Huan Gu

RKIP and 14-3-3ε Exert an Opposite Effect on Human Gastric Cancer Cells SGC7901 by Regulating the ERK/MAPK Pathway Differently

BackgroundRaf-1 kinase inhibitor protein (RKIP) inhibits Raf (a key element in the ERK/MAPK pathway) and is regarded as anti-tumoral. In contrast, 14-3-3 is considered protumoral. However, the pathogenetic role of RKIP and 14-3-3ε in gastric cancer is unclear.AimThe purpose of this study was to examine the influence of 14-3-3ε and RKIP on SGC7901, the regulation of the ERK/MAKP pathway by both, and the interaction between the two proteins.MethodsRKIP and 14-3-3ε genes were introduced into SGC7901 cells using gene cloning technique, then, the bioactivities including the proliferation, migration and invasion of the cells were assessed by MTT and migration assays. ERK/MAKP pathway’s activity was examined using real-time quantitative RT–PCR, western blot, immunoprecipitation and 3D-immunolocalization techniques.ResultsOur results showed that RKIP inhibited SGC7901 cells’ bioactivities whereas 14-3-3ε upregulated them through the involvement of the ERK/MAPK pathway. RKIP inactivated this pathway, but 14-3-3ε activated it. RKIP and 14-3-3ε were co-localized in the cells and interacted with each other; this attributed to their opposite influence on the ERK/MAPK pathway and the cells bioactivities.ConclusionsThe ERK/MAPK pathway is involved in the pathogenesis of gastric cancer; RKIP and 14-3-3ε exert an opposite effect on this pathway and the cells possibly via both direct and indirect reactions with the elements in this pathway. The interaction between RKIP and 14-3-3ε may also contribute to their pathogenetic roles in gastric cancer.

The value of serum pepsinogen levels for the diagnosis of gastric diseases in Chinese Han people in midsouth China

BackgroundSerum pepsinogen (PG) levels are valuable in the diagnosis of gastric diseases. However, PG levels are affected by many factors such as the area and race. This study aimed to investigate serum PG levels in patients with different gastric diseases who were Chinese Han people in Hunan Province, midsouth China.MethodsA total of 248 gastric disease patients and 34 healthy controls were enrolled. The patients included those with non-atrophic and chronic atrophic gastritis, gastric and duodenal ulcer, early and advanced gastric cancer. Serum PG I and II levels were detected by Biohit ELISA kit (Finland), and PG I/II ratio was calculated. Differences in patients with gastric disease and healthy controls were analyzed using paired t-test.ResultsCompared with controls, patients with early and advanced gastric cancer had a significantly lower PG I level and PG I/II ratio (p <0.005). In contrast, patients with gastric and duodenal ulcer had a significantly higher PG I level (p <0.005). Compared with atrophic gastritis patients, patients with early and advanced carcinoma of the stomach had a significantly lower PG I/II ratio (p < 0.001). Combination of the cut-off levels of PG I (70 μg/L) and PG I/II ratio (6) provided 62.1% sensitivity of and 94.2% specificity for the diagnosis of gastric cancer.ConclusionsDecreased PG I level and PG I/II ratio are risk factors for gastric cancer. Combined use of serum PG I level and PG I/II ratio may help the early diagnosis of gastric cancer.

eIF4E promotes tumorigenesis and modulates chemosensitivity to cisplatin in esophageal squamous cell carcinoma

Patients with esophageal squamous cell cancer are often diagnosed with advanced diseases that respond poorly to chemotherapy. Overexpression of eIF4E leads to enhance the translation of key malignancy-related proteins and enabling tumor growth and chemoresistance in a variety of human malignancies, but whether it has a role in ESCC remains obscure. We hypothesized that eIF4E promoted ESCC tumorigenesis and facilitated the development of acquired resistance to the cisplatin-based chemotherapy. In this study, we showed that eIF4E expression was increased significantly in clinical ESCC tissues and and ESCC cell lines and its expression level was correlated with lymph node metastasis, TNM stage, as well as overall and disease-free survival of ESCC. We also showed here that knockdown of eIF4E in EC9706 would dramatically reduced cell proliferation, colony formation, migration and invasion, apoptosis in vitro as well as in vivo, and vice versa. Moreover, “weak mRNAs” were demonstrated to be regulated by eIF4E in ESCC, which might interpret the above function. Overexpression of eIF4E decreased the efficacy of cisplatin-induced cell growth inhibition in ESCC cell line and xenograft model (P < 0.05). eIF4E knockdown by shRNA increased cisplatin-induced cytotoxicity in ESCC cell lines, and enhanced chemosensitivity to cisplatin in xenograft tumor models. Furthermore, we found that the PI3K/AKT pathway and Bcl-2/Bax ratio might be responsible for the eIF4E-induced cisplatin resistance in ESCC. Our data collectively show association of eIF4E expression with chemotherapeutic response in ESCC, and suggest that therapeutically targeting eIF4E may be a viable means of improving chemotherapy response in ESCC.

