Spartina alterniflora invasion altered phosphorus retention and microbial phosphate solubilization of the Minjiang estuary wetland in southeastern China.

Spartina alterniflora invasion is considered a critical event affecting sediment phosphorus (P) availability and stock. However, P retention and microbial phosphate solubilization in the sediments invaded with or without S. alterniflora have not been fully investigated. In this study, a sequential fractionation method and high-throughput sequencing were used to analyze P transformation and the underlying microbial mechanisms in the sediments of no plant (NP) zone, transition (T) zone, and plant (P) zone. Results showed that except for organic phosphate (OP), total phosphate (TP), inorganic phosphate (IP), and available phosphate (AP) all followed a significant decrease trend from the NP site to the T site, and to the P site. The vertical decrease of TP, IP, and AP was also observed with an increase in soil depth. Among the six IP fractions, Fe-P, Oc-P, and Ca10-P were the predominant forms, while the presence of S. alterniflora resulted in an obvious P depletion except for Ca8-P and Al-P. Although S. alterniflora invasion did not significantly alter the alpha diversity of phosphate-solubilizing bacteria (PSB) harboring phoD gene, several PSB belonging to p_Proteobacteria, p_Planctomycetes, and p_Cyanobacteriota showed close correlations with P speciation and IP fractions. Further correlation analysis revealed that the reduced soil pH, soil TN and soil EC, and the increased soil TOC mediated by the invasion of S. alterniflora also significantly correlated to these PSB. Overall, this study elucidates the linkage between PSB and P speciation and provides new insights into understanding P retention and microbial P transformation in the coastal sediment invaded by S. alterniflora.

Value of multidetector-row computed tomography in the preoperative T and N staging of gastric carcinoma: a large-scale Chinese study.

OBJECTIVES: To investigate the value of multidetector-row computed tomography (MDCT) in the preoperative T and N staging of gastric carcinoma and to further investigate the clinicopathological factors affecting the diagnostic accuracy. METHODS: Seven hundred ninety gastric carcinoma patients underwent preoperative MDCT examination. The results of MDCT were compared with surgical and pathological findings. RESULTS: Early gastric carcinoma patients whose primary tumor was detected by MDCT had higher incidence of lymph node metastasis, larger tumor size, and deeper invasion. The overall accuracy of MDCT in determining T stage of gastric carcinoma was 73.80% (T1 45.93%, T2 53.03%, T3 86.49%, and T4 85.79%). The overall accuracy of MDCT in preoperative N staging was 75.22% (N0 76.17%, N1 68.81%, and N2 80.63%). The overall diagnostic sensitivity, specificity, and accuracy of MDCT for determining lymph node metastasis was 86.26%, 76.17%, and 82.09%, respectively. Multivariate analysis showed that the diagnostic sensitivity of MDCT in determining lymph node metastasis related with tumor size, N stage, and number of metastatic lymph nodes. CONCLUSIONS: The clinical value of MDCT in the preoperative T and N staging of gastric carcinoma is relatively high. MDCT can be the first choice for the preoperative evaluation of patients with gastric carcinoma.

Effect of surgical work volume on postoperative complication: superiority of specialized center in gastric cancer treatment.

PURPOSE: We investigated the risk factors for early postoperative complications after gastric cancer surgery. MATERIALS AND METHODS: The data from a total of 273 patients with gastric cancer were analyzed by univariate and multivariate analysis. We applied physiological and operative severity score for the enumeration of morbidity and mortality (POSSUM) to compare risk-adjusted surgical outcomes among different surgical units. RESULTS: Among the preoperative variables, patient gender, chronic obstructive pulmonary disease, surgical unit, and intraoperative blood loss were independent risk factors for a higher rate of postoperative complications. There were significant differences in complication rates among different surgical units (P = 0.001). The observed-to-expected morbidity ratio (O-to-E ratio) ranged from 0.81 to 1.63. Units with low surgical work volume had higher complication rates. Postoperative length of stay was significantly shorter (P = 0.000) and the rate of moderate and severe complications was significantly lower (P = 0.001) in specialized unit. CONCLUSIONS: POSSUM is a valid system for risk-adjusted evaluation of surgical outcomes. We conclude that surgical experience and work volume greatly influence the outcome, with overall surgical outcome in specialized centers superior to that in other units. Hence, gastric cancer surgery should be performed in specialized centers. Risk factors identified in this study need further confirmation by a prospective study involving a larger cohort.

Inhibition of the proliferation of human gastric cancer cells SGC-7901 in vitro and in vivo using Bcl-2 siRNA.

