of ESCC. Frequent silencing of Gadd45G but not Gadd45A and Gadd45B were found in esophageal cancer cell lines and the silencing of Gadd45G may be reversed by 5-Aza-dC or TSA treatment in Eca109 cell line. The aberrant proximal promoter methylation of Gadd45G induces silencing of Gadd45G expression in Eca109 cell line. Gadd45A mRNA and protein expression in ESCC tumor tissues was significantly different compared to corresponding normal tissues. Decreased mRNA and protein expression of Gadd45G was observed in ESCC tumor tissues and was associated with Gadd45G proximal promoter methylation. Gadd45A or Gadd45B expression was not correlated with ESCC patients survival, while Gadd45G methylation status and protein expression were independently associated with ESCC patients' survival. These data indicated that Gadd45G may be a functional tumor suppressor and its inactivation through proximal promoter methylation may play an important role in ESCC carcinogenesis and reactivation of Gadd45G gene may has therapeutic potential and may be used as a prognostic marker for ESCC patients.

Methylation‐mediated repression of GADD45A and GADD45G expression in gastric cardia adenocarcinoma

The growth arrest DNA damage-inducible gene (GADD45) family, which is composed of GADD45A, GADD45B, and GADD45G, may play similar but not identical roles in tumorigenesis. Genetic changes associated with or responsible for their dysregulation are in general uncommon. This study was to detect the role of GADD45 gene family in gastric cardia adenocarcinoma (GCA) and the relationship of GADD45A and GADD45G methylation to a series of pathological parameters in a large GCA sample, in order to elucidate more information on the role of GADD45 gene family with regard to the pathogenesis of GCA. Decreased mRNA and protein expression of GADD45A and GADD45G but not GADD45B were found in 138 GCA tumor tissues. The methylation frequency of 5' 4 CpG region located in distal promoter of GADD45A and proximal promoter of GADD45G in GCA tumor tissues was significantly higher than that in corresponding normal tissues. The expression levels of GADD45A and GADD45G were inversely correlated with methylation levels. GADD45B expression was not correlated with GCA patients survival, while GADD45A and GADD45G methylation status and protein expression were independently associated with GCA patients' survival. These results suggest that GADD45A and GADD45G gene may act as functional tumor suppressor but being frequently inactivated epigenetically in patients with GCA. Silencing of GADD45A and GADD45G, negative regulator of cell growth, is most likely responsible for conferring a selective growth advantage during GCA evolution and outgrowth.

Abnormal expression of GADD45B in human colorectal carcinoma

BackgroundGADD45B is a member of the growth arrest DNA damage-inducible gene family associated with cell growth control, apoptosis, and DNA damage repair response. The aim of this study is to detect the role of GADD45B in colorectal carcinoma (CRC); the area not studied in depth to date.MethodsThe mRNA and protein levels of GADD45B were examined by Real-Time quantitative PCR (RT-qPCR) and immunohistochemistry (IHC) in CRC tissues and adjacent noncancerous tissues (ANCT). Over-expression plasmids and SiRNA were used to regulate GADD45B expression in CRC cell lines in vitro and flow cytometry and Western blotting were used to detect apoptotic changes.ResultsThe mRNA and protein levels of GADD45B were significantly higher in CRC tissues than those in ANCT (P<0.05). Up-regulation of GADD45B was also correlated with relapse and death of CRC patients (P<0.05). The Kaplan-Meier survival curves indicated that disease-free survival (DFS) was significantly worse in CRC patients who showed GADD45B overexpression. A Cox multivariate analysis revealed that GADD45B overexpression and TNM stage were significant factors affecting patients’ survival. On the other hand, as a tumor suppressor gene, GADD45B amplified from normal colorectal tissues could induce apoptosis in CRC cell lines and may be associated with the p53-mediated apoptotic pathways.ConclusionGADD45B, a tumor suppressor gene potentially through the p53-mediated apoptotic pathways, is paradoxically overexpressed in CRC and as such may play an unappreciated role in tumorigenesis. The exact mechanism of GADD45B inactivation and overexpression requires further investigation. GADD45B could be a potential therapeutic target for CRC treatment in future.

