Significance of the expression of green fluorescent protein on detection of glioma invasion in vivo

Objective: To investigate the invasion and metastasis of glioma in vivo by xenotransplanted tumor established by implanting C6 glioma cells transfected with green fluorescent protein (GFP) gene in vitro into the brain of SD rats. Methods: C6 cells were transfected with a plasmid vector (pEGEP-N3) containing the GFP gene. Stable GFP-expressing clones were isolated and performed examination by flow cytometry and electron microscope. GFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later rats were killed and continuous brain sections respectively were examined by HE staining, immunohistochemistry method and fluorescence microscopy for detection of tumor cell invasion. Xenotransplanted tumor was primarily cultured to determine the storage of exotic GFP gene in vivo. Results: There were not obvious changes in cell cycle and ultrastructure for the cells transfected with GFP gene. C6 cells transfected with GFP gene maintained stable high-level GFP expression in the central nervous system during their growth in vivo. GFP fluorescence clearly demarcated the primary tumor margin and readily allowed for the detection of distant invasion on the single-cell level, which was evidently superior to HE and immunohistochemistry staining. There was not GFP gene loss of transfected cells in vivo. Conclusions: It is suggested that C6 cells transfected with GFP gene can be visualized by fluorescent microscopy after intracranial implantation. This model is an excellent experimental animal model in research on invasion of glioma. Foundation item: This work was supported by a grant from the National Natural science foundation of China (No. 39970752). Biography: LI Xia (1975–), master of neurosurgery, Xijing Hospital, the Fourth Military Medical University, majors in gene diagnosis and gene therapy of brain malignant tumor.     

Reversal of transformed phenotypes by herbimycin A in src oncogene expressed rat fibroblasts

We studied the effectiveness of herbimycin A, an inhibitor of the function of the src oncogene, to reverse the various transformed phenotypes in normal rat kidney (NRK) cells integrating temperature-sensitive v-src (ts/NRK). Elevated glucose transport in ts/NRK cells at a permissive temperature (33 degrees C) was decreased by herbimycin in 8 h to near the level that was observed either in ts/NRK grown at a nonpermissive temperature (39 degrees C) or in untransformed NRK cells at either temperature. Herbimycin caused no significant decrease in glucose uptake in ts/NRK cells grown at 39 degrees C. The effects of herbimycin on serum- and anchorage-independent growth properties of ts/NRK cells and of NRK cells integrating K-ras (KNRK) were also examined. With ts/NRK cells grown at 33 degrees C, the inhibition of cell growth by herbimycin became more pronounced when the serum concentration in the medium was lowered. With KNRK cells, in contrast, almost the same extent of cell growth inhibition was exerted by herbimycin irrespective of the serum concentration. Furthermore, with ts/NRK cells grown at 33 degrees C, herbimycin inhibited the colony formation in the soft agar medium more strongly than on a solid support. No such differential effects were observed with KNRK cells under similar conditions. These results suggest that herbimycin specifically acts on cells expressing the src oncogene and reverses various transformed characteristics to the normal ones.     

Aggregation of platelets and cell membrane vesiculation by rat cells transformed in vitro by Rous sarcoma virus.

Primary rat embryo cells and established normal rat kidney cells transformed in vitro by Rous sarcoma virus induced the aggregation of rat platelets in vitro. The aggregating activity was shown to be specific for the transformed cells and was absent in the normal parent cells. The aggregation reaction is accompanied by the release of serotonin from the platelets. Further analysis and purification of this activity from the transformed cells demonstrated that the activity is shed from the cells growing in culture and is associated with membrane vesicles of heterogenous size. The normal cells also produced vesicles in culture; however, the level of vesicle productio was less than that from transformed cells, and the platelet aggregation and serotonin release activities were greatly reduced or absent in these vesicles.     

