Feasibility of Gastric Tumor Xenograft (GTX)-derived Cell Lines for Individualized Anti-cancer Drug Screening.

BACKGROUND/AIM: Because there are ongoing efforts to identify and develop novel drugs in the treatment of refractory gastric cancer, it is necessary to develop effective preclinical studies. Here, the preclinical efficacy of gastric tumor xenograft (GTX)-derived cell line models for the personalized treatment of gastric cancer was investigated. MATERIALS AND METHODS: Anti-cancer drugs were scanned with high-throughput screening (HTS) using pre-established GTX-derived cell lines. The efficacy of a selected drug (afatinib) was re-confirmed in vivo and intracellular signaling pathways were investigated in xenograft tumor cell lysates using western blotting. Validation studies, such as cell proliferation and caspase activity assays, were also conducted in vitro with GTX-derived cell lines. RESULTS: HTS indicated that afatinib was effective in one of the five GTX-derived cell lines (GTX-087). A xenograft mouse model was established from GTX-087, and administration of afatinib at 1 mg/20 g body weight/day per oral resulted in tumor-suppressive activity in vivo. The RAS-ERK pathway was inactivated by an increase in Bax and cleaved caspase-3 in this xenograft model. In vitro cell proliferation assay also revealed that afatinib was able to suppress the growth of the GTX-087 cell line. Caspase activity assay confirmed that afatinib had an apoptotic role on GTX-087 and showed that caspase-3/7 activity increased in a time dependent manner. CONCLUSION: The GTX-derived cell line model might be useful for estimating novel drug responses and could be an alternative to patient-derived xenograft animal models.

Prognostic Value of Highly Expressed Type VII Collagen (COL7A1) in Patients With Gastric Cancer.

Collagen is a major component in the tumor microenvironment. This study reveals a novel biomarker candidate, type VII collagen (COL7A1), in patients with gastric cancer. To identify genes differentially expressed in gastric cancer tissue, we analyzed cancerous (n = 20) and noncancerous tissues (n = 13) using a DNA microarray. To perform immunohistochemistry and validate the upregulation of COL7A1 expression, we collected 200 more gastric cancer tissues and 100 normal gastric tissues from 200 randomly selected patients who underwent gastrectomy for gastric cancer between January 2010 and December 2013. The correlations between COL7A1 expression and clinicopathological parameters and patients' overall survival (OS) were analyzed. In the microarray, COL7A1 was upregulated in gastric cancer tissue compared with normal tissue. In the immunohistochemistry study, COL7A1 was more highly expressed in cancer tissue than in normal tissue (p = 0.001). Patients with intracellular COL7A1 expression had significantly poorer five-year OS than those with only extracellular expression (41.5 versus 69.7%, p = 0.001), and the site of expression was an independent prognostic factor of OS (hazard ratio 2.00, 95% CI 1.26-3.16, p = 0.003). Also, we found a significant association between the COL7A1 immunohistochemistry score and distant metastasis (high versus low, odds ratio 4.45, 95% CI 1.40-14.16, p = 0.011). The site and total immunohistochemistry score of COL7A1 expression in gastric cancer showed prognostic significance for OS and distant metastasis, respectively. COL7A1 could be a novel biomarker with diagnostic and therapeutic value.

Proteins associated with reproductive disorders in testes of human erythropoietin gene-harboring transgenic boars.

To investigate reproductive disorder in human erythropoietin (EPO)-expressing pig, we performed comparative proteomic analyses of testicular tissues from human erythropoietin (hEPO) gene-harboring transgenic pigs and wild type pigs born from natural conception. In hEPO TG pigs, we found relatively low sperm motility and higher death rate indicating impaired sperm development. Consistently, plasma concentration of testosterone was significantly lower in the transgenic post-pubertal boars compared with wild type boars. Normalized protein spots showing higher than 2-fold differential expression intensity in two-dimensional polyacrylamide gel electrophoresis were selected for matrix associated laser desorption/ionization time-to-flight mass spectrometry analysis. Specific proteins were identified by searching the NCBI protein sequence databases. Among 55 proteins selected, 12 proteins were identified as those differentially expressed between transgenic and wild type pigs. Three downregulated proteins (ß-globin, carbonyl reductase 1, and peroxiredoxin 6) and nine upregulated proteins (cytoskeletal ß-actin, α 2,3-sialyltransferase, apolipoprotein A-I, tubulin α-1A chain, tropomodulin 3, thioredoxin, heat shock Protein 70.2, ch4/domains of swine IgM, and albumin), all of which are closely related to apoptosis and cytoskeletal development, were found in the transgenic boar testes. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay confirmed the increased occurrence of apoptosis in the transgenic boar testes compared with the wild type boar testes. Reproductive defects of the hEPO-expressing transgenic pigs may be caused by the abnormal expression of the genes identified in this study.

Mel-18 negatively regulates INK4a/ARF-independent cell cycle progression via Akt inactivation in breast cancer.