BACKGROUND: Bcl-2, the anti-apoptotic protein is overexpressed in the majority of gastric cancers and associated with its pathogenesis. To better understanding of the role of Bcl-2, RNA interference (RNAi) was used to inhibit Bcl-2 expression in the human gastric cancer cells in vitro and in vivo. METHODS: Bcl-2 small interfering RNA (siRNA) was transfected into human gastric cancer cells SGC-7901, and Bcl-2 expression was monitored by real-time polymerase chain reaction (PCR) and Western blot. Cell proliferation, apoptosis, and telomerase activity were examined by MTT, flow cytometry, and TRAP assay, respectively. Gastric cancer cells treated with 100 nmol/L Bcl-2 siRNA were subcutaneously transplanted into nude mice and tumor growth was assessed. RESULTS: Bcl-2 siRNA significantly inhibited the expression of Bcl-2 in human gastric cancer cells at both mRNA and protein levels in a time- and dose-dependent manner. Bcl-2 siRNA also decreased telomerase activity (by 78.76%) and increased the rate of apoptosis (by 37.47%). SGC-7901 cell growth was also significantly suppressed in vivo and in vitro. CONCLUSIONS: Bcl-2 expression knockdown suppressed the growth of gastric cancer cells. Thus, Bcl-2 may play a very important role in carcinogenesis of gastric cancer and its knockdown may offer a new potential gene therapy approach for human gastric cancer in future.

The impact of hyperthermic chemotherapy on human gastric cancer cell lines: preliminary results.

In this preliminary study, we evaluated the impact of hyperthermia (HT) and hyperthermic chemotherapy (HTCT) on six human gastric cancer cell lines and explored the mechanisms of cell-killing effect under HTCT. Treatment conditions were categorized into 4 modes: i) normothermic control (NT), ii) HT, iii) normothermic chemotherapy (NTCT) and iv) HTCT. According to the data of MTT and LM observations, isolated HT only temporarily inhibited cell proliferation and had no cell-killing effect on gastric cancer cell lines employed in our study except for SNU-1. Combining with HT enhanced the cytotoxicity of CDDP in all gastric cell lines and the concentration inhibiting cell proliferation and inducing cell death of HTCT was much lower than that of NTCT. There was a synergistic effect of HT and chemotherapy on inhibiting proliferation in each cell line in a certain range of CDDP concentration. The data of TEM and FCM proved that HTCT induced cell death with two modes - apoptosis and necrosis, and apoptosis was the major type. Microarray illustrated that, under HTCT, a total of 58 gene expressions were regulated according to the filtering criteria, including 10 extra genes with an expression change below the threshold or even unchanged when treated with either HT or CDDP alone. Five of these 10 genes were verified by QRT-PCR. These genes may include the target genes for the enhancing effect of HT on chemotherapy and their effects should be further validated by functional analysis.

[High-resolution analysis of chromosome 5 and identification of candidate genes in gastric cancer].

OBJECTIVE: To explore the relationship between loss of heterozygosity (LOH) of 5p with the histological phenotype in gastric cancer. METHODS: Eighty pairs of tumor and adjacent normal mucosa samples were collected and genomic DNA was extracted. Total of 17 polymorphic microsatellite markers for 5p were used for LOH analysis. A part of samples were fixed in 10% buffered formalin and stained with H&E. Histological type of gastric cancer was defined according to Lauren's classification. RESULTS: The average informative rate of all seventeen markers was 60.0%. The LOH at least in one locus was detected in 28 of the 80 (35.0%) cases. The highest LOH frequency occurring at D5S2849 (7.77 cM), with LOH frequency of 35.2% (19/54). The minimal LOH region was spanned from 6.67 to 9.41 cM (1.18 Mb, covering 2.7 cM), including D5S417, D5S2849, D5S1492 and D5S2088. In 28 with LOH, 24 (85.7%) cases were of intestinal type, and only 4 cases (14.3%) were of diffuse type. There is significant difference between LOH frequency in intestinal-type and diffuse-type gastric cancers (P < 0.01). Searching the NCBI database disclosed that this minimal deletion region at 5p15.33 covered 3 candidate genes, IRX1, IRX2, and CEI. CONCLUSION: The molecular events in 5p 15.33 may be related with the morphological differentiation and development of gastric cancer. Gastric cancer with LOH of 5p15.33 locus tends to develop in to intestinal type. The cluster of candidate genes in 5p15.33 may be closely implicated in carcinogenesis of intestinal type gastric carcinoma.

