Expression of survivin in primary and metastatic gastric cancer cells obtained by laser capture microdissection

AIM:Survivin, a recently identified member of the inhibitor of apoptosis protein family, is expressed during development and in various human cancers. However, its expression in normal tissues and clinical relevance in cancers are still debated. In the present study, we analyzed the expression of the survivin gene in human primary and metastatic gastric cancer cells as well as in paired epithelial cells from normal gastric mucosa by means of a novel laser capture microdissection (LCM) technique coupled with reverse transcription -polymerase chain reaction (RT-PCR).METHODS: Thirty patients who had undergone gastrectomy with lymph node dissection for gastric cancer without preoperative treatments were included. Neoplastic tissue, metastatic lymph nodes, and apparently uninvolved normal tissue were collected from each patient. LCM-captured 'pure' cell groups were respectively subjected to RT-PCR analysis with primers specific for the survivin gene.RESULTS: Of the paired samples from 30 gastric cancer patients studied, 24 (80%) primary gastric cancer cell groups and 7 (23%) adjacent morphologically 'normal' gastric epithelial cell groups were shown to have a detectable survivin expression. There was a statistically significant difference in suvivin expression between these two groups (P<0.01). Meanwhile, 95% (19/20) of the metastatic gastric cancer cell groups from lymph nodes had a clear expression of the survivin gene. However, no significant correlation between survivin expression and clinicopathological features of gastric cancer was observed in the present study. CONCLUSION: Survivin expression is present in the majority of gastric cancer cell groups obtained by LCM techniques. The high expression rate in metastatic lesions suggests a possible role of survivin in cancer invasiveness and metastasis. It may contribute to the detection of gastric cancer micrometastasis as a potential molecular marker. In addition, the high expression percentage renders survivin a potential target in the therapy for gastric cancer.

Effect of mitogen-activated protein kinase signal transduction pathway on multidrug resistance induced by vincristine in gastric cancer cell line MGC803

AIM:To investigate the correlation between mitogen-activated protein kinase (MAPK) signal transduction pathway and multidrug resistance (MDR) in MGC803 cells.METHODS:Western blot was used to analyze the expression of MDR associated gene in transient vincristine (VCR) induced MGC803 cells, which were treated with or without the specific inhibitor of MAPK, PD098059.Morphologic analysis of the cells treated by VCR with or without PD098059 was determined by Wright-Giemsa staining. The cell cycle analysis was performed by using flow cytometric assay and the drug sensitivity of MGC803 cells which were exposed to VCR with or without PD098059 was tested by using MTT assay.RESULTS:Transient exposure to VCR induced P-gp butnot MRP1 or GST-π expression in MGC803 cells and the expression of P-gp was inhibited by PD098059.Apoptotic bodies were found in the cells treated with VCR or VCR+PD098059. FCM results indicated that more MGC803 cells showed apoptotic phenotype when treated by VCR and PD098059 (rate:31.23%) than treated by VCR only (rate:18.42%) (P<0.05).The IC50(284±13.2 μg/L) of MGC803 cells pretreated with VCR was 2.24-fold as that of negative control group (127±17.6μg/L) and 1.48-fold as that of the group treated with PD098059 (191±27.9μg/L).CONCLUSION:This study shows that the expression of P-gp can be induced by transient exposure to VCR and this induction can be prevented by PD098059, which can block the activity of MAPK. MAPK signal transduction pathway may play some roles in modulating MDR1 expression in gastric cancer.

Expression of ST13 in colorectal cancer and adjacent normal tissues

AIM: To investigate the in situ expression of suppressionof tumorigenecity 13 (ST13) mRNA in both colorectal cancer and adjacent normal tissues.METHODS: Colorectal cancer cell lines SW1116, SW620and CoLo205 were enrolled to confirm the feasibility of the in situ hybridization procedure. Seven colorectal cancer and adjacent normal tissues were included for RNA-RNA in situ hybridization.RESULTS: The expression of ST13 in the seven normal colon tissues was positive and the positive signals appeared in mucosal cells. Only three of the seven colorectal cancer tissues had positive hybridization signals that appeared in adenocarcinoma cells.CONCLUSION: The expression of ST13 decreases in colorectal cancer tissue compared with that in adjacent normal tissue. ST13 is mostly expressed in colorectal epithelia and adenocarcinoma cells.