Dose-dependent and gender-related radiation-induced transcription alterations of Gadd45a and Ier5 inhuman lymphocytes exposed to gamma ray emitted by 60Co

Growth arrest DNA damage-inducible 45a gene (Gadd45a) and immediate early response gene 5 (Ier5) have been emphasised as ideal radiation biomarkers in several reports. However, some aspects of radiation-induced transcriptional alterations of these genes are unknown. In this study, gender-dependency and dose-dependency as two factors that may affect radiation-induced transcription of Gadd45a and Ier5 genes were investigated. Human lymphocyte cells from six healthy voluntary blood donors (three women and three men) were irradiated in vitro with doses of 0.5-4.0 Gy from a (60)Co source and RNA isolated 4 h later using the High Pure RNA Isolation Kit. Dose and gender dependency of radiation-induced transcriptional alterations of Gadd45a and Ier5 genes were studied by quantitative real-time polymerase chain reaction. The results showed that as a whole, Gadd45a and Ier5 gave responses to gamma rays, while the responses were independent of radiation doses. Therefore, regardless of radiation dose, Gadd45a and Ier5 can be considered potential radiation biomarkers. Besides, although radiation-induced transcriptional alterations of Gadd45a in female and male lymphocyte samples were insignificant at 0.5 Gy, at other doses, their quantities in female samples were at a significantly higher level than in male samples. Radiation-induced transcription of Ier5 of females samples had a reduction in comparison with male samples at 1 and 2 Gy, but at doses of 0.5 and 4 Gy, females were significantly more susceptible to radiation-induced transcriptional alteration of Ier5.

Abstract B52: Design and synthesis of GADD45β agonists for sorefenib-resistance hepatocellular carcinoma cells

Abstract Background: Growth arrest DNA damage-inducible gene 45β (GADD45β) have been reported in regulating cellular stress response, survival, senescence, and apoptosis in cancer cells. Our previous data showed that GADD45β was induced in sorafenib-treated hepatocellular carcinoma (HCC) cell line and xenograft models. Sorafenib induction was not induced after the treatment ERK kinase inhibitor (U1206) and Raf inhibitor (ZM336372), suggesting that Raf/MEK/ERK kinase signaling pathway did not involve in induction of GADD45β by sorafenib. Methods: We have designed and synthesized a series of SC-compounds. These compounds were treated with and analyzed in terms of GADD45β expression in HCC cell lines (Huh-7, Huh7R). Also, Effects of SC-compounds and sorafenib induced cell apoptosis were evaluated. Results: In seeking new GADD45β agonists, we have screened a series of compounds with GADD45β expression assay. SC-20, a small molecule with N-(4-(quinolin-4-yloxy)phenyl)benzamide skeleton, has shown significantly in induction of GADD45β expression. Interestingly, SC-20 has no inhibition in phosphorylation of ERK, indicating that GADD45β induction by SC-20 is independent of ERK signaling pathway. In addition, SC-20 is able to inhibit HCC cell growth with MTT assay and further induce cell apoptosis. Moreover, SC-20 is able to overcome sorafenib resistance cell line by activating GADD45β expression. Conclusions: SC-20 provides a new concept that increasing GADD45β along will be applicable in sorafenib-sensitive and resistant HCC therapy. Grant support: NSC-100-2325-B-002-042, DOH100-TD-B-111-001, NSC-100-2325-B-010-007

Role of p53 in induction of growth arrest DNA damage-inducible gene 45 β in human hepatoma cells by hydroxyurea

Objective To identify the role of p53 in the induction of growth arrest DNA damageinducible gene 45 (βGADD45β) in hepatocellular carcinoma (HCC) cells by hydroxyurea.Methods Hep3B +p53 clone was established by the transfection of the full-length p53 sequence to Hep3B.Following hydroxyurea administration,quantitative real-time polymerase chain reaction (PCR) was employed to validate the expression changes of GADD45β.pGL3 basic luciferase plasmids including promoter fragments were synthesized in vitro and transfected into cells.The effects on promoter activity,cell growth and the cleavage of Caspase-3 were further focused on.Results Hep3B +p53 could express p53 protein stably.The transfection of p53 could enhance the induction of GADD45β in Hep3B by hydroxyurea.The promoter activity of fragments constructing nuclear factor-κB (NF-κB) and E2F-1 binding sites could be induced about 1.1 and 0.8 folds by transfection of p53.The colony formation and DNA syntheses were inhibited apparently in Hep3B +p53 with p53 by hydroxyurea (37.15％ and 85.29％ by 10 mmol/L Hydroxyurea,respectively).Moreover,the p53 transfection could trigger the cleavage of Caspase-3 more rapidly.Conclusion p53 may play a role in the induction of GADD45β in Hep3B by ribonucleotide reductase inhibitor. Key words: Carcinoma,hepatocellular; Hydroxyurea; p53; Promoter

