Progress of N-myc downstream regulated gene 2 / 国际儿科学杂志

The full name of NDRG2 is N-myc downstream regulated gene 2,which is a member of NDRG.It has a high degree of homology,mainly involved in cell proliferation and differentiation.At present it has been researched mainly as a tumor suppressor gene,in addition,it has participated in the development of embryonic and nervous system,the regulation of water and sodium metabolism in renal proximal tubule and collecting duct by aldosterone aldosterone.It is also involved in the response to stress.This article rewiews the mechanism of transcriptional regulation and molecular interaction.

Role of NDRG2 in the dorsal horn of the spinal cord in rats with spinal nerve ligation of neuropathic pain / 临床麻醉学杂志

Objective To investigate the effect of NDRG2 on neuropathic pain model rats with spinal cord ligation(SNL)in the dorsal horn of the spinal cordby using intrathecal NDRG2 adenovirus (AD-Ndrg2-RNAi).Methods Forty-two male SD rats weighing 180-230 g were randomly divided into sham operation group (n=6)and SNL group (n=3 6).SNL group underwent lumbar spinal dor-sal ligation.The sham operation group only exposed the spinal nerve without ligation.The mechanical withdraw threshold (MWT)and thermal withdrawlantency (TWL)were measured at 1 d before op-eration and at 1,3,7,10,14 and 21 d after operation.After the behavioral testing,the rats in SNL group were sacrificed and the lumber segment of the spinal cord was removed to test theprotein and mRNA level of NDRG2.Another forty-eight male SD rats were randomly divided into 4 groups after intrathecal catheterization (n=12):group sham operation with saline (group CS);group sham oper-ation with adenovirus (group CA);group SNL with saline (group SS);group SNL with adenovirus(group SA).The rats in groups CS and SS were treated with intrathecal injection of normal saline 10 μl on the third day after operation,while groups CA and SA received the single injection titer of 2× 109PFU/ml of AD-Ndrg2-RNAi on the same day.The rat plantar MWT and TWL were measured at 1 d before SNL and at 1,3,7 and 10 d after SNL.The rats were sacrificed after the lastbehavioral test.Lumbar enlargement of the spinal cord was removed to test the level of NDRG2 and GFAP pro-tein.The expression of NDRG2 mRNA was detected by real-time fluorescence quantitative PCR. Results Compared with sham group,the MWT and TWL was significantly decreased and reduced in SNL group 1,3,7,10,14,21 d and 3,7,10,14,21 d after surgery respectively(P<0.01).Com-pared with 1 d before surgery,protein content and mRNA expression of NDRG2 in the spinal dorsal horn of the SNL group were significantly decreased 1 d after surgery,and they were significantly in-creased on the 7,10,14,and 21 d after operation(P<0.01).Compared with group CS,the MWT and TWL were significantly decreased and reduced in group SS and group SA 1,3,7 and 10 d after surgery(P<0.05).MWT and TWL on 7 and 10 d were increased significantly in group SA (P<0.05)compared with group SS.Compared with group CS,protein content and mRNA expression of NDRG2 in the spinal dorsal horn of group CA were significantly decreased,and increased significantly in group SS 10 d after surgery(P<0.05).Compared with group SS,protein content and mRNA ex-pression of NDRG2 in the spinal dorsal horn of group SA were significantly decreased.Compared with group CS,the protein content of GFAP in spinal dorsal horn of group CA,group SS and group SA were increased significantly 10 d after surgery (P<0.05).Conclusion Intrathecal injection of AD-Ndrg2-RNAi significantly inhibits neuropathic pain caused by spinal cord ligation in rats,suggesting that NDRG2 in astrocytes is involved in the development and progression of neuropathic pain.

