Effects of exogenous AGR2 on the proliferation and invasion abilities of colon cancer cells / 国际肿瘤学杂志

Objective:To detect the expressions of anterior gradient protein 2 (AGR2) in the cultures of three colon cancer cell lines SW480, SW620 and COLO205, and to investigate the effects of different concentrations of exogenous AGR2 on the proliferation, migration and invasion abilities of SW620 cells.Methods:Western blotting was used to detect the expression levels of AGR2 protein in SW480, SW620 and COLO205 colon cancer cell cultures. SW620 cells were divided into blank control group, anterior gradient protein 2 homologous human recombinant protein (rAGR2) low concentration group (100 μg/ml) and rAGR2 high concentration group (200 μg/ml), and CCK-8 assay, cell scratch assay and Transwell migration and invasion assay were used to detect the effects of different concentrations of rAGR2 on the biological behaviors of SW620 cells.Results:Western blotting results showed that the expression levels of AGR2 protein in SW480, SW620 and COLO205 cells were 0.545±0.097, 0.662±0.040 and 0.882±0.156 respectively, with a statistically significant difference ( F=7.46, P=0.024). The level of AGR2 protein in COLO205 cell line was significantly higher than that in SW480 and SW620 cell lines ( P=0.009; P=0.047). The results of CCK-8 experiment showed that the proliferative activities of SW620 cells in the blank control group, rAGR2 low concentration group and rAGR2 high concentration group were 0.422±0.031, 0.542±0.040 and 0.574±0.033 respectively, with a statistically significant difference ( F=26.35, P<0.001), and the rAGR2 low concentration group and rAGR2 high concentration group were significantly higher than the blank control group (both P<0.001). The results of cell scratching assay showed that the percentage of 36 h cell scratching area was (28.029±2.107)%, (20.642±0.983)% and (16.951±1.608)% for the three groups of cells respectively, with a statistically significant difference ( F=35.85, P<0.001), the rAGR2 low concentration group was higher than the blank control group ( P=0.001), and the rAGR2 high concentration group was higher than the rAGR2 low concentration group ( P=0.032). The results of cell migration assay showed that the number of cells migrated in the three groups was 447.1±32.3, 513.1±55.8 and 632.4±50.3 respectively, with a statistically significant difference ( F=35.62, P<0.001), the rAGR2 low concentration group was more than the blank control group ( P=0.007), and the rAGR2 high concentration group was more than the rAGR2 low concentration group ( P<0.001). The results of the invasion assay showed that the number of cells invaded in the three groups was 369.1±56.1, 505.1±34.4 and 579.0±71.5 respectively, with a statistically significant difference ( F=32.40, P<0.001), the rAGR2 low concentration group was more than the blank control group ( P<0.001), and the rAGR2 high concentration group was more than the rAGR2 low concentration group ( P=0.010). Conclusion:The expression of AGR2 protein varies in the extracellular fluid of different invasive colon cancer cells and increases with the invasive ability. AGR2 protein can increase the proliferation, migration and invasive abilities of colon cancer cells SW620.

Preliminary bioinformatics analyses of the expression and function of AGR2 in human breast cancer / 中国肿瘤生物治疗杂志

@#Objective: To explore the expression of AGR2 gene in breast cancer as well as to predict its relevant biological functions and molecular signaling pathways with bioinformatics tool. Methods: The expression of AGR2 in breast cancer tissues and normal tissues was analyzed in Oncomine and GEPIA databases, and the expression of AGR2 in breast cancer cell lines was evaluated in CCLE database. Meanwhile, HPA database was used to analyze the expression of AGR2 protein in normal and breast cancer tissues. Besides, the gene expression microarray data download from CCLE database was analyzed by using R software to obtain genes co-expressed withAGR2. Functional annotation ofAGR2 co-expressed genes was performed by using GO Enrichment and KEGG pathway analyses. Results: Oncomine and GEPIA databases showed that AGR2 gene was highly expressed in breast cancer tissues, and CCLE database analysis showed that AGR2 was highly expressed in all breast cancer cell lines. Immunohistochemistry results from the HPA database showed that the expression of AGR2 protein was significantly higher in breast cancer tissues compared with normal tissues. A total of 946 genes co-expressed with AGR2 in breast cancer were screened out with the R software. With the GO function Enrichment analysis, the co-expressed genes were demonstrated to be mainly involved in biological functions, such as protein localization to cell periphery, protein localization to plasma membrane, cell junction assembly, cell-substrate adhesion, and cell junction organization etc. In addition, the KEGG analysis results showed that co-expressed genes were mainly involved in the progression of gastric cancer and breast cancer, and were associated with proteoglycans in cancer, as well as proline, leucine and isoleucine degradation pathways. Conclusions:AGR2 is highly expressed in breast cancer tissues, which may be a potential oncogenic gene and a new therapeutic target of breast cancer.

Effect of EGFR-TKI combined with chemotherapy on serum IGF-1 and AGR2 levels in patients with advanced non-small cell lung cancer / 国际检验医学杂志