Osteopontin expression is associated with hepatopathologic changes in Schistosoma japonicum infected mice

AIM To investigate osteopontin expression and its association with hepatopathologic changes in BALB/C mice infected with Schistosoma japonicum. METHODS The schistosomal hepatopathologic mouse model was established by abdominal infection with schistosomal cercaria. Liver samples were obtained from mice sacrificed at 6, 8, 10, 14, and 18 wk after infection. Liver histopathological changes were observed with hematoxylin-eosin and Masson trichrome staining. The expression of osteopontin was determined with immunohistochemistry, reverse transcription-polymerase chain reaction, and Western blotting. The expression of α-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1) were determined by immunohistochemistry. Correlations of osteopontin expression with other variables (α-SMA, TGF-β1, hepatopathologic features including granuloma formation and degree of liver fibrosis) were analyzed. RESULTS Typical schistosomal hepatopathologic changes were induced in the animals. Dynamic changes in the expression of osteopontin were observed at week 6. The expression increased, peaked at week 10 (P < 0.01), and then gradually decreased. Positive correlations between osteopontin expression and α-SMA (r = 0.720, P < 0.01), TGF-β1 (r = 0.905, P < 0.01), granuloma formation (r = 0.875, P < 0.01), and degree of liver fibrosis (r = 0.858, P < 0.01) were also observed. CONCLUSION Osteopontin may play an important role in schistosomal hepatopathology and may promote granuloma formation and liver fibrosis through an unexplored mechanism.

Construction of a novel vector expressing the fusion suicide gene yCDglyTK and hTERT-shRNA and its antitumor effects

This study aimed to construct a novel recombinant expression vector, pcDNA3.1(-)hTERT-shRNA/yCDglyTK. Its bioactivity and antitumor effects were investigated in the SGC7901 human gastric cancer cell line. Interfering RNA (RNAi) targeting human telomerase reverse transcriptase (hTERT) was applied to construct the pYr1.1-hTERT-shRNA vector. The shRNA expression cassette (including U6 promoter) was subcloned into the pcDNA3.1(-) CV-yCDglyTK vector to build a new vector, pcDNA3.1(-) hTERT-shRNA/yCDglyTK, which was identified by restriction enzyme digestion and gene sequencing. All the plasmids were delivered into SGC7901 cells using calcium phosphate nanoparticles (CPNPs). Expression of yCDglyTK and hTERT was detected by immunofluorescence, real-time PCR and western blot analysis. MTT assays were applied to measure the cytotoxic effect of the plasmids with 5-fluorocytosine (5-FC). Cell apoptosis was detected by flow cytometry. Restriction enzyme digestion and gene sequencing confirmed that the recombinant vector pcDNA3.1(-)hTERT-shRNA/yCDglyTK had been successfully constructed. Immunofluorescence, real-time PCR and western blot analysis showed that yCDglyTK was expressed, and that hTERT expression was inhibited in cells transfected with the recombinant vector. The cells transfected with the recombinant vector were the most sensitive to 5-FC and the apoptosis rates of the cells were also increased. The pcDNA3.1(-)hTERTshRNA/yCDglyTK vector was constructed successfully; it was confirmed that targeting hTERT through RNAi could synergize with suicide gene therapy.