Inducible nitric oxide synthase, nitrotyrosine and apoptosis in gastric adenocarcinomas and their correlation with a poor survival

AIM: To detect the presence of inducible nitric oxide synthase (iNOS), nitrotyrosine (NT) and apoptosis in gastric adenocarcinomas and their possible correlations with the clinicopathological characteristics and prognosis of gastric adenocarcinoma.METHODS: Sixty-six specimens of gastric adenocarcinoma and corresponding adjacent normal gastric tissues were studied. Immunohistochemistry was employed to localize iNOS and NT protein and an immunohistochemical scoring system was used. The occurrence of apoptotic cell death (apoptotic index [AI]) was analyzed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL) method. RESULTS: Results showed that iNOS expression wasdetected at an intermediate or high level in 41 of 66 (62%) specimens of gastric adenocarcinoma. NT expression was 58%. Neither of them was found in the normal gastric tissues; there were significant positive correlations among iNOSexpression, NT expression and AI. Many clinicopathologic characteristics of gastric adenocarcinoma, such as tumor size, depth of invasion, lymph node metastasis and TNM staging, were related to iNOS and NT expressions (P＜0.05). In 66 surviving patients, the 5-year survival rate of 41 patients who had tumors with intermediate or high iNOS expressions and high Ais (4.09%; 19.96%) was significantly lower than that of 25 patients who had tumors with negative or low iNOS expressions and low Ais (0.79%; 47.14%) (P= 0.001). COX's multivariate analysis revealed that the iNOS expression was identified as one of the significant independent prognostic factors predictive of a poor survival (relative risk [RR] = 2.69).CONCLUSION: NO produced by iNOS may play a stronger role in promoting gastric adenocarcinoma growth than in suppressing its growth. iNOS and NT expressions by gastric adenocarcinoma may correlate with a poor survival.

Correlation between metastatic potential and variants from colorectal tumor cell line HT-29

AIM: To evaluate the relationship between uPA, PAI-1,CEA, PI3K and metastatic potential in three colorectal tumor cell lines.METHODS: Metastatic model in nude rats was established by variants HT-29c and HT-29d cell lines and the metastatic potential of two tumor cell variants was compared.Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) were determined using ELISA in colorectal carcinoma WiDr, HT29 and HT-29d cell lines with different metastatic potentials.Expression of carcinoembryonic antigen (CEA) and phosphoinositide 3-kinase (PI3-Kinase) was analyzed using immunohistochemistry (IHC) in these cell lines in vitro and in vivo. CEA expression was compared using fluorescence activated cell sorter (FACS)in vitro.RESULTS: The number of HT-29d cells arrested in liver dramatically decreased within the initial 24 hours after injection. The taking rate of liver metastases in the variant HT-29d increased as compared with parental HT-29 cells (70 % versus 50 %) and a variant HT-29b cells (70 % versus 60 %), and extensive organs were synchronously involved in metastases. The uPA concentration of variant HT-29d cell line was significantly higher than that of the non-metastatic WiDr and the low metastatic HT-29 cell lines. The variant HT-29d cells produced stronger PI3-kinase expression as compared with the non-metastatic WiDr cells and the low metastatic HT-29 cellsin vivo.CONCLUSION: The selected variant HT-29d cell exhibited an enhanced metastatic potential. The level of uPA and PAI-1 is positively correlated with the metastatic capacity of tumor cells. The expression of PI3-kinase correlates with tumor development and metastasis.