Mel-18, a polycomb group (PcG) protein, has been suggested as a tumor suppressor in human breast cancer. Previously, we reported that Mel-18 has antiproliferative activity in breast cancer cells. However, its functional mechanism has not been fully elucidated. Here, we investigated the role of Mel-18 in human breast cancer. We saw an inverse correlation between Mel-18 and phospho-Akt, which were expressed at low and high levels, respectively, in primary breast tumor tissues from 40 breast cancer patients. The effect of Mel-18 on cell growth was examined in two breast cancer cell lines, SK-BR-3 and T-47D, which express relatively low and high levels of endogenous Mel-18, respectively. On Mel-18 overexpression in SK-BR-3 cells, cell growth was attenuated and G(1) arrest was observed. Likewise, suppression of Mel-18 by antisense expression in T-47D cells led to enhanced cell growth and accelerated G(1)-S phase transition. In these cells, cyclin-dependent kinase (Cdk)-4 and Cdk2 activities were affected by Mel-18, which were mediated by changes in cyclin D1 expression and p27(Kip1) phosphorylation at Thr(157), but not by INK4a/ARF genes. The changes were both dependent on the phosphatidylinositol 3-kinase/Akt signaling pathway. Akt phosphorylation at Ser(473) was reduced by Mel-18 overexpression in SK-BR-3 cells and enhanced by Mel-18 suppression in T-47D cells. Akt-mediated cytoplasmic localization of p27(Kip1) was inhibited by Mel-18 in SK-BR-3 cells. Moreover, Mel-18 overexpression showed reduced glycogen synthase kinase-3beta phosphorylation, beta-catenin nuclear localization, T-cell factor/lymphoid enhancer factor promoter activity, and cyclin D1 mRNA level. Taken together, we established a linear relationship between Mel-18-->Akt-->G(1) phase regulators.

Id-1 regulates Bcl-2 and Bax expression through p53 and NF-kappaB in MCF-7 breast cancer cells.

Although increasing evidence supports the protective role of inhibitor of differentiation and DNA binding-1 (Id-1) against anticancer drug-induced apoptosis, the underlying molecular mechanisms seem to vary depending on the tumor system. Here, we examined the direct role of Id-1 in MCF-7 breast cancer cells by ectopically overexpressing Id-1 under serum-free condition, where the endogenous Id-1 expression was suppressed. Id-1 expression resulted in increased number of viable cells, reduced Bax expression, enhanced Bcl-2 expression, but no change in Bcl-xL expression. The expression of nuclear factor-kappaB (NF-kappaB) was augmented, while those of p53 and IkappaB were reduced. Such changes in p53 and NF-kappaB pathways were also functional, as assessed by real-time polymerase chain reactions and reporter assays of their known downstream targets, p21 and Il-6, as well as Bax and Bcl-2 genes. Finally, Id-1 played a protective role against taxol-induced apoptosis in breast cancer cells as assessed by MTT assay and apoptotic cell count upon taxol treatment (0-200 nM). Reduced Bax expression and enhanced Bcl-2 expression by Id-1 were also noted in the presence of taxol. Taken together, we present a molecular mechanism where Id-1 regulates p53 and NF-kappaB pathways, which in turn regulates Bax and Bcl-2 genes, thus providing a survival advantage under exogenous stress such as serum-free or taxol treatment in MCF-7 breast cancer cells. In this regard, inactivation of Id-1 may provide a potential therapeutic strategy leading to inhibition of breast cancer progression and anti-cancer drug resistance.

Anticancer effects of wogonin in both estrogen receptor-positive and -negative human breast cancer cell lines in vitro and in nude mice xenografts.

Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. Herein, we investigated the in vitro and in vivo anticancer effects and associated mechanisms of wogonin in human breast cancer. Effects of wogonin were examined in estrogen receptor (ER)-positive and -negative human breast cancer cells in culture for proliferation, cell cycle progression, and apoptosis. The in vivo effect of oral wogonin was examined on tumor xenograft growth in athymic nude mice. The molecular changes associated with the biological effects of wogonin were analyzed by immunoblotting. Cell growth was attenuated by wogonin (50-200 microM), independently of its ER status, in a time- and concentration-dependent manner. Apoptosis was enhanced and accompanied by upregulation of PARP and Caspase 3 cleavages as well as proapoptotic Bax protein. Akt activity was suppressed and reduced phosphorylation of its substrates, GSK-3beta and p27, was observed. Suppression of Cyclin D1 expression suggested the downregulation of the Akt-mediated canonical Wnt signaling pathway. ER expression was downregulated in ER-positive cells, while c-ErbB2 expression and its activity were suppressed in ER-negative SK-BR-3 cells. Wogonin feeding to mice showed inhibition of tumor growth of T47D and MDA-MB-231 xenografts by up to 88% without any toxicity after 4 weeks of treatment. As wogonin was effective both in vitro and in vivo, our novel findings open the possibility of wogonin as an effective therapeutic and/or chemopreventive agent against both ER-positive and -negative breast cancers, particularly against the more aggressive and hormonal therapy-resistant ER-negative types.