[Gene expression profiling of diffuse-type gastric cancer by cDNA microarray].

OBJECTIVE: To identify cancer-related genes in diffuse-type gastric cancer and to explore its molecular mechanism by cDNA microarray analysis. METHODS: A total of 22 pairs of diffuse-type gastric cancer tissue and the corresponding normal mucosa were taken and freshly frozen. cDNA microarray with 14,592 genes/ESTs was used. Genes were considered to be up- or down-regulated when the fluorescent intensity ratio between tumor and normal mucosa was over 2-fold in over 50% of the samples (P < 0.05). Hierarchical clustering of regulated genes was performed as a measure to study expressional similarity. Validation of array results was carried out by real time quantitative PCR (QPCR). RESULTS: Compared with those of corresponding normal mucosa, there were a total of 153 genes/ESTs up-regulated and 204 down-regulated in diffuse-type gastric cancer. Hierarchical clustering demonstrated that the genes belonging to the same subgroup displayed similar function. Most of the overexpressed genes were those related to cell adhesion, cell motility, matrix reconstruction, cell proliferation and/or signal transduction; while genes related to defense response, toxicoid metabolism, DNA repairing, nuclear-cytoplasmic transport and/or anti-apoptosis made up the main list of the underexpressed genes. Seven genes showed higher expression in TNM (T I + T II) group than in (T III + T IV) group. QPCR confirmed the array analysis results. CONCLUSION: Gene expression profiling by cDNA microarray analysis provides not only molecular understanding of biological properties of cancer, but may also be helpful in discovering new diagnostic markers and therapeutic targets in gastric adenocarcinoma.

[The relationship between frameshift mutations of transforming growth factor-beta type II receptor, insulin growth factor II receptor, bcl-2 associated X protein, E2F4 and microsatellite instability in gastric carcinoma].

OBJECTIVE: To determine the microsatellite instability in gastric carcinomas, examine the frameshift mutations of transforming growth factor-beta type II receptor (TGFbetaRII), insulin growth factor II receptor (IGFIIR), bcl-2 associated X protein (BAX) and E2F4, and investigate whether or how alterations of the TGFbetaRII, IGFIIR, BAX and E2F4 gene are associated with MSI in gastric cancer. METHODS: Formalin-fixed, paraffin-embedded gastrectomy specimens and matching normal tissues of 65 cases of gastric carcinomas were retrieved from shanghai Ruijin Hospital and Shanghai East Hospital. DNA was extracted from tissue sections using phenol chloral isoamyl alcohol. Sections with no more than 50% of tumor cell areas were isolated by microdissection. DNA was amplified by PCR-based single strand conformation polymorphism (SSCP) for microsatellite analysis and was sequenced directly. Frameshift mutations in the coding regions, repetitive mononucleotide tracts of (A)10 for TGFbetaRII, (G)8 for IGFIIR, (G)8 for BAX, and trinucleotide repeats of (AGC)13 for transcription factors E2F4 were detected using PCR. Tumors were classified as being microsatellite stable (MSS) or having a low frequency of MSI (MSI-L, one of markers different in the tumor) or a high frequency of MSI (MSI-H, two or more of markers different). RESULTS: Eleven cases (18.0%) showed MSI-L, 12 (19.7%) showed MSI-H and 38 (62.3%) cases showed MSS. The mutation rates of TGFbetaRII, IGFIIR, BAX and E2F4 gene were 19.7%, 4.9%, 6.6% and 16.4% respectively. Among the 12 MSI-H gastric cancers, there were 10 TGFbetaRII mutations, 3 IGFIIR mutations, 4 BAX mutations and 10 E2F4 gene mutations. The alterations in the repeats of the related genes presented polymorphisms. Associations of MSI-H status and mutations of the 4 genes were highly significant (P < 0.01, respectively). No repeat tracts mutations in TGFbetaRII, IGFIIR, BAX and E2F4 gene were found in MSS tumors. CONCLUSIONS: The repeat coding regions within TGFbetaRII, IGFIIR, BAX and E2F4 gene are the targets of microsatellite instability. Frameshift mutations of the 4 genes play an important role in the development and progression of gastric cancers with microsatellite instability.

Transcription factor Sp1 expression in gastric cancer and its relationship to long-term prognosis.