Expression of merlin, NDRG2, ERBB2, and c-MYC in meningiomas: relationship with tumor grade and recurrence

Meningiomas are common, usually benign tumors of the central nervous system that have a high rate of post-surgical recurrence or regrowth. We determined expression of the proteins merlin, NDRG2, ERBB2, and c-MYC in meningiomas using immunohistochemistry and assessed relationships between protein expression and gender, age, tumor grade, and recurrence or regrowth. The study sample comprised 60 patients, (44 women and 16 men) with a mean age of 53.2±12.7 years. Tumors were classified as grade I (n=48) or grades II and III (n=12). Expression of merlin, NDRG2, ERBB2, and c-MYC was not significantly different statistically with relation to gender, age, or meningioma recurrence or regrowth. Merlin was expressed in 100% of the cases. No statistically significant difference between tumor grade and recurrence or regrowth was identified. Statistically significant differences were identified between the mean age of patients with grade I (54.83±11.60) and grades II and III (46.58±15.08) meningiomas (P=0.043), between strong c-MYC expression and grades II and III (P<0.001), and between partial surgical resection and tumor recurrence or regrowth (P<0.001). These findings reveal the lower mean age among grades II and III meningioma patients than grade I patients, the influence of the protein merlin on tumorigenesis, the association of c-MYC with aggressive meningiomas, and that partial surgical resection is associated with tumor recurrence or regrowth.

NDRG2 Expression Decreases Tumor-Induced Osteoclast Differentiation by Down-regulating ICAM1 in Breast Cancer Cells

Bone matrix is properly maintained by osteoclasts and osteoblasts. In the tumor microenvironment, osteoclasts are increasingly differentiated by the various ligands and cytokines secreted from the metastasized cancer cells at the bone metastasis niche. The activated osteoclasts generate osteolytic lesions. For this reason, studies focusing on the differentiation of osteoclasts are important to reduce bone destruction by tumor metastasis. The N-myc downstream-regulated gene 2 (NDRG2) has been known to contribute to the suppression of tumor growth and metastasis, but the precise role of NDRG2 in osteoclast differentiation induced by cancer cells has not been elucidated. In this study, we demonstrate that NDRG2 expression in breast cancer cells has an inhibitory effect on osteoclast differentiation. RAW 264.7 cells, which are monocytic preosteoclast cells, treated with the conditioned media (CM) of murine breast cancer cells (4T1) expressing NDRG2 are less differentiated into the multinucleated osteoclast-like cells than those treated with the CM of 4T1-WT or 4T1-mock cells. Interestingly, 4T1 cells stably expressing NDRG2 showed a decreased mRNA and protein level of intercellular adhesion molecule 1 (ICAM1), which is known to enhance osteoclast maturation. Osteoclast differentiation was also reduced by ICAM1 knockdown in 4T1 cells. In addition, blocking the interaction between soluble ICAM1 and ICAM1 receptors significantly decreased osteoclastogenesis of RAW 264.7 cells in the tumor environment. Collectively, these results suggest that the reduction of ICAM1 expression by NDRG2 in breast cancer cells decreases osteoclast differentiation, and demonstrate that excessive bone resorption could be inhibited via ICAM1 down-regulation by NDRG2 expression.

Effects of tumor suppressor NDRG2 gene on proliferation and invasive ability of colon cancer cell line SW620 / 国际肿瘤学杂志

Objective To observe the influence of N-myc downstream-regulated gene 2 (NDRG2) on the growth and invasive ability of human colon cancer cell line SW620,and to explore its mechanism.Methods pcDNA3.1-NDRG2 and siRNA-NDRG2 were transfected transiently respectively into SW620 by Lipofectamine TM 2000,untreated cells as the control group.Western blotting was used to investigate the expression of NDRG2 and matrix metalloproteinase-2 (MMP-2).Matrigel invasion assay was used to study the invasive abilities of SW620 cells in all groups.The growth curve was determined through 3-(4,5-dimethyl-2-thiazoly)-2,5-diphenyl-2H-tetrazolium bromide method.Result After transfecting pcDNA3.1-NDRG2 into the SW620 cells,the protein level of NDRG2 increased and the expression of MMP-2 declined markedly.After transfecting siRNA-NDRG2 into the SW620 cells,the protein level of NDRG2 declined and the expression of MMP-2 increased markedly.In addition,compared with the control group (75.80 ± 4.82),the numbers of transmembrane cells in pcDNA3.1 group (56.20 ± 7.40) and in siRNA group (94.20 ± 9.23) were significantly different (t =13.102,P =0.000;t =11.820,P =0.000).The growth curve showed that:compared with the control group (0.67 ±0.01),the absorbance of the fifth day after transfection in pcDNA3.1 group (0.46 ±0.01) and in siRNA group (0.91 ± 0.02) were different significantly (t =9.561,P =0.000;t =10.922,P =0.000).Conclusion NDRG2 can reduce the invasion and proliferation ability of colon cancer cell SW620,and its mechanism may be related to the down-regulation of MMP-2 expression.