Portal vein embolization before major hepatectomy

To discuss the rationale, techniques and the unsolved issues regarding preoperative portal vein embolization (PVE) before major hepatectomy. After a systematic search of Pubmed, we reviewed and retrieved literature related to PVE. Preoperative PVE is an approach that is gaining increasing acceptance in the preoperative treatment of selected patients prior to major hepatic resection. Induction of selective hypertrophy of the nondiseased portion of the liver with PVE in patients with either primary or secondary hepatobiliary, malignancy with small estimated future liver remnants (FLR) may result in fewer complications and shorter hospital stays following resection. Additionally, PVE performed in patients initially considered unsuitable for resection due to lack of sufficient remaining normal parenchyma may add to the pool of candidates for surgical treatment. The results suggest that PVE is recomm-endable in treating the cirrhotic patients before major liver resection.

Expression of a novel immunoglobulin gene SNC73 in human cancer and non-cancerous tissues

AIM: To investigate the expression of immunoglobulin gene SNC73 in malignant tumors and non-cancerous normal tissues.METHODS: Expression level of SNC73 in tumors and non-cancerous tissues from the same patient was determined by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay (RT-PCR-ELISA) in 90 cases of malignant tumors, including colorectal cancer, gastric cancer, breast cancer, lung cancer and liver cancer, Analysis on the correlation of SNC73 expression with sex, age, site,grade of differentiation, depth of invasion, and metastases in colorectal cancer patients was made.RESULTS: Expression level of SNC73 in non-cancerous colorectal mucosa and colorectal cancerous tissues was 1.234±0.842 and 0.737±0.731, respectively (P<0.01), with the mean ratio of 7.134±14.092 (range, 0.36-59.54).Expression of SNC73 showed no significant difference among gastric cancer, breast cancer, lung cancer and liver cancer when compared with non-cancerous tissues (P>0.05). No correlation was found between SNC73 expression level and various clinicopathological factors, including sex, age, site,grade of differentiation, depth of invasion and metastases of CRC patients.CONCLUSION: Down-regulation of SNC73 expression may be a relatively specific phenomenon in colorectal cancer.SNC73 is a potential genetic marker for the carcinongenesis of colorectal cancer. The relationship of SNC73 expression and carcinogenesis of colorectal cancer merits further study.

Amino acid uptake in arterio-venous serum of normal and cancerous colon tissues

AIM:To investigate the difference of amino acid uptake between normal and cancerous colon tissues.METHODS:Sixteen patients with colon cancer were enrlled in our study.Blood samples were taken during operations,serum amion acid concentrations of blood from cancerous or normal colon were analyzed.Amino acid uptake rate was calculated by the A-V difference and evaluated statistically.RESULTS:Except for methionine,the uptake rate of amino acids in cancer was higher than that in normal colon (25.01% vs -2.29%,p<0.01).The amino acid uoptake rate did not correlate to the size of tumor mass (p>0.05).There was no staistical significance in the amino acid uptake rate according to the Dukes stage,though it was higher in patients with Dukes stage C or D than that with Dukes stage B(P>0.05).CONCLUSION:Abnormal synthetic metabolism of colon cancer may contribute to its higher amino caid uptake rate than that of normal colon.

Ability of a Specific ERK Signal-Pathway Inhibitor to Reverse P-Glycoprotein- Mediated Vincristine Resistance in Colon Cancer Cell Unes

OBJECTIVE To investigate the effect of a specific inhibitor PD098059 of the extracellular-signal regulated protein kinase (ERK) pathway on the P-glycoprotein (P-gp)-mediated resistance of colon cancer cell lines SW480/VCR and CoLo205NCR.METHODS SW480NCR and CoLo205NCR cells were generated byexposuring SW480 and CoLo205 cells to vincristine (VCR) (30 ng/ml) for 72h, which resulted in a comparatively higher level of P-gp expression.Western blotting was used to analyze P-gp, MRP, LRP, GST-'rr and TOPOIIexpression after exposuring the SW480 and CoLo205 cells to VCR (30 ng/ml)for 72 hrs. P-gp and pERK1/2 expressions was analyzed in SW480NCR andCoLo205/VCR cells treated with or without the specific inhibitor of MEK,PD098059. The MTT assay was used to determine the susceptibility ofSW480NCR and CoLo205NCR cells to VCR, treated with or withoutPD098059.I^F.SULI"S The results showed that VCR induced a comparatively higher levelof P-gp expression in the cell lines, but not that of MRP, LRP, GST-n- orTOPOII. P-gp expression levels were depressed significantly in SW480/VCR and COLO205/VCR cells by the specific inhibitor of MEK, PD098059.The IC50 (248 +19.6 and 215 +10.7 ng/ml) to VCR of SW480/VCR andCoLo205/VCR cells exhibited a 2.16 and 2.03-fold higher resistancecompared to the negative control group (SW480 and CoLo205 cells)(115+15.6 and 106 +11.9 ng/ml), but a 1.35 and 1.21 -fold higher resistance thanthe group treated with VCR (30 ng/ml)+ PD098059 (184 + 21.8 and 177+19.4 ng/ml).CONCLUSION This study shows that the expression of P-gp can beinduced by exposuring cells to VCR, and that this induction can be reversedby inhibiting the ERK signaling pathway at the point of MEK by its specificinhibitor, PD098059. The ERK signal-transduction pathway may play a rolein modulating mdrl expression in colon cancer.