14-3-3ε functions as an oncogene in SGC7901 gastric cancer cells through involvement of cyclin E and p27kip1

Investigation into the highly conserved 14‑3‑3ε protein has become increasingly important in cell biology due to its involvement in cell survival signaling, cell cycle control and apoptosis. The 14‑3‑3ε protein has been found to exert an impact on the development of various tumor types. However, the functional role and the possible mechanism of 14‑3‑3ε in gastric cancer remains to be elucidated. A previous study by our group indicated a negative correlation between 14‑3‑3ε expression levels and gastric cancer tissue differentiation and a positive correlation between 14‑3‑3ε expression levels and tumor infiltration, lymph node metastasis and tumor, nodes and metastasis staging. In the present study, 14‑3‑3ε suppression in the SGC7901 gastric cancer cell line was demonstrated to inhibit cell proliferation in vitro and tumor growth in vivo and the cell cycle‑associated proteins cyclin E and p27kip1 may have contributed to this antitumor effect. The present study showed for the first time that reducing the expression of 14‑3‑3ε may inhibit the proliferation and progression of gastric cancer and inhibition of this protein may provide a potential strategy for gastric cancer therapy in the future.

Clinical Characteristics and Prognostic Factors of Small Intestine Angiosarcoma: a Retrospective Clinical Analysis of 66 Cases

Background/Aims: Primary angiosarcoma of the small intestine is a rare neoplasia, and there are limited data from systematic analyses. The aim of this study is to describe the clinical and pathological characteristics in addition to the prognostic factors for this rare neoplasia. Methods: We retrospectively collected the clinical records and prognostic information of 66 patients with small intestine angiosarcoma reported between 1970 and 2017. We used the Chi-square test, the log-rank test, and Cox regression analyses to evaluate the data. Results: There were 66 patients diagnosed with small intestine angiosarcoma. The onset age ranged from 24–92 years old. There were 24 patients diagnosed before the year 2000, and 42 patients were diagnosed after 2000. The data indicated that 49 cases were diagnosed as primary disease, and the remaining 15 cases were secondary disease. The main clinical symptoms were nonspecific and included gastrointestinal (GI) bleeding and abdominal pain. Additionally, we found multi-center foci were one of the characteristics of this disease. Radiation-induced small intestine angiosarcoma (RSIA) is a special type of disease with a similar prognosis. This type was more frequent in females and decreased after the year 2000. We also found that GI bleeding was less common in RSIA cases. The log-rank test results revealed that old-age, poor differentiation, and GI bleeding were associated with worse prognosis. Surgical treatment showed a trend toward a prolonged survival time. However, the result was not statistically significant. Our results show treatment with adjuvant therapy improved prognosis. The multivariate Cox analysis demonstrated adjuvant therapy was an independent indicator of a favorable outcome in small intestine angiosarcoma patients. Conclusion: Pay attention to the unexplained gastrointestinal bleeding could lead to a faster diagnosis and control of small intestine angiosarcoma. Furthermore, treatments including adjuvant therapy can effectively improve the prognosis.

Mapping the interactome of overexpressed RAF kinase inhibitor protein in a gastric cancer cell line

BackgroundGastric cancer (GC) is a threat to human health with increasing incidence and mortality worldwide. Down-regulation or absence of RAF kinase inhibitor protein (RKIP) was associated with the occurrence, differentiation, invasion, and metastasis of GC. This study aims to investigate the molecular mechanisms and biological functions of RKIP in the GC biology.MethodsThe fusion expression plasmid pcDNA3.1-RKIP-3xFLAG was transfected into SGC7901 cells, the RKIP fusion proteins were purified with anti-flag M2 magnetic beads, and the RKIP-interacting proteins were identified with tandem mass spectrometry (MS/MS), and were analyzed with bioinformatics tools. Western blot and co-immunoprecipitation were used to confirm the interaction complex.ResultsA total of 72 RKIP-interacting proteins were identified by MS/MS. Those proteins play roles in enzyme metabolism, molecular chaperoning, biological oxidation, cytoskeleton organization, signal transduction, and enzymolysis. Three RKIP-interaction protein network diagrams were constructed with Michigan Molecular Interactions, functional linage network, and Predictome analysis to address the molecular pathways of the functional activity of RKIP. The MS/MS-characterized components of the existing interaction complex (RKIP, HSP90, 14-3-3ϵ, and keratin 8) were confirmed by Western blot analysis and co-immunoprecipitation.ConclusionThis study is the first discovery of the interaction of RKIP with HSP90, 14-3-3, and keratin. The present data would provide insight into the molecular mechanisms of how RKIP inhibits the occurrence and development of GC.