Ying-Yu Ma

Recurrent TERT promoter mutations identified in a large-scale study of multiple tumour types are associated with increased TERT expression and telomerase activation

BACKGROUND Several somatic mutation hotspots were recently identified in the telomerase reverse transcriptase (TERT) promoter region in human cancers. Large scale studies of these mutations in multiple tumour types are limited, in particular in Asian populations. This study aimed to: analyse TERT promoter mutations in multiple tumour types in a large Chinese patient cohort, investigate novel tumour types and assess the functional significance of the mutations. METHODS TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumour types and 799 tumour tissues from Chinese cancer patients. Thymic epithelial tumours, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), telomerase activity by the telomeric repeat amplification protocol (TRAP) assay and promoter activity by the luciferase reporter assay. RESULTS TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%) and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in gastrointestinal stromal tumour (GIST), thymic epithelial tumours, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter. CONCLUSIONS TERT promoter mutations are frequent in multiple tumour types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumourigenesis, making them potential therapeutic targets.

Up-regulated miR-199a-5p in gastric cancer functions as an oncogene and targets klotho

BackgroundRecent studies have shown that miR-199a-5p plays opposite roles in cancer initiation and progression of different cancer types, acting as oncogene for some cancer types but as tumor suppressor gene for others. However, the role and molecular mechanism of miR-199a-5p in gastric cancer are largely unknown.MethodsIn this study, miR-199a-5p expression level in gastric cancer was first analyzed by qPCRand then validated in 103 gastric cancer patients by in situ hybridization (ISH). Gastric cancer cell lines were transfected with miR-199a-5p inhibitor and mimic, and underwent in vitro transwell assays. Target genes (klotho) were identified using Luciferase reporter assay. Immunohistochemical staining was also used to investigate on how miR-199a-5p regulates the tumour-suppressive effects of klotho in gastric cancer.ResultsIn our present study, we found that miR-199a-5p level was significantly increased in gastric cancer tissues compared to paired normal tissues. We observed that miR-199a-5p could promote migration and invasion of gastric cancer cells. In situ hybridization of miR-199a-5p also confirmed that higher miR-199a-5p expression level was associated with increased likelihood of lymph node metastasis and later TNM stage. Luciferase reporter assay and immunohistochemistry revealed that klotho might be the downstream target of miR-199a-5p.ConclusionsOur present study suggests that miR-199a-5p acts as an oncogene in gastric cancer and functions by targeting klotho.

Oridonin induces apoptosis in gastric cancer through Apaf-1, cytochrome c and caspase-3 signaling pathway