Augmentation of endoplasmic reticulum stress in cerebral ischemia/reperfusion injury associated with comorbid type 2 diabetes

<title/>Objective: Diabetes is one of the major risk factors for ischemic stroke and is reported to aggravate the ischemic brain damage in different experimental models as well as clinical situations. However, the mechanisms underlying the exacerbated ischemia/reperfusion (I/R) brain injury associated with comorbid diabetes are still not clear. This study investigated the role of endoplasmic reticulum (ER) stress in pathophysiology of aggravated I/R brain injury associated with diabetes.Methods: Focal cerebral ischemia was induced by middle cerebral artery occlusion for 2 hours followed by 22 hours of reperfusion in high-fat diet-fed and low-dose streptozotocin-treated type 2 diabetic rats. Immunohistochemistry and western blotting analysis were performed to detect the changes in expression of various ER stress and apoptotic markers such as 78 kDa glucose-regulated protein (GRP78), CCAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153), and caspase-12. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed to detect the extent of DNA fragmentation and cell death.Results: The diabetic rats subjected to I/R manifested significantly larger brain infarct volume and severe deterioration in neurological deficits than their normal, non-diabetic counterparts. There was a marked upregulation of GRP78 observed in brains of diabetic rats after 22 hours of reperfusion. Furthermore, augmentation of CHOP/GADD153 expression and activation of caspase-12 (ER stress-induced apoptotic factors) were observed in parallel with enhanced TUNEL-positive cells or DNA fragmentation in diabetic rats compared to normal rats following cerebral I/R.Discussion: Taken together, the current experimental findings demonstrate that diabetes exacerbates brain I/R injury which may be mediated through enhanced ER stress and cell death involving CHOP/GADD153 and caspase-12 activation.

Edaravone Offers Neuroprotection in a Diabetic Stroke Model via Inhibition of Endoplasmic Reticulum Stress

Recent investigations have postulated a link between oxidative stress and endoplasmic reticulum (ER) dysfunction in cerebral ischaemic/reperfusion (I/R) injury. Diabetes is common amongst elderly patients with stroke and has been postulated to aggravate brain I/R damage by triggering oxidative as well as ER stress. We investigated whether treatment with edaravone (1-10 mg/kg), a potent free radical scavenger protects against cerebral I/R injury in rats associated with comorbid type 2 diabetes. Diabetic rats exposed to 2-hr middle cerebral artery occlusion (MCAO) and 22 hr of reperfusion significantly had increased infarct, oedema volume and functional neurological deficits as compared to sham-operated rats. Also, the massive DNA fragmentation accompanied by significant increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) positive cells was noticed in the ipsilateral penumbral brain region of diabetic I/R rats. The effects of I/R injury were associated with significant up-regulation of 78 kDa-glucose-regulated protein (GRP78), CCAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153) and activation of caspase-12, markers of ER stress/apoptosis. Treatment with edaravone (3 and 10 mg/kg) significantly diminished the cerebral infarct, oedema volume and improved functional recovery of neurological deficits. In addition, edaravone treatment ameliorated the DNA fragmentation concomitantly with a significant decrease in induction of GRP78, CHOP/GADD153 immunoreactivity/expression and activation of caspase-12 in the ischaemic brain hemispheres. Overall, the present data indicate that edaravone offers good neuroprotection against diabetic stroke by interrupting the ER stress-mediated apoptotic pathways involving CHOP/GADD153 and caspase-12.