Gene transfer and expression of enhanced green fluorescent protein in variant HT-29c cells

AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT29 cells.METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (selected from HT-29 cells) were transduced by a retroviral vector encoding the GEFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transduced with the EGFP bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter (FACS) analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransduced and transduced cells in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo.RESULTS: After transduced, HT-29c cells displayed a stable and long-term EGFP expression under the nonselective conditionsin vitro. After cells were successively cultured to passage 50 in vitro, EGFP expression was still at a high level. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransduced parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed.CONCLUSION: An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo.These cells with EGFP are a valuable tool forin vivo research of tumor metastatic spread.

Clinicopathological and molecular genetic analysis of HNPCC in China

AIM: To explore the clinicopathological and molecular genetic features of hereditary nonpolyposis colorectal cancer (HNPCC) in Chinese population. METHODS: We collected 16 Chinese HNPCC families from Wenzhou, Zhejiang Province, China. Tumor tissues and peripheral white blood cells were studied using microdissection, microsatellite analysis, immunostaining of hMSH2 and hMLH1 proteins and direct DNA sequencing of hMSH2 and hMLH1 genes. RESULTS: (1) A total of 50 patients had CRC. Average age at diagnosis of the first CRC was 45.7 years; 40.9% and 28.7% of the CRCs were located proximal to the splenic flexure and in the rectum, respectively. Thirty-eight percent of the colorectal cancer patients had synchronous and metachronous CRC. 34.4% and 25% of the CRCs were poor differentiation cancer and mucinous adenocarcinoma, respectively. Fourteen extracoloni tumors were found, and the hepatic cancer was the most common tumor type. Twenty-one patients whose median survival time was 5.7 years died during 1-23 years. Twenty-nine patients have survived for 1-28 years, 58.6%, 41.4% and 24.1% patients have survived for more than 5, 10 and 15 years, respectively; (2) All nine tumor-tissues showed microsatellite instability (MSI) at more than two loci. Four tumor-tissues lost hMSH2 protein expression and one lost hMLH1 protein expression. Three pathological germline mutations were identified from five genetically analyzed families; two of three mutations had not been reported previously as they were a transition from C to A in exon 14 (codon 743) of hMSH2 and a TIC deletion in exon 14 (codon 530)of hMLH1. CONCLUSION: Chinese HNPCC have specific clinicopathological features, such as early onset, propensity to involve the proximal colon, and high frequency of multiple CRCs, liver cancer more frequent than endometrial cancer. Chinese HNPCC showed relatively frequent germline mutation of mismatch repair (MMR) genes that correlated closely with high-level MSI and loss of expression of MMR genes protein.