AIM To investigate the effect and mechanism of oridonin on the gastric cancer cell line HGC-27 in vitro. METHODS The inhibitory effect of oridonin on HGC-27 cells was detected using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. After treatment with 10 μg/mL oridonin for 24 h and 48 h, the cells were stained with acridine orange/ethidium bromide. The morphologic changes were observed under an inverted fluorescence microscope. DNA fragmentation (a hallmark of apoptosis) and lactate dehydrogenase activity were examined using DNA ladder assay and lactate dehydrogenase-release assay. After treated with oridonin (0, 1.25, 2.5, 5 and 10 μg/mL), HGC-27 cells were collected for anexin V-phycoerythrin and 7-amino-actinomycin D double staining and tested by flow cytometric analysis, and oridonin- induced apoptosis in HGC-27 cells was detected. After treatment with oridonin for 24 h, the effects of oridonin on expression of Apaf-1, Bcl-2, Bax, caspase-3 and cytochrome c were also analyzed using reverse-transcript polymerase chain reaction (RT-PCR) and Western blotting. RESULTS Oridonin significantly inhibited the proliferation of HGC-27 cells in a dose- and time-dependent manner. The inhibition rates of HGC-27 treated with four different concentrations of oridonin for 24 h (1.25, 2.5, 5 and 10 μg/mL) were 1.78% ± 0.36%, 4.96% ± 1.59%, 10.35% ± 2.76% and 41.6% ± 4.29%, respectively, which showed a significant difference (P < 0.05). The inhibition rates of HGC-27 treated with oridonin at the four concentrations for 48 h were 14.77% ± 4.21%, 21.57% ± 3.75%, 30.31% ± 4.91% and 61.19% ± 5.81%, with a significant difference (P < 0.05). The inhibition rates of HGC-27 treated with oridonin for 72 h at the four concentrations were 25.77% ± 4.85%, 31.86% ± 3.86%, 48.30% ± 4.16% and 81.80% ± 6.72%, with a significant difference (P < 0.05). Cells treated with oridonin showed typical apoptotic features with acridine orange/ethidium bromide staining. After treatment with oridonin, the cells became round, shrank, and developed small buds around the nuclear membrane while forming apoptotic bodies. Lactate dehydrogenase (LDH) release assay showed that after treated with 1.25 μg/mL and 20 μg/mL oridonin for 24 h, LDH release of HGC-27 caused by apoptosis increased from 22.94% ± 3.8% to 52.68% ± 2.4% (P < 0.001). However, the change in the release of LDH caused by necrosis was insignificant, suggesting that the major cause of oridonin-induced HGC-27 cell death was apoptosis. Flow cytometric analysis also revealed that oridonin induced significant apoptosis compared with the controls (P < 0.05). And the apoptosis rates of HGC-27 induced by the four different concentrations of oridonin were 5.3% ± 1.02%, 12.8% ± 2.53%, 28.5% ± 4.23% and 49.6% ± 3.76%, which were in a dose-dependent manner (P < 0.05). After treatment for 24 h, DNA ladder showed that oridonin induced a significant increase in DNA fragmentation in a dose-dependent manner. RT-PCR revealed that mRNA expression levels were up-regulated compared with the controls in caspase-3 (0.917 ± 0.103 vs 0.357 ± 0.019, P < 0.05), cytochrome c (1.429 ± 0.111 vs 1.002 ± 0.014, P < 0.05), Apaf-1 (0.688 ± 0.101 vs 0.242 ± 0.037, P < 0.05) and Bax (0.856 ± 0.101 vs 0.278 ± 0.027, P < 0.05) (P < 0.05), whereas down-regulated in Bcl-2 (0.085 ± 0.012 vs 0.175 ± 0.030, P < 0.05). Western blotting analysis also confirmed this result. CONCLUSION Apoptosis of HGC-27 induced by oridonin may be associated with differential expression of Apaf-1, caspase-3 and cytochrome c, which are highly dependent upon the mitochondrial pathway.

Inducible nitric oxide synthase expression correlates with angiogenesis, lymphangiogenesis, and poor prognosis in gastric cancer patients ☆

Increased nitric oxide synthase expression plays a key role in tumor progression. To examine inducible nitric oxide synthase expression and its correlation with clinical variables, such as tumor progression, angiogenesis, lymphangiogenesis, and prognosis in gastric cancer, we studied inducible nitric oxide synthase expression in gastric cancer samples from 211 patients with 5-year follow-up. CD105 and D2-40 were adopted as biomarkers for tumor angiogenesis and lymphangiogenesis, respectively. Inducible nitric oxide synthase staining was mainly found in the cytoplasm of gastric cancer tumor cells. Positive inducible nitric oxide synthase immunoreactivity was seen in 54.03% of gastric cancer specimens, which was correlated with lymph node metastasis, vascular invasion, distant metastasis, and TNM stage. Compared with inducible nitric oxide synthase negative patients, inducible nitric oxide synthase-positive patients had significantly shorter survival times and higher microvessel density and lymphatic vessel density. Intratumor and peritumor blood microvessel density and lymphatic vessel density correlated with inducible nitric oxide synthase expression (Spearman ρ test, P < .05). We conclude that inducible nitric oxide synthase expression correlates with lymph node metastasis, vascular invasion, distant metastasis, TNM stage, and poor survival rate in gastric cancer. We propose that synthesized inducible nitric oxide synthase increases angiogenesis, and lymphangiogenesis thus promotes tumor progression. Inducible nitric oxide synthase expression may be a good biomarker for poor prognosis in gastric cancer.