Sodium phenylbutyrate ameliorates focal cerebral ischemic/reperfusion injury associated with comorbid type 2 diabetes by reducing endoplasmic reticulum stress and DNA fragmentation

Endoplasmic reticulum (ER) stress has been postulated to play a crucial role in the pathophysiology of cerebral ischemic/reperfusion (I/R) injury and diabetes. Diabetes is a major risk factor and also common amongst the people who suffer from stroke. In this study, we have investigated the neuroprotective potential of sodium 4-phenylbutyrate (SPB; 30–300 mg/kg), a chemical chaperone by targeting ER stress in a rat model of transient focal cerebral ischemia associated with comorbid type 2 diabetes. Intraperitoneal treatment with SPB (100 and 300 mg/kg) significantly ameliorated brain I/R damage as evidenced by reduction in cerebral infarct and edema volume. It also significantly improved the functional recovery of various neurobehavioral impairments (neurological deficit score, grip strength and rota rod) evoked by I/R compared with vehicle-treatment. Further, SPB (100 mg/kg) significantly reduced the DNA fragmentation as shown by prominent reduction in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells. This effect was observed concomitantly with significant attenuation in upregulation of 78 kDa glucose regulated protein (GRP78), CCAAT/enhancer binding protein homologous protein or growth arrest DNA damage-inducible gene 153 (CHOP/GADD153) and activation of caspase-12, specific markers of ER stress/apoptosis. The neuroprotection observed with SPB was independent of its effect on cerebral blood flow and blood glucose. In conclusion, this study demonstrates the neuroprotective effect of SPB owing to amelioration of ER stress and DNA fragmentation. It also suggest that targeting ER stress might offer a promising therapeutic approach and benefits against ischemic stroke associated with comorbid type 2 diabetes.

Hexahydro-β-acids induce apoptosis through mitochondrial pathway, GADD153 expression, and caspase activation in human leukemia cells

Hexahydro-β-acids (HBA) and β-acids (BA) displayed strong growth inhibitory effects against human leukemia HL-60 cells and were able to induce apoptosis in a concentration- and time-dependent manner and the morphological changes associated with apoptotic cell death; however, BA was less effective. Treatment with HBA caused a rapid loss of mitochondrial trans-membrane potential, release of mitochondrial cytochrome c into cytosol. The levels of Bad and Bax were dramatically increased in cells treated with HBA. In addition, the results showed that HBA promoted the up-regulation of Fas prior to the processing and activation of pro-caspase-8 and cleavage of Bid, suggesting the involvement of a Fas-mediated pathway in HBA-induced cells. Moreover, the changes occurred after single breaks in DNA were detected, suggesting that HBA induced irreparable DNA damage, which in turn triggered the process of apoptosis. HBA markedly enhanced the growth arrest DNA damage-inducible gene 153 (GADD153) protein in a concentration- and time-dependent manner. These findings suggest that HBA creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn triggers apoptosis in HL-60 cells. Our study identified the novel mechanisms of HBA-induced apoptosis and indicated that HBA may be used as a potential chemopreventive and chemotherapeutic agent.

Induction of DNA Damage-Inducible Gene GADD45β Contributes to Sorafenib-Induced Apoptosis in Hepatocellular Carcinoma Cells

Markers that could accurately predict responses to the general kinase inhibitor sorafenib are needed to better leverage its clinical applications. In this study, we examined a hypothesized role in the drug response for the growth arrest DNA damage-inducible gene 45β (GADD45β), which is commonly underexpressed in hepatocellular carcinoma (HCC) where sorafenib may offer an important new therapeutic option. The anticancer activity of sorafenib-induced GADD45β expression was tested in a panel of HCC cell lines and xenograft models. We found that GADD45β mRNA and protein expression were induced relatively more prominently in HCC cells that were biologically sensitive to sorafenib treatment. GADD45β induction was not found after treatment with either the mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 or the Raf inhibitor ZM336372, suggesting that GADD45β induction by sorafenib was independent of Raf/MEK/ERK signaling activity. However, c-Jun NH2-terminal kinase (JNK) kinase activation occurred preferentially in sorafenib-sensitive cells. Small interfering RNA-mediated knockdown of GADD45βor JNK kinase limited the proapoptotic effects of sorafenib in sorafenib-sensitive cells. We defined the -339/-267 region in the GADD45β promoter containing activator protein-1 and SP1-binding sites as a crucial region for GADD45β induction by sorafenib. Together, our findings suggest that GADD45β induction contributes to sorafenib-induced apoptosis in HCC cells, prompting further studies to validate its potential value in predicting sorafenib efficacy.