Adenovirus-mediated NDRG2 inhibits the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro

OBJECTIVE@#To investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro.@*METHOD@#NDRG2 was harvested by RT-PCR, confirmed by DNA sequencing, and then cloned into the eukaryotic expression vector pIRES2-EGFP, which encodes green fluorescent protein (GFP), to construct pIRES2-EGFP-NDRG2 plasmid. OS-RC-2 cells with NDRG2 negative expression were transfected with pIRES2-EGFP-NDRG2 plasmid. The growth of transfected OS-RC-2 cells was observed under light and fluorescence microscopes. After colony-forming cell assays, cell proliferation detection and MTT assays, the growth curves of cells in each group were plotted to investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of OS-RC-2 cells. Cell cycle was determined by flow cytometry. Confocal laser scanning microscopy showed that NDRG2 protein was specifically located on subcellular organelle.@*RESULTS@#A eukaryotic expression vector pIRES2-EGFP-NDRG2 was successfully constructed. After NDRG2 transfection, the growth of OS-RC-2 cells was inhibited. Flow cytometry showed that cells were arrested in S phase but the peak of cell apoptosis was not present, and confocal laser scanning microscopy showed that NDRG2 protein was located in mitochondrion.@*CONCLUSIONS@#NDRG2 can significantly inhibit the proliferation of OS-RC-2 cells in vitro and its protein is specifically expressed in the mitochondrion.

Adenovirus-mediated NDRG2 inhibits the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro

Objective: To investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro. Method: NDRG2 was harvested by RT-PCR, confirmed by DNA sequencing, and then cloned into the eukaryotic expression vector pIRES2-EGFP, which encodes green fluorescent protein (GFP), to construct pIRES2-EGFP-NDRG2 plasmid. OS-RC-2 cells with NDRG2 negative expression were transfected with pIRES2-EGFP-NDRG2 plasmid. The growth of transfected OS-RC-2 cells was observed under light and fluorescence microscopes. After colony-forming cell assays, cell proliferation detection and MTT assays, the growth curves of cells in each group were plotted to investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of OS-RC-2 cells. Cell cycle was determined by flow cytometry. Confocal laser scanning microscopy showed that NDRG2 protein was specifically located on subcellular organelle. Results: A eukaryotic expression vector pIRES2-EGFP-NDRG2 was successfully constructed. After NDRG2 transfection, the growth of OS-RC-2 cells was inhibited. Flow cytometry showed that cells were arrested in S phase but the peak of cell apoptosis was not present, and confocal laser scanning microscopy showed that NDRG2 protein was located in mitochondrion. Conclusions: NDRG2 can significantly inhibit the proliferation of OS-RC-2 cells in vitro and its protein is specifically expressed in the mitochondrion.

AKT inhibitor suppresses hyperthermia-induced Ndrg2 phosphorylation in gastric cancer cells