A correlative study of Ki67 and vascular endothelial growth factor and their value in laryngeal squamous cell carcinoma

Objective： To study the correlation between Ki67 and vascular endothelial growth factor（VEGF） and the significance of their expression in laryngeal squamous cell carcinoma（LSCC）. Methods： The expressions of Ki67 and VEGF in 40 cases of LSCC and 5 cases of normal laryngeal mucosa were examined by immunohistochemical staining. Results： The expression levels of Ki67 and VEGF in LSCC tissue were higher than in normal laryngeal mucosa （Ki67： P〈0.001, VEGF： P〈0.001）. The two indexes＇ levels in patients of different age or different sex had no significant difference （P〉0.05）. They were higher in LSCC with metastasis of lymph nodes than in patients without metastasis （Ki67： P=0.034, VEGF： P=0.006）. The expressions of the two genes elevated correspondingly along with the development of LSCC T stage （P〈0.05）. In addition, correlation analysis indicated that the expression of Ki67 had a positive correlation with VEGF in LSCC（r=0.823, P〈0.01）. Conclusion： Ki67 and VEGF are objective indexes for the biological behavior of LSCC, and they might be helpful to the diagnosis, treatment and prognosis of laryngocarcinoma.     

体外模拟CO2气腹环境对宫颈癌细胞生长与转移的影响

 目的 研究体外模拟CO₂气腹环境对宫颈癌细胞生长与浸润转移的影响。方法 利用宫颈癌细胞作为研究对象,建立体外CO₂气腹模型,将宫颈癌细胞经不同压强、不同时间CO₂作用后, 测定细胞生长曲线、细胞集落形成、细胞生长周期、细胞侵袭黏附迁移能力。观察癌细胞生长浸润转移的变化。结果 经CO₂作用后,宫颈癌细胞的生长增殖在受到短暂抑制后,增殖能力明显增强,但与CO₂压力的改变无关;宫颈癌细胞的侵袭、迁移和黏附能力显著下降。结论 CO₂对宫颈癌细胞的生长增殖能力起先抑制后促进的作用;对宫颈癌细胞的浸润转移能力是抑制的。     

THERPEUTIC EFFECTS ON EXPERIMENTAL METASTIC TUMOR- BEARING MICE BY QI-LE(启乐) POLYSACCHARIDE ANTITUMOR METASTASES

Objective: To study the efficiency and the mechanism of QI-LE polysaccharide (QI-LE), a new compound extracted from one kind of Chinese herbs with antiplatelet activity, against tumor metastasis in mice. Methods: QI-LE was prepared by our Lab. C57BL/6 female mice transplanted with H₂₂ liver cancer cells were administered with the agent. Then the tumor nodules, pulmonary metastasis rate, and tumor cell proliferation activity were checked. Proliferation changes of tumor cells treated with different concentrations of QI-LE were also tested. Results: High and middle dosage of QI-LE could inhibit both the lymphatic and blood systemic tumor pulmonary metastases significantly, but not the tumor cell proliferation in vitro. Conclusion: QI-LE has antitumor metastasis activity, thus may be a new candidates for antitumor metastases agents. Foundation item: This work was supported by the Science Foundation from Health Department of Shandong Province (No. 2001CAICDAI) and Chinese Medicine Administration Bureau of Shandong Province(No. 99-46). Biography: Zheng Guang-juan(1964–), female, doctor of medicine, associate professor, Shandong University of Traditional Chinese Medicine, majors in tumor pathology and immunology.     

Exosomal miR-30a and miR-222 derived from colon cancer mesenchymal stem cells promote the tumorigenicity of colon cancer through targeting MIA3