Objective To observe the effect of EGFR‐TKI combined with chemotherapy on the changes of serum insulin‐like growth factor1(IGF‐1)and anterior gradient‐2(AGR2)levels in the patients with advanced non‐small cell lung cancer(NSCLC) ,and to investigate whether IGF‐1 and AGR2 can serve as a potential indicator of the prognosis and efficacy of chemotherapy in NSCLC . Methods Sixty‐eight patients with advanced NSCLC were selected as the experimental group treated by EGFR‐TKI combined chemotherapy and 30 healthy people served as the healthy control group .(treatment group) .The levels of serum IGF‐1 and AGR2 before chemotherapy and at 3 weeks after chemotherapy were detected by ELISA .The influence of serum IGF‐1 and AGR2 levels on the prognosis was analyzed by using Kanplan‐Meier method .Results (1)The disease control rate(DCR)in the EGFR‐TKI com‐bined chemotherapy was 52 .9% ;(2)the level of serum IGF‐1 before treatment in the experimental group was (329 .35 ± 88 .13)μg/L ,which was significantly higher than (146 .36 ± 41 .27)μg/L in the healthy control group(P<0 .01);the level of serum AGR2 in experimental group was(16 .72 ± 6 .23)ng/mL ,which was significantly higher than (4 .38 ± 2 .17)ng/mL in the healthy control group(P<0 .01);serum levels of IGF‐1 and AGR2 after treatment were(211 .53 ± 52 .31)μg/L and (9 .72 ± 3 .56)ng/mL respec‐tively ,which were significantly lower than those before treatment (P<0 .01);serum IGF‐1 and AGR2 in NSCLC patients were pos‐itively correlated(r=0 .489 ,P<0 .01);(3)serum levels of IGF‐1 and AGR2 after chemotherapy were(128 .62 ± 48 .24)μg/L and (7 .22 ± 4 .27)ng/mL respectively ,which were obviously lower than(334 .23 ± 82 .11)μg/L and(18 .43 ± 6 .17)ng/mL before chem‐otherapy(all P<0 .01) .The Kanplan‐Meier analysis revealed that serum IGF‐1 and AGR2 levels in advanced NSCLC had an obvi‐ous influence on the prognosis .Conclusion Serum IGF‐l and AGR2 levels may have a potential clinical value to assess the therapeu‐tic efficacy of EGFR‐TKI combined chemotherapy and prognosis in advanced NSCLC .

Expression and its clinical significance of AGR2 and Lgr5 in colorectal cancer / 中国现代医生

Objective To investigate the expression and clinical significance of AGR2 and Lgr5 in CRC and the corre-lation with clinical pathologic features. To evaluate the significance with combined detection of the clinical and patho-logical diagnosis of CRC. Methods AGR2 and Lgr5 expression were assessed in 70 cases of CRC and 20 cases of nor-mal tissues near the cancer by immunohistochemistry. Results ①The expression of AGR2 was significantly higher in CRC than that in normal tissue(68.57%vs 25.00%,P<0.01). The AGR2 expression in CRC was correlated with the de-gree of tumor differentiation and lymph node metastasis(P<0.05).②The expression of Lgr5 was significantly higher in CRC than that in normal tissue (74.29%vs 40.00%, P<0.01). The Lgr5 expression in CRC was correlated with the de-gree of tumor differentiation﹑depth of invasion and lymph node metastasis(P<0.05). ③The Area Under Curve(AUC) of combined detection of AGR2 and Lgr5 were significantly higher than detection of both alone. the difference was statis-tically significant (P all <0.05). Conclusion AGR2 and Lgr5 played an important role in the occurrence and develop-ment of CRC, the combined detection not only contribute to the judgment of tumor progression, but also provide theo-retical support for molecular diagnosis and treatment of patients with CRC .

In vitro induction of anterior gradient-2-specific cytotoxic T lymphocytes by dendritic cells transduced with recombinant adenoviruses as a potential therapy for colorectal cancer

Anterior gradient-2 (AGR2) promotes tumor growth, cell migration, and cellular transformation, and is one of the specific mRNA markers for circulating tumor cells in patients with gastrointestinal cancer. We investigated the feasibility of AGR2 as a potent antigen for tumor immunotherapy against colorectal cancer (CRC) cells using dendritic cells (DCs) transduced with a recombinant adenovirus harboring the AGR2 gene (AdAGR2). DCs transduced with a recombinant adenovirus encoding the AGR2 gene (AdAGR2/DCs) were characterized. These genetically-modified DCs expressed AGR2 mRNA as well as AGR2 protein at a multiplicity of infection of 1,000 without any significant alterations in DC viability and cytokine secretion (IL-10 and IL-12p70) compared with unmodified DCs as a control. In addition, AdAGR2 transduction did not impair DC maturation, but enhanced expression of HLA-DR, CD80, and CD86. AdAGR2/DCs augmented the number of IFN-gamma-secreting T-cells and elicited potent AGR2-specific cytotoxic T lymphocytes capable of lysing AGR2-expressing CRC cell lines. These results suggest that AGR2 act as a potentially important antigen for immunotherapy against CRC in clinical applications.

AGR2, a mucinous ovarian cancer marker, promotes cell proliferation and migration

Ovarian cancer is a leading cause of death in women. Early detection of ovarian cancer is essential to decrease mortality. However, the early diagnosis of ovarian cancer is difficult due to a lack of clinical symptoms and suitable molecular diagnostic markers. Thus, identification of meaningful tumor biomarkers with potential clinical application is clearly needed. To search for a biomarker for the early detection of ovarian cancer, we identified human anterior gradient 2 (AGR2) from our systematic analysis of paired normal and ovarian tumor tissue cDNA microarray. We noted a marked overexpression of AGR2 mRNA and protein in early stage mucinous ovarian tumors compared to normal ovarian tissues and serous type ovarian tumors by Western blot analysis and immunohistochemistry. To further elucidate the role of AGR2 in ovarian tumorigenesis, stable 2774 human ovarian cancer cell lines overexpressing AGR2 were established. Forced expression of AGR2 in 2774 cells enhanced the growth and migration of ovarian cancer cells. AGR2 protein was detected in the serum of mucinous ovarian cancer patients by Western blot and ELISA analysis. Thus, AGR2 is a potential biomarker for the diagnosis of mucinous ovarian cancer and an ELISA assay may facilitate the early detection of mucinous ovarian cancer using patient serum.