Hyperthermia is one of the most effective adjuvant treatments for various cancers with few side effects. However, the underlying molecular mechanisms still are not known. N-myc downstream-regulated gene 2 (NDRG2), a tumor suppressor, has been shown to be involved in diverse cellular stresses including hypoxia, lipotoxicity, etc. In addition, Ndrg2 has been reported to be related to progression of gastric cancer. In the current study, our data showed that the apoptosis rate of MKN28 cells increased relatively rapidly to 13.4% by 24 h after treatment with hyperthermia (42°C for 1 h) compared to 5.1% in control cells (P < 0.05). Nevertheless, there was no obvious change in the expression level of total Ndrg2 during this process. Further investigation demonstrated that the relative phosphorylation levels of Ndrg2 at Ser332, Thr348 increased up to 3.2- and 1.9-fold (hyperthermia group vs control group) at 3 h in MKN28 cells, respectively (P < 0.05). We also found that heat treatment significantly increased AKT phosphorylation. AKT inhibitor VIII (10 µM) decreased the phosphorylation level of Ndrg2 induced by hyperthermia. Accordingly, the apoptosis rate rose significantly in MKN28 cells (16.4%) treated with a combination of AKT inhibitor VIII and hyperthermia compared to that (6.8%) of cells treated with hyperthermia alone (P < 0.05). Taken together, these data demonstrated that Ndrg2 phosphorylation could be induced by hyperthermia in an AKT-dependent manner in gastric cancer cells. Furthermore, AKT inhibitor VIII suppressed Ndrg2 phosphorylation and rendered gastric cancer cells susceptible to apoptosis induced by hyperthermia.

NDRG2 Promotes GATA-1 Expression through Regulation of the JAK2/STAT Pathway in PMA-stimulated U937 Cells

BACKGROUND: N-myc downstream-regulated gene 2 (NDRG2), a member of a newly described family of differentiation-related genes, has been characterized as a regulator of dendritic cells. However, the role of NDRG2 on the expression and activation of transcription factors in blood cells remains poorly understood. In this study, we investigated the effects of NDRG2 overexpression on GATA-1 expression in PMA-stimulated U937 cells. METHODS: We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 on GATA-1 expression. RESULTS: NDRG2 overexpression in U937 cells significantly induced GATA-1 expression in response to PMA stimulation. Interestingly, JAK2/STAT and BMP-4/Smad pathways associated with the induction of GATA-1 were activated in PMA-stimulated U937-NDRG2 cells. We found that the inhibition of JAK2 activation, but not of BMP-4/Smad signaling, can elicit a decrease of PMA-induced GATA-1 expression in U937-NDRG2 cells. CONCLUSION: The results reveal that NDRG2 promotes the expression of GATA-1 through activation of the JAK2/STAT pathway, but not through the regulation of the BMP-4/Smad pathway in U937 cells. Our findings further suggest that NDRG2 may play a role as a regulator of erythrocyte and megakaryocyte differentiation during hematopoiesis.

NDRG2-mediated Modulation of SOCS3 and STAT3 Activity Inhibits IL-10 Production

BACKGROUND: N-myc downstream regulated gene 2 (NDRG2) is a member of the NDRG gene family. Our previous report indicated a possible role for NDRG2 in regulating the cytokine, interleukin-10 (IL-10), which is an important immunosuppressive cytokine. Several pathways, including p38-MAPK, NF-kappaB, and JAK/STAT, are used for IL-10 production, and the JAK/STAT pathway can be inhibited in a negative feedback loop by the inducible protein, SOCS3. In the present study, we investigated the effect of NDRG2 gene expression on IL-10 signaling pathway that is modulated via SOCS3 and STAT3. METHODS: We generated NDRG2-overexpressing U937 cell line (U937-NDRG2) and treated the cells with PMA to investigate the role of NDRG2 in IL-10 production. U937 cells were also transfected with SOCS3- or NDRG2-specific siRNAs to examine whether the knockdown of SOCS3 or NDRG2 influenced IL-10 expression. Lastly, STAT3 and SOCS3 induction was measured to identify the signaling pathway that was associated with IL-10 production. RESULTS: RT-PCR and ELISA assays showed that IL-10 was increased in U937-mock cells upon stimulation with PMA, but IL-10 was inhibited by overexpression NDRG2. After PMA treatment, STAT3 phosphorylation was decreased in a time-dependent manner in U937-mock cells, whereas it was maintained in U937-NDRG2 cells. SOCS3 was markedly reduced in U937-NDRG2 cells compared with U937-mock cells. IL-10 production after PMA stimulation was reduced in U937 cells when SOCS3 was inhibited, but this effect was less severe when NDRG2 was inhibited. CONCLUSION: NDRG2 expression modulates SOCS3 and STAT3 activity, eventually leading to the inhibition of IL-10 production.