BackgroundMultipotent mesenchymal stem cells (MSCs) derived from virus tumors have been reported to contribute to malignant cell growth, invasion, and metastasis. However, the mechanism of communication between MSCs and colon cancer cells is poorly understood. Recent studies have suggested that exosomes are an important player in crosstalk between cells and could significantly suppress the invasion ability of human cancer cells (hCCs) when transfected with a microRNA inhibitor. However, to date, no study has illuminated the miRNA changes in exosomes derived from hCC-MSCs.MethodsColon cancer stem cells were cultured in medium and passaged to develop fibroblast-like morphology. Exosomes were collected using ExoQuick precipitation and exosome morphology was visualized by transmission electron microscopy. Small RNA sequencing was analyzed using an Illumina HiSeq4000 analyzer, and the expression of MIA3 was assessed by real-time PCR and Western blot. The functional roles of miR-30a and miR-222 in colon cancer cells were evaluated through cell and animal experiments.ResultsOur results showed that the characteristics of MSC-like cells (hCC-MSCs) derived from human colon cancer stem cells were comparable to those of bone marrow-derived MSCs, including surface antigens and the ability to multi-differentiate to osteocytes and adipocytes. Furthermore, we screened the microRNA (miRNA) profiles of exosomes derived from hCC-MSCs and the corresponding parent hCC-MSCs. We found a significant enrichment in the miR-30a and miR-222 level in hCC-MSC-derived exosomes. Furthermore, in vitro and in vivo experiments demonstrated that miR-30a and miR-222 bound to their shared downstream target, MIA3, to promote the ability of colon cells to proliferate, migrate, and metastasize, thus evidencing their functional roles as oncogenic miRNAs.ConclusionsThese data suggest that hCC-MSC-secreted exosomes promote colon cancer cell proliferation and metastasis through delivering miR-30a and miR-222. Subsequently, exosomal miR-30a and miR-222 simultaneously target MIA3, suppress its expression, and promote colon cell proliferation, migration, and metastasis.     

Studies on the Expression of MMP-9 and Significance ofa Macrophage Assay in Oral Squamous Cell Carcinoma

OBJECTIVE To investigate the significance and relationship between matrix metaloproteinase 9(MMP-9)and infiltration of macrophages in the process of invasion and metastasis in oral squamous cel carcinoma(OSCC). METHODS The immunohistochemical SABC method was used to detect the expression of MMP-9 and CD68(for labeling macrophages)in 42 cases of OSCC and in 10 normal tissues. RESULTS The expression of MMP-9 and macrophage counts in the OSCC cases were significantly higher compared to normal tissues(P<0.05). The expression of MMP-9 and macrophage counts were related to lymphnode metastasis and the TNM stage(P<0.05),showing that there was a positive correlation among these parameters(γ=0.443,P<0.01). CONCLUSION Both MMP-9 and macrophages may play an important role in the process of invasion and metastasis in OSCC,and this cel ular activity may relate to the macrophages which affect the tumor cells and upregulate the expression of MMP-9.     

Detecting expression of MRP-1/CD9 mRNA in lung cancers using tissue microarrays and fluorescence in situ hybridization methods

Objective: The aim of this study was to investigate the MRP-1/CD9mRNA expression in lung cancer and normal lung tissues and the relationship between its expression and pathologic grades, clinical stages, metastasis and prognosis. Methods: To observe MRP-1/C9mRNA expression, tissue microarray (TMA) containing 54 lung cancers and 10 normal lung tissues was prepared and Fluorescence in situ hybridization was used. Results: The positive rate of MRP-1/CD9 expression was 48.1% in lung cancer, lower than that of normal lung tissues. The statistical difference was significant (P<0.05). Its protein expression had no relationship with the patients’ ages, sex and the macroscopic type of tumor, but had relationships with the histological type, clinical stage, differentiated degree and metastasis. The expression in non-small cell lung cancer (NSCLC) was higher than that in small cell lung cancer (SCLC); in well-moderately differentiated group was higher than that in poorly differentiated group; Earlier period group (I+II) was higher than in later period group (III+IV); and in group without lymphoid metastasis was higher than in patients with lymphoid metastasis. Conclusion: The progression of the lung cancer maybe related with the descended MRP-1/Cd9 expression, which may be useful in evaluating the prognosis of cancer patients. Foundation item: This work was supported by a grant from Tianjin Science and Technology Committee (No. 033804211) Biography: WANG Xin-yun(1945–), female, professor, Tianjin Medical University, majors in molecular biology of lung cancer.     

