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Objective:The purpose of this study was to investigate the expression and role of N-myc downstream regulatory gene 2 (NDRG2) in radiation resistance of bladder cancer cells.Methods:T24 cells were cultured in vitro and irradiated with different doses of X-ray (0, 2, 4, 8, 10 and 20 Gy). The best dose of X-ray was selected for subsequent treatment. The radioresistant BCa cell line T24/R was established. The cytotoxicity of T24/R cells was detected by counting kit-8 (CCK-8) method. The proliferation and invasion ability of T24/R cells and T24 cells were detected by flow cytometry and transwell, respectively. Western blot was used to detect the expression of epithelial mesenchymal transition (EMT) related proteins. The survival rate of T24/R group (control group) and T24/R-NDRG 2 group was detected, and the migration ability of T24/R-NDRG 2 cells was detected after 2 Gy treatment. Results:The cell viability was inhibited significantly when the dose of X-ray was ≥2 Gy X-ray, so 2 Gy X-ray irradiation was chosen as the best condition for BCa cytotoxicity and T24/R radiation resistance cell line was successfully established; Apoptosis test showed that the number of S-phase cells was increased in T24/R group, and the proportion of S-phase cells in T24/R vs T24 was (26.49±4.5)% vs (14±2.6)% ( P<0.05); Transwell test showed that T24/R cells showed stronger migration ability than control group ( P<0.05), but there was no significant difference in EMT related protein expression between the two groups ( P>0.05). Overexpression of NDRG2 can significantly decreased the activity and migration ability of radiation-resistant T24/R cells ( P<0.05) when the radiation dose was gradually increasing in both groups. Conclusions:The radiation resistance of BCa cells is one of the causes of local tumor recurrence. Up-regulation of NDRG2 expression can inhibit the radiation resistance of T24 cells, so it can be used as a candidate for treatment of radiation-resistant BCa patients.
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OBJECTIVE@#To investigate the effects of Shengmai Injection (, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2 (NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2.@*METHODS@#LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×10 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI (0.6, 1.2, 2.4, 4.8 or 6 μL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of SMI on different cell growth states (cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% conflfluence, apoptosis was detected by flflow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot.@*RESULTS@#When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 μL/mL of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment (P<0.05). And 2.4 μL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h (P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h (P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 μL/mL (P<0.05).@*CONCLUSION@#The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI (2.4, 4.8 and 6 μL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.
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Humans , Apoptosis , Cell Proliferation , Cells, Cultured , Drugs, Chinese Herbal , Pharmacology , Hepatic Stellate Cells , Physiology , Injections , Liver Cirrhosis , Drug Therapy , Tumor Suppressor Proteins , GeneticsABSTRACT
Objective To investigate the expression of N-myc downstream regulated gene-2 (NDRG2) on renal fibrosis in unilateral ureteral obstruction(UUO) rat model and the mechanism of renal fibrosis.Methods Forty-eight male Wistar rats (120-150g)were randomly divided into two groups:the sham-operation group (n =24)underwent the left ureteral dissection,the UUO group (n =24)underwent the left ureteral ligation.At the 3,7,14,21day after the operation,6 rats from each of the group were sacrificed and the obstructive kidneys were collected.The histopathological changes were observed through HE and Masson staining.E-cadherin and α-SMA were detected by Western blot and immunohistochemistry.NDRG2 were detected by Western blot,immunohistochemistry and real-time PCR.Results There is not diffenrence in the sham group at anytime.Compared with the sham-group,the fibrosis was obvious in UUO group.The protein of E-cadherin decreased(P ＜ 0.05) and α-SMA increased(P ＜0.05).The expression of NDRG2 in UUO group was lower than that in the sham group.The OD of immunohistochemistry was detected by ImagePro Plus 6.0.OD of NDRG2 was the lowest[(14.33 ± 2.45) x 10-3] in 21d group.The western blot showed that the ratio of NDRG2 to β-actin was the lowest(0.03 ± 0.01) in 21d group.The mRNA of NDRG2 decreased obverously in UUO group (P ＜ 0.05).Conclusion NDRG2 decreases in UUO group and NDRG2 might be involved in the pathologic process of renal fibrosis.