NDRG2 gene transfection inhibits proliferation of breast cancer cell / 基础医学与临床

Objective To explore the effect of gene NDRG2 (N-myc downstream regulated gene 2) transfection on the proliferation and apoptosis of breast cancer cell. Methods Bcap37 cell line expressing low level product encoded by NDRG2 gene is transfected with recombinating adenovirus expressing high level NDRG2 gene product to improve NDRG2 gene expression level. NDRG2 expression was detected by Western blot and the proliferation activity of the cell lines was detected by MTT and flow cytometry. Results High level expression of NDRG2 inhibits cells growth and results in G_1 phase arrest in Bcap37 cell line. Compared with non-transfected Bcap37 cells and Bcap37 cells transfected by empty vector, apoptosis cells obviously increase in Bcap37 cell line transfected by recombinating adenovirus expressing high level NDRG2 gene, 12% after 48 hours and 21. 5% after 72 hours. Conclusion Overexpres-sion of NDRG2 in Bcap37 cells effectively inhibites cell proliferation and induces cell cycle arrest and apoptosis.

Expression of NDRG2 is related to tumor progression and survival of gastric cancer patients through Fas-mediated cell death

Although N-myc downstream regulated gene 2 (NDRG2) has been known to be a tumor suppressor gene, the function of this gene has not been elucidated. In the present study, we investigated the expression and function of NDRG2 in human gastric cancer. Among seven gastric cancer and two non-cancer cell lines, only two gastric cancer cell lines, SNU-16 and SNU-620, expressed NDRG2, which was detected in the cytoplasm. Interestingly, NDRG2 was highly expressed in normal gastric tissues, but gastric cancer patients were divided into NDRG2-positive and -negative groups. The survival rate of NDRG2-negative patients was lower than that of NDRG2-positive patients. We confirmed that the loss of NDRG2 expression was a significant and independent prognostic indicator in gastric carcinomas by multivariate analysis. To investigate the role of NDRG2 in gastric cancer cells, we generated a NDRG2-silenced gastric cancer cell line, which stably expresses NDRG2 siRNA. NDRG2-silenced SNU-620 cells exhibited slightly increased proliferation and cisplatin resistance. In addition, inhibition of NDRG2 decreased Fas expression and Fas-mediated cell death. Taken together, these data suggest that inactivation of NDRG2 may elicit resistance against anticancer drug and Fas-mediated cell death. Furthermore, case studies of gastric cancer patients indicate that NDRG2 expression may be involved in tumor progression and overall survival of the patients.

A Study for Expression and Biological Function of N-myc Downstream Regulated Gene 2 in Breast Cancer / 한국유방암학회지

PURPOSE: It is important to identify a potential tumor marker that is associated with pathophysiologic processes of breast cancer. N-Myc downstream regulated genes (NDRG) are composed of four subtypes (NDRG 1-4) and NDRG2 gene has been reported as a specifically expressed gene in the human solid tumor including breast cancer. Although NDRG2 inhibits cell proliferation and promote differentiation, the molecular basis of the tumor-suppressor activity of NDRG2 in breast cancer is unknown. Herein, we tried to reveal the correlations between the expression of NDRG2 and the various clinicopathologic prognostic factors and evaluate its functional and pathophysiological roles in tumorigenesis of breast cancer. METHODS: We were obtained the 67 breast cancers and paired normal tissue samples from patients who operated for breast cancer between June 2002 and June 2004. The expression of NDRG2 were measured with immunohistochemistry using monoclonal antibody and it was used eukaryotic transfection to manipulate the expression in MDA-MB-231 breast cancer cell line. Cell proliferation analysis were evaluated with trypan blue stain and status of differentially-expressed genes by NDRG2 overexpression were investigated with oligo microarray chip analysis. RESULTS: Significant difference of NDRG2 mRNA expression between breast cancer and normal tissue was not detected. However, NDRG2 was significantly down-regulated in breast cancer tissue, compared to normal tissue (p<0.0001). It was a inverse-correlation between the NDRG2 expression and tumor size, histologic grade although other clinicopathological parameters such as axillary lymph node metastasis were not correlated. Overexpression of NDRG2 in MDA-MB-231 cell showed a decrease of cell proliferation compared with Mock control. Of the 24,000 genes, 64 genes were increased in expression while 256 genes including cyclin D1 were repressed by NDRG2 overexpression. CONCLUSION: Our results suggest that NDRG2 can function as a regulator of cell differentiation and cell cycle (as a tumor suppressor gene) in the early stage of breast cancer. In addition, NDRG2 protein indicates a prognostic tumor marker for breast cancer.