Predictive Factors Relating to Tumor-Free Survival Rates and the Clinical Management of Small Cancer of the Liver after Hepatectomy

OBJECTIVE To investigate factors associated with tumor-free survival rates and methods of treatment for small cancer of the liver. METHODS A total 105 cases of small cancer of the liver (maximum diameter ≤5 cm in a solitary nodule or the sum of maximum diameters in double nodules ≤5 cm) were studied between 1983 and 2000. Patients were divided into an invasive group (primary tumor accompanied by any one or more of the following features: satellite nodules, venous invasion, adjacent organ involvement and double nodules) and a non-invasive group. RESULTS Three patients died from liver failure within 30 days after operation and 100 of the other patients were followed-up. The 1, 3, 5, 7 and 10-year survival rates after the first resection were 95.8%, 64.8%, 48.8%, 39.4% and 34.3% respectively. The main factors influencing tumor-free survival after radical resection were tumor size, presence or absence of satellite nodules or vascular invasion, the incisal edge, the UICC TNM stage, and the number of tumor nodules. The survival rate of the invasive group was significantly lower than that of the non-invasive group. Tumors of fifty-one cases recurred after radical resection. For the recurrent patients, treatments included a repeated resection for 17 cases, transcatheter artery chemotherapy and embolization (TACE) for 18 cases and no treatment or chemotherapy for 18 cases. The 1,3 and 5-year survival rates after repeated hepatectomy for recurrent patients were 82.4% , 51.3% and 34.2% respectively, which were higher than those in the non-resected group.CONCLUSIONS Factors that influence postoperative tumor-free survival rate were concluded to be early stage detection, tumor invasive or noninvasive traits and the incisal edge. For the recurrent patients, active treatment especially a second hepatectomy, is safe and feasible, and can improve the 5-year survival rate by 10 percent. The categorization of invasive and non-invasive groups for small cancer of the liver is useful in clinical work. For patients with invasive small cancer of the liver, postoperative close surveillance and follow-up is the key to improve the prognosis.

TROP2与肿瘤的关系及其作为肿瘤治疗靶点的价值

恶性肿瘤是全球最主要的死亡原因.据WHO统计,2008年全球死于癌症的人数为760万人,占全球所有死亡人数的13％[1].恶性肿瘤的早期诊断是提高患者长期生存率、甚至治愈的关键,探索有效的早期诊断和对患者预后判断的可靠指标显得非常重要.癌基因的过表达是导致恶性肿瘤发生、侵袭和转移的重要因素,针对癌基因表达进行检测对疾病的治疗效果判断和预后评估起着越来越重要的作用,而靶向治疗则有可能使患者获得生命的延长甚至治愈.TROP2（tumor-associated calcium signal transducers 2）是一种与恶性肿瘤发生、侵袭和转移有关的癌基因,本文对TROP2与恶性肿瘤的关系及其潜在的靶向治疗价值的研究进展进行综述.

Human γδT-cell subsets and their involvement in tumor mmunity

γδT cells are a conserved population of innate lymphocytes with diverse structural and functional heterogeneity that participate in various immune responses during tumor progression. γδT cells perform potent immunosurveillance by exerting direct cytotoxicity, strong cytokine production and indirect antitumor immune responses. However, certain γδT-cell subsets also contribute to tumor progression by facilitating cancer-related inflammation and immunosuppression. Here, we review recent observations regarding the antitumor and protumor roles of major structural and functional subsets of human γδT cells, describing how these subsets are activated and polarized, and how these events relate to subsequent function in tumor immunity. These studies provide insights into the manipulation of γδT-cell function to facilitate more targeted approaches for tumor therapy.

Positive association of RhoC gene overexpression with tumour invasion and lymphatic metastasis in gastric carcinoma

Worldwide estimates establish gastric carcinoma as the second most frequent cause of cancer deaths. Tumour invasion and metastasis is the biggest impediment to gastric carcinoma cure. Active migration of tumour cells is now considered as the pivotal step in cancer invasion and metastasis. RhoC is a member of the Ras-superfamily of small guanosine triphosphatases that can regulate many cellular functions, especially cytoskeletal organization and cell locomotion. Overexpressing RhoC in vitro in the poorly metastatic cell line from human melanoma may induce a highly metastatic phenotype.~1 The recent development of laser capture microdissection (LCM) affords the opportunity to further evaluate the role RhoC plays in the invasion and metastasis of gastric carcinoma cells in their native tissue environment.