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Objective To investigate the effect of N-myc downstream regulated gene 2 (NDRG2) on the apoptosis of bladder cancer cells by regulating the signal transducer and activator of transcription 3(STAT3) signaling pathway.Methods The expression level of NDRG2 in human bladder cancer cell BIU-87 and immortalized cell SV-HUC-1 was detected by Western blot.NDRG2 over expression vector and empty vector control (pcDNA3.1),siRNA-NDRG2,siRNA control were transfected into BIU-87 cells.After transfected 48 h,the expression level of NDRG2,Cleaved caspase 3,STAT3,p-STAT3,JAK2,p-JAK2 were detected by Western blot,the cell proliferation and apoptosis were measured by MTT and flow cytometry.After adding inhibitor AG490 of 45 μmol/L in cultured BIU-87 cells,MTT assay was used to detect cell proliferation and flow cytometry was used to detect the cell apoptosis,Western blot to detect the expression level of Cleaved caspase 3,STAT3,p-STAT3,JAK2,p-JAK2.Results The expression level of NDRG2 in bladder cancer cells was higher than that in bladder epithelial cells.The cell survival rate of pcDNA3.1/NDRG2 group was lower than that of pcDNA3.1 group,the difference was statistically significant (P ＜ 0.01).The cell survival rate of siRNA-NDRG2 group was higher than that of siRNA control group (P＜ 0.01).The apoptosis rate of pcDNA3.1/NDRG2 group was higher than that of pcDNA3.1 group (P ＜ 0.01).The apoptosis rate of NDRG2 siRNA group was lower than that of siRNA control group (P ＜ 0.01).The level of p-STAT3 and p-JAK2 in pcDNA3.1/NDRG2 group was lower than that in pcDNA3.1 group (P＜ 0.01).The survival rate and apoptosis rate of BIU-87 cells cultured with AG490 were in agreement with the trend of pcDNA3.1/NDRG2 after transfection.Conclusions NDRG2 could promote the apoptosis of bladder cancer cells,and its mechanism may be related to STAT3 signaling pathway.
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<p><b>OBJECTIVE</b>To investigate the effects of Salvia miltiorrhiza and Ligustrazine Injection (SML) on proliferation and apoptosis of human hepatic stellate cell LX-2 and the expression of N-myc downstreamregulated gene 2 (NDRG2, a tumor suppressor gene).</p><p><b>METHODS</b>HSCs from the LX-2 cell line were cultured in vitro. The proliferative state of different initial LX-2 cell numbers was measured using a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. LX-2 cells were plated in 96-well plates at an approximate density of 2.50×10cells/mL and cultured for 24 h followed by the application of different concentrations of SML (1, 2, 4 and 8 μL/mL). Cell proliferation was measured using the MTT assay at 24 and 48 h. Apoptosis was detected by flow cytometry at 24 h. LX-2 cells were treated with different concentrations of SML and extracted with protein lysis buffer. The levels of NDRG2 and β-catenin were measured by Western blot.</p><p><b>RESULTS</b>With the exception of the 1 and 2 μL/mL concentrations, 4 and 8 μL/mL SML inhibited cell proliferation in a concentration-dependent manner at 24 and 48 h (P<0.05). With the exception of the 1 and 2 μL/mL concentrations, the NDRG2 expression level was greatly increased in a concentration-dependent manner. However, the level of β-catenin was unaffected.</p><p><b>CONCLUSION</b>SML inhibit LX-2 cell proliferation in a concentration-dependent manner, and the mechanism may be associated with NDRG2 over-expression.</p>
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Background and purpose:Breast cancer is one of the most common malignant diseases in women and its malignant proliferation is the major cause of death. To investigate the effects of N-myc downstream regulated gene 2 (NDRG2) on proliferation of breast cancer cells by using two parallel cell lines (MCF-7 and LM-MCF-7) with different metastatic abilities.Methods:The expression level of NDRG2 in breast cancer cells was detected by Western blot. The effects of overexpressing (or down-regulating) NDRG2 on proliferation of breast cancer cells were investigat-ed by lfow cytometry. The expression and location of β-catenin were detected by Western blot and immunolfuorescence respectively. NDRG2 blocking the transcription activity of β-catenin was investigated via co-transfecting MCF-7 cells with NDRG2 siRNA and pCMV-Tcfδ (lacking the portion responsible for the protein binding to DNA).Results:The expression level of NDRG2 was negatively related to the proliferation ability of breast cancer cells. Over-expressing NDRG2 (or down-regulating) via transfecting LM-MCF-7 (or MCF-7) cells with pCMV-NDRG2 (or NDRG2 siRNA) could inhibit (or promote) cell proliferation. Interestingly, the results of Western blot, immunolfuorescence and lfow cytometry revealed that down-regulation of NDRG2 resulted from the down-regulation of β-catenin and blocking its nuclear translocation, which led to losing control of the proliferation of breast cancer cells.Conclusion:NDRG2 inhibit the proliferation of breast cancer cells via down-regulating the expression of β-catenin and blocking its nuclear translo-cation, which is signiifcant for exploring the molecular mechanism of proliferation of breast cancer cells.
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The pancreatic tissues from patients with islet cell hyperplasia,insulinoma,and pancreatic adenocarcinoma,as well as normal pancreatic tissues were embedded with paraffin,serial sections were cut and mounted on glass slides.Immunohistochemical staining was carried out with N-myc down-stream regulated gene 2 (Ndrg2) monoclonal antibody by means of ABC method,and Western blotting was carried out to detect the expression and distribution of Ndrg2.The results showed that Ndrg2 positive immunoreactivity was mainly localized in the cytoplasm of islet cell,being similar to the localization of insulin positive immunoreactivity.The number and volume of pancreatic islets were increased in the patients with islet cell hyperplasia,and Ndrg2 expression was also increased.Western blotting results showed that the expression of Ndrg2 in the pancreas of patients with islet cell hyperplasia was increased compared with normal group.The above results suggest that Ndrg2 may play an important role in performing physiological function of islet cells.