A Study for Expression and Biological Function of N-myc Downstream Regulated Gene 2 in Breast Cancer / 한국유방암학회지

PURPOSE: It is important to identify a potential tumor marker that is associated with pathophysiologic processes of breast cancer. N-Myc downstream regulated genes (NDRG) are composed of four subtypes (NDRG 1-4) and NDRG2 gene has been reported as a specifically expressed gene in the human solid tumor including breast cancer. Although NDRG2 inhibits cell proliferation and promote differentiation, the molecular basis of the tumor-suppressor activity of NDRG2 in breast cancer is unknown. Herein, we tried to reveal the correlations between the expression of NDRG2 and the various clinicopathologic prognostic factors and evaluate its functional and pathophysiological roles in tumorigenesis of breast cancer. METHODS: We were obtained the 67 breast cancers and paired normal tissue samples from patients who operated for breast cancer between June 2002 and June 2004. The expression of NDRG2 were measured with immunohistochemistry using monoclonal antibody and it was used eukaryotic transfection to manipulate the expression in MDA-MB-231 breast cancer cell line. Cell proliferation analysis were evaluated with trypan blue stain and status of differentially-expressed genes by NDRG2 overexpression were investigated with oligo microarray chip analysis. RESULTS: Significant difference of NDRG2 mRNA expression between breast cancer and normal tissue was not detected. However, NDRG2 was significantly down-regulated in breast cancer tissue, compared to normal tissue (p<0.0001). It was a inverse-correlation between the NDRG2 expression and tumor size, histologic grade although other clinicopathological parameters such as axillary lymph node metastasis were not correlated. Overexpression of NDRG2 in MDA-MB-231 cell showed a decrease of cell proliferation compared with Mock control. Of the 24,000 genes, 64 genes were increased in expression while 256 genes including cyclin D1 were repressed by NDRG2 overexpression. CONCLUSION: Our results suggest that NDRG2 can function as a regulator of cell differentiation and cell cycle (as a tumor suppressor gene) in the early stage of breast cancer. In addition, NDRG2 protein indicates a prognostic tumor marker for breast cancer.

NDRG2 gene transfection inhibits proliferation of breast cancer cell / 基础医学与临床

Objective To explore the effect of gene NDRG2(N-myc downstream regulated gene 2) transfection on the proliferation and apoptosis of breast cancer cell.Methods Bcap37 cell line expressing low level product encoded by NDRG2 gene is transfected with recombinating adenovirus expressing high level NDRG2 gene product to improve NDRG2 gene expression level. NDRG2 expression was detected by Western blot and the proliferation activity of the cell lines was detected by MTT and flow cytometry. Results High level expression of NDRG2 inhibits cells growth and results in G1 phase arrest in Bcap37 cell line. Compared with non-transfected Bcap37 cells and Bcap37 cells transfected by empty vector,apoptosis cells obviously increase in Bcap37 cell line transfected by recombinating adenovirus expressing high level NDRG2 gene,12% after 48 hours and 21.5% after 72 hours. Conclusion Overexpression of NDRG2 in Bcap37 cells effectively inhibites cell proliferation and induces cell cycle arrest and apoptosis.