Claudin-1, -3 and -4 proteins and mRNA expression in benign and malignant breast lesions: a research study

IntroductionWe compared levels of protein and mRNA expression of three members of the claudin (CLDN) family in malignant breast tumours and benign lesions.MethodsAltogether, 56 sections from 52 surgically resected breast specimens were analyzed for CLDN1, CLDN3 and CLDN4 expression by immunohistochemistry. mRNA was also analyzed using real-time PCR in 17 of the 52 cases.ResultsCLDNs were rarely observed exclusively at tight junction structures. CLDN1 was present in the membrane of normal duct cells and in some of the cell membranes from ductal carcinoma in situ, and was frequently observed in eight out of nine areas of apocrine metaplasia, whereas invasive tumours were negative for CLDN1 or it was present in a scattered distribution among such tumour cells (in 36/39 malignant tumours). CLDN3 was present in 49 of the 56 sections and CLDN4 was present in all 56 tissue sections. However, CLDN4 was highly positive in normal epithelial cells and was decreased or absent in 17 out of 21 ductal carcinoma grade 1, in special types of breast carcinoma (mucinous, papillary, tubular) and in areas of apocrine metaplasia. CLDN1 mRNA was downregulated by 12-fold in the sample (tumour) group as compared with the control group using GAPDH as the reference gene. CLDN3 and CLDN4 mRNA exhibited no difference in expression between invasive tumours and surrounding tissue.ConclusionsThe significant loss of CLDN1 protein in breast cancer cells suggests that CLDN1 may play a role in invasion and metastasis. The loss of CLDN4 expression in areas of apocrine metaplasia and in the majority of grade 1 invasive carcinomas also suggests a particular role for this protein in mammary glandular cell differentiation and carcinogenesis.     

EBV-LMP1上调Ezrin表达对鼻咽癌细胞转移潜能的影响

背景与目的:细胞骨架相关蛋白Ezrin的异常表达与肿瘤侵袭转移密切相关,既往研究已证实EB病毒潜伏膜蛋白1(Epstein-Barr virus latent membrane protein1,EBV-LMP1)可促进鼻咽癌细胞的转移能力。本研究旨在进一步探讨EBV-LMP1是否通过改变Ezrin的表达来影响鼻咽癌细胞的转移能力。方法:采用免疫细胞化学和Western blot检测两种鼻咽癌细胞株-CNE1细胞(EBV阴性)和CNE1-GL细胞(稳定转染EBV-LMP1)中LMP1和Ezrin的表达;细胞-基质粘附实验检测CNE1、CNE1-GL和经Ezrin抗体预处理的CNE1-GL细胞(AntiEzrin-CNE1-GL)对细胞外基质的粘附力;Transwell小室法检测上述3种细胞的运动和对重组基底膜侵袭能力。结果:CNE1细胞中Ezrin阴性表达,而CNE1-GL细胞中Ezrin强阳性表达。CNE1-GL细胞对细胞外基质的粘附率[(89.38±6.12)%]强于CNE1细胞[(49.42±5.37)%](P<0.001),而AntiEzrin-CNE1-GL细胞对基质的粘附率[(56.94±4.08)%]明显下降,与CNE1-GL相比,差异有统计学意义(P<0.05)。运动实验和侵袭实验均提示CNE1-GL细胞运动、侵袭能力(107±11和179±25)强于CNE1细胞(27±3和46±6),差异均有统计学意义(P<0.001),而AntiEzrin-CNE1-GL细胞的运动、侵袭能力(38±4和51±5)较CNE1-GL细胞显均显著下降(P<0.001)。结论:CNE1细胞中EBV-LMP1可通过上调Ezrin表达来促进细胞转移。     

Cloning and high expression of full length hTRF1 in E. Coli

Telomere and telomerase is a new hot spot in tumor and leukemia study field. Human telomeric repeat binding factor (hTRF1) is a new telomeric repeat binding protein reported on Science in 1995, it plays an important role in maintaining telomere length and regulating telomerase activity.[1] Its excessive expression could induce gradually shortening of telomere length in HT1080 cell line. But the mechanism is still unknown.[2] There have been no reports on monoclone antibody to TRF1, nor rep…     