Expressions of Neutrophil Gelatinase‐Associated Lipocalin in Gastric Cancer: A Potential Biomarker for Prognosis and an Ancillary Diagnostic Test

The aim of this study was to explore the clinical significance of neutrophil gelatinase‐associated lipocalin (NGAL) in the development and prognosis of gastric cancer. NGAL tumor levels were determined in 333 GC patients by immunohistochemistry. NGAL in blood samples from 63 healthy donors and 60 gastric cancer patients were also determined by enzyme‐linked immunosorbent assay. Rate of NGAL expression was correlated with the size of tumor (69.3% in >4 cm tumors vs. 46.1% in ≤4 cm tumors), Laurens classification (84.3% in diffuse type vs. 28.2% in intestinal type), lymph node metastasis (75.6% vs. 16.4% with no metastasis), vascular invasion (74.9% vs. 26.8% with no invasion), distant metastasis (94.3% vs. 50.3% with no distant metastasis), and TNM stage (81.8% in TNM III+IV vs. 20.5% in TNM I+II). NGAL expression can be used as an independent prognostic factor in gastric cancer as indicated by multivariate analysis. Positivity for serum NGAL was higher than that for carbohydrate antigen determinant, CA19‐9 (38.1% vs. 12.5%) in TNM I, and higher than that for carcinoembryonic antigen, CEA (58.3% vs. 8.3%) and CA19‐9 (58.3% vs. 8.3%) in TNM II. In conclusion, serum NGAL has great potential to be used as an ancillary test for diagnosis of gastric cancer. Increased expression of NGAL in tumors suggests gastric cancer is likely to be at an advanced stage with invasion and metastasis, and also poor prognosis. Anat Rec, 2010.

Suppression of selenium-binding protein 1 in gastric cancer is associated with poor survival.

Gastric cancer is one of the leading causes of death in Asian countries, especially in China. Because of the lack of suitable biomarkers for early detection, most patients are diagnosed at late stages, and the 5-year survival rate is low. In this study, we used proteomic analysis to identify specific disease-associated proteins as potential clinical biomarkers in gastric cancer. Protein spots were determined and identified by 2-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry in 12 paired specimens. Twenty distinct proteins displaying a difference in expression of at least 1.8-fold between the tumor and compared adjacent normal mucosa were detected by 2-dimensional polyacrylamide gel electrophoresis, and one of the spots was selenium-binding protein 1 identified by matrix-assisted laser desorption ionization time of flight mass spectrometry. Selenium-binding protein 1 was significantly decreased or even absent in gastric cancer tissue compared with the adjacent normal mucosa in 44 paired specimens detected by reverse transcriptase-polymerase chain reaction and in 28 paired specimens detected by Western blot. Immunohistochemical staining result of 126 cases showed that the selenium-binding protein 1 level was correlated with differentiation, TNM stage, and lymph node metastasis (P < .05). The 3-year survival rate of patients with high expression of selenium-binding protein 1 was significantly higher than that of patients with low expression. Our results provided the first evidence of selenium-binding protein 1 as a potentially novel biomarker for prognosis of gastric cancer.

Interaction of coagulation factors and tumor-associated macrophages mediates migration and invasion of gastric cancer

Abundant macrophage infiltration and increased expression of coagulation factors have been observed in cancer patients. The aim of the present study was to determine how the interaction between activated coagulation factors and monocytes/macrophages contributes to gastric cancer (GC) cell migration and invasion. We assessed cytokine/chemokine production of coagulation‐factor‐treated macrophages by ELISA. The effects of the interaction between coagulation factors and tumor‐associated macrophages (TAM) on GC cell migration and invasion were determined by in vitro migration and invasion assay. In addition, we used an in vitro co‐culture system of GC cells/TAM treated by coagulation factors to evaluate the effect of coagulation factor/TAM interaction on the human umbilical vein endothelial cell line (HUVEC). We found that the M2‐like phenotype of interleukin (IL)‐4high, IL‐10high, transforming growth factor (TGF)‐βhigh, tumor necrosis factor (TNF)‐αhigh was exhibited when the human monocytic cell line THP‐1 was stimulated by coagulation factors III (TF), VIIa (FVIIa) and XIIa (FXIIa). For the migration assay, the GC cells (BGC‐823 or SGC‐7901) that were co‐cultured with activated coagulation factor/TAM both showed increased migration. For the invasion assay, both BGC‐823 and SGC‐7901 cells co‐cultured with TF/TAM showed increased invasion. We also found that TAM activated by coagulation factors could induce vascular endothelial growth factor/MMP‐9 expression, which could promote invasion of GC cells. The HUVEC co‐cultured with TAM (PMA‐treated THP‐1 macrophages co‐cultured with GC cells) expressed high levels of FXIIa. In conclusion, coagulation factors might facilitate GC cell migration and invasion by transforming macrophages toward TAM‐like cells. Interaction of coagulation factors and TAM mediates migration and invasion of GC. (Cancer Sci 2011; 102: 336–342)