PEBP4 gene expression and its significance in invasion and metastasis of non-small cell lung cancer

The goal of this study was to investigate the function of phosphatidylethanolamine-binding protein 4 (PEBP4) in invasion and metastasis of non-small cell lung cancer (NSCLC). PEBP4 mRNA and protein expression in 56 cases of NSCLC tissues were detected using RT-PCR and Western blot, and the relationship between PEBP4 expression and invasion and metastasis of NSCLC was analyzed. The change in the invasive ability of human NSCLC cell line HCC827 was observed after knocking down PEBP4 expression using RNA interference. PEBP4 mRNA and protein expression in cancer tissues of patients with lymph node metastasis were significantly higher than those in patients without lymph node metastasis (p < 0.05). PEBP4 expression significantly decreased in HCC827 cells after transfection with PEBP4 siRNA (p < 0.01), and the number of HCC827 cells that migrated through Transwell chambers was significantly lower than that of non-transfected control and transfected control cells (p < 0.01). PEBP4 over-expression may promote the invasion and metastasis of NSCLC.     

Relationship between fibrinolysis of cultured cells and malignancy.

Cells cultured from various human and nonhuman malignant and normal tissues as well as mammalian cells transformed in vitro were examined for their ability to induce fibrinolysis. Generally, except for normal cells derived from lung or kidney, malignant cells had a greater ability to induce fibrinolysis than did their normal counterparts. A correlation existed between the abilities of the cells to induce fibrinolysis, grow in soft agar, and form tumors in immunosuppressed hosts.     

EXPRESSION OF β-CATENIN AND C-MYC IN THYMOMA AND THE ASSOCIATION WITH BIOLOGICAL FEATURES

Objective： β-catenin is closely associated with proliferation, differentiation, invasion and metastasis of tumor cells. C-myc is proved to be abnormally expressed in various kinds of malignant neoplasmas. This study is aimed to investigate the features of β-catenin and C-myc expressions in thymoma and the corresponding clinical significance. Method： Immunochemistry was used to detect the features of expressions of β-catenin and C-myc in thymoma and normal thymus. Of the 105 samples, according to Beratz classification, there were： epithelial type 34; lymphocyte type 35; mixed type 31 and shuttle cell type 5. According to Masaoka classification, there were： stage 1： 38; stage 2： 32; stage 3：20 and stage 4： 15. According to WHO classification, there were： type A 20; type AB 19; type B1 21; type B2 17; type B3 13 and type C 15. Results： Different ways of expression was revealed in different stages of thymomas. With the progress of thymoma, the expression of β-catenin on cell membrane decreased, while increased in cytoplasma and nucleus, even with nucleus shift. The expression of C-myc increased in invasive thymoma. Abnormal expression of β-catenin and C-myc increased, with significant difference（P〈0.05）. Conclusion： The different ways of expression and diversity of expression level may indicate the biological characters of thymoma, and could be used as an objective index for invasive thymoma.     

Claudin-1 enhances tumor proliferation and metastasis by regulating cell anoikis in gastric cancer

Claudin-1 (CLDN1) is overexpressed in gastric cancer and correlated with tumor invasion, metastasis and poor outcome. Here, we both down and up regulated CLDN1 expression in gastric cancer cells to elucidate its role in gastric carcinogenesis and tumor progression. We found that deficiency of CLDN1 inhibited cells migration, invasion, and colony formation in vitro and tumorigenicity, metastasis in vivo. Also, CLDN1 promoted cell aggregation and increased anoikis resistance. Down or up regulation of CLDN1 was accompanied with changes of membrane β-catenin expression as well as Akt and Src activities. When β-catenin was up-regulated in CLDN1-KD cells, cell aggregation and anoikis resistance were restored, and Akt and Src signal pathways were re-activated. Taken together, these findings suggest that CLDN1 is oncogenic in gastric cancer and its malignant potential may be attributed in part to regulation of anoikis, by mediating membrane β-catenin-regulated cell-cell adhesion and cell survival.     

Significance of circulating tumor cells in the portal vein regarding metastases and vascular invasion in hepatocellular carcinoma patients

BackgroundVascular invasion is an important risk factor of poor prognosis in hepatocellular carcinoma (HCC) patients. The detection of circulating tumor cells (CTCs) in the blood is direct evidence of tumor presence. There are few reports on CTCs and metastasis and vascular invasion of HCC. The purpose of this study was to analyze the significance of CTCs in the portal vein regarding metastases and vascular invasion in HCC patients.MethodsA total of 104 HCC patients diagnosed and treated in Zhengzhou University People’s Hospital were enrolled. Surgery was performed in 60 individuals. Portal vein blood samples were collected before treatment for CTCs detection. We used the isolation by size of epithelial tumor cells (ISET) and fluorescence in situ hybridization (FISH) to enrich and classify CTCs from blood samples. The patients were divided into metastasis and nonmetastasis groups according to the metastasis status before treatment. Differences in clinical indicators such as alpha-fetoprotein (AFP) levels, tumor size, CTCs count, and macrovascular tumor thrombus between the two groups were analyzed as well as the associations of CTCs count with the above indicators. For individuals with postoperative pathology, the relationship between CTCs counts and microvascular invasion (MVI) was analyzed.ResultsThe amounts of portal vein CTCs were higher in patients with metastases compared with the nonmetastases group (20 vs. 7; z=3.795; P<0.001). Multivariate logistic regression analysis showed that the CTC count was a risk factor for HCC metastasis [odds ratio (OR) =1.044; 95% CI: 1.011–1.079]. The sensitivity and specificity of CTC count in predicting HCC metastasis were 82.93% and 52.38%, respectively. CTC count was significantly correlated with tumor size (r_s=0.308; P=0.001), vascular invasion (z=4.211; P<0.001), and MVI (z=12.763; P=0.002). A threshold CTC count of seven showed the most significant power for predicting metastasis.ConclusionsVascular invasion positivity was closely related to HCC metastasis. Portal vein CTC count before treatment was correlated with vascular invasion and could be considered one of the factors affecting HCC metastasis. However, the ability of CTC count was limited in predicting HCC metastasis due to insufficient specificity.     

Clinical implications of mRNA expression of KAI1 in ovarian cancer with or without metastasis

Background. KAI1 is a potential metastasis suppressor gene for prostate cancer. Decreased expression of KAI1 mRNA has been shown to be associated with the formation of metastasis and the progression of prostate, lung, breast, pancreatic, and bladder cancer. It has also been reported, however, that KAI1 expression is unchanged in metastatic and nonmetastatic esophageal and gastric cancer. We performed the present study to investigate the function of KAI1 in the progression and/or metastasis of ovarian cancer. Methods. We investigated the mRNA expression levels of the KAI1 gene, using quantitative polymerase chain reaction (PCR), in 29 ovarian tumors (1 adenoma, 2 low- malignant potential tumors, 9 adenocarcinomas without metastasis, and 17 adenocarcinomas with metastasis), seven ovarian cancer cell lines, and two normal ovaries. Using a thermal cycler, we found that the KAI1 gene was amplified in parallel with an internal control gene, β-Actin. The relative expression ratio (KAI1/β-Actin) as measured by densitometry was used to evaluate gene expression. Immunohistochemical localization of the KAI1 protein in ovarian cancer tissues was confirmed by the avidin-biotin peroxidase complex (ABC) method. Results. The mRNA expression levels of KAI1 were consistent in normal ovary, ovarian tumor samples, and ovarian cancer cell lines. No statistically significant difference in the KAI1 mRNA expression level was found in ovarian cancer samples with or without metastasis. Immunohistochemistry revealed that the KAI1 protein was expressed in the cell membranes of ovarian cancer cells. Conclusions. Our results suggest that reductions in KAI1 mRNA expression are not involved in either the progression or metastasis of ovarian carcinomas. Received: April 22, 1999 / Accepted: July 26, 1999