Therapeutic effects of amniotic fluid-derived mesenchymal stromal cells on lung injury in rats with emphysema

BackgroundIn chronic obstructive pulmonary disease (COPD), two major pathological changes that occur are the loss of alveolar structure and airspace enlargement. To treat COPD, it is crucial to repair damaged lung tissue and regenerate the lost alveoli. Type II alveolar epithelial cells (AECII) play a vital role in maintaining lung tissue repair, and amniotic fluid-derived mesenchymal stromal cells (AFMSCs) possess the characteristics of regular mesenchymal stromal cells. However, it remains untested whether transplantation of rat AFMSCs (rAFMSCs) might alleviate lung injury caused by emphysema by increasing the expression of surfactant protein (SP)A and SPC and inhibiting AECII apoptosis.MethodsWe analyzed the phenotypic characteristics, differentiation potential, and karyotype of rAFMSCs, which were isolated from pregnant Sprague–Dawley rats. Moreover, we examined the lung morphology and the expression levels of SPA and SPC in rats with emphysema after cigarette-smoke exposure and intratracheal lipopolysaccharide instillation and rAFMSC transplantation. The ability of rAFMSCs to differentiate was measured, and the apoptosis of AECII was evaluated.ResultsIn rAFMSCs, the surface antigens CD29, CD44, CD73, CD90, CD105, and CD166 were expressed, but CD14, CD19, CD34, and CD45 were not detected; rAFMSCs also strongly expressed the mRNA of octamer-binding transcription factor 4, and the cells could be induced to differentiate into adipocytes and osteocytes. Furthermore, rAFMSC treatment up-regulated the levels of SPA, SPC, and thyroid transcription factor 1 and inhibited AECII apoptosis, and rAFMSCs appeared to be capable of differentiating into AECII-like cells. Lung injury caused by emphysema was alleviated after rAFMSC treatment.ConclusionsrAFMSCs might differentiate into AECII-like cells or induce local regeneration of the lung alveolar epithelium in vivo after transplantation and thus could be used in COPD treatment and lung regenerative therapy.

Overexpression of FOXC1 correlates with poor prognosis in gastric cancer patients.

The aim of this study was to determine FOXC1 expression in gastric tissues, and the clinical significance of FOXC1 in the development, progression and metastasis of gastric cancer (GC).

Evaluation of REG4 for early diagnosis and prognosis of gastric cancer

We explored the correlation between the development of gastric cancer and the concentration of REG4 and hence the suitability of REG4 as an indicator of the prognosis of patients with GC. Real-time polymerase chain reaction was conducted to detect REG4 messenger RNA expression. The amount of the REG4 protein was measured by immunohistochemistry staining of tissue and enzyme-linked immunosorbent assay of serum. Serum carcinoembryonic antigen and carbohydrate antigen 19-9 concentrations were measured using a commercial automated immunoassay. Real-time polymerase chain reaction results confirmed that REG4 was significantly up-regulated in gastric cancer compared with paired normal mucosa (P < .001). Immunohistochemistry staining revealed that high expression of REG4 correlated with diffuse type, poor differentiation, lymph node metastasis, distant metastasis, and TNM stage III or IV. The mean survival time for patients in the REG4-positive group was significantly less than that in the REG4-negative group (P = .013). The percentage of serum samples that were REG4 positive was 44.0%, which was higher than that for serum carcinoembryonic antigen (P = .039) or carbohydrate antigen 19-9 (P = .012) in TNM stage I and was significantly higher (P = .031) than that in TNM stage II. Thus, REG4 may be not only a prognostic indicator but also a better serum marker than carcinoembryonic antigen and carbohydrate antigen 19-9 for early diagnosis of gastric cancer.