AIM: To explore the expression of Sp1 in gastric carcinoma as well as its association with other clinicopathologic features, and to evaluate the role of Sp1 as a prognostic indicator of gastric carcinoma. METHODS: By using immunohistochemistry, we examined the Sp1 expression patterns in 65 cases of human gastric cancer, and 40 normal gastric mucosa specimens. Simultaneously, the correlation between Sp1 expression and clinical outcome or clinicopathologic features was investigated. RESULTS: The percentage of Sp1 expression was 12.5% (5/40) in normal gastric mucosa, and the Sp1 protein was mainly expressed in the nuclei of cells located in the mucous neck region. In sharp contrast, strong Sp1 expression was detected in tumor cells, whereas no or faint Sp1 staining was detected in stromal cells and normal glandular cells surrounding the tumors. The expression rate of Sp1 in gastric cancer lesions was 53.85% (35/65). The medium survival duration in patients who had a tumor with negative, weak and strong Sp1 expressions was 1 700, 1 560 and 1 026 d, respectively (P<0.05). Sp1 protein expression was closely related to the depth of tumor infiltration (chi(2) = 13.223, P<0.01) and TNM stage (chi(2) = 11.009, P<0.05), but had no relationship with the number of lymph nodes and Lauren's classification (P>0.05). Cox regression model for multivariate analysis revealed that high Sp1 expression (P<0.05) and advanced stage (P<0.01) were independent predictors of poor survival. CONCLUSION: Normal and malignant gastric tissues have unique Sp1 expression patterns. Sp1 might serve as an independent prognostic factor, by influencing the tumor infiltration and progression.

Hepatic preconditioning of doxorubicin in stop-flow chemotherapy: NF-kappaB/IkappaB-alpha pathway and expression of HSP72.

AIM: To provide hepatic protection through administration of doxorubicin before stop-flow chemotherapy (SFC) and to investigate the expression of heat shock protein 72 (HSP72) and role of nuclear factor kappa B (NF-kappaB) in this effect. METHODS: The hepatic preconditioning of doxorubicin was established in a porcine model by injection of doxorubicin (1 mg/kg) before SFC. The experimental animals were randomized into two groups: groups receiving doxorubicin (DOX) and normal saline (NS). Serial serum and tissue samples were taken from both groups to evaluate the protection of doxorubicin. Western blot and immuno-precipitation were applied to detect the expression of HSP72, NF-kappaB p65 protein, inhibitor kappaB-alpha (IkappaB-alpha) and phosphorylated IkappaB-alpha as well. The expression of tumor necrosis factor alpha (TNF-alpha) was estimated by semiquantitative RT-PCR. And the extent of the hepatic injury was estimated with the level of serum aminotransferases. RESULTS: An abundance production of HSP72 in porcine liver was observed after 24 h of intravenous administration of doxorubicin, but without any change in the expression of NF-kappaB p65 subunit in cytoplasm. NF-kappaB p65 subunit accumulated in nuclei at the end of SFC and reached its highest level at 30 min after the restoration of the abdominal circulation and decreased gradually during the 6 h after SFC in NS group, while there was little change in DOX group. There was also a slight decrease of IkappaB-alpha at 30 min after the restoration of the abdominal circulation in NS group accompanying with the appearance of phosphorylated IkappaB-alpha. The expression of TNF-alpha was significantly higher in NS group than that in DOX group (average 1.40+/-0.17 vs 0.62+/-0.22, P<0.01) at serial time points after SFC. Serum ALT and AST levels of NS group were higher after 24 h than those of DOX group (93.2+/-7.8 IU/L vs 53.3+/-13.9 IU/L, 217.0+/-29.4 IU/L vs 155.0+/-15.6 IU/L for ALT and AST respectively, P<0.05) and after 48 h than those of DOX group (66.6+/-18.1 IU/L vs 43.3+/-16.7 IU/L, 174.4+/-21.3 IU/L vs 125.7+/-10.5 IU/L for ALT and AST respectively, P<0.05). CONCLUSION: Doxorubicin renders the liver to be tolerant to the hepatic influence in SFC in a porcine model through the NF-kappaB/IkappaB-alpha pathway with the expression of HSP72.

[Transcription factor Sp1 expression in gastric cancer and its prognostic value].

OBJECTIVE: To investigate the expression of transcription factor Sp1 in human gastric cancer tissues and normal gastric mucosa, and its prognostic significance. METHODS: By using immunohistochemistry, we studied the Sp1 expression patterns in 65 cases of gastric cancer with various clinico-pathologic characteristics, and 40 normal gastric mucosa specimens obtained from patients who underwent partial gastrectomy for benign gastric diseases. The significance of Sp1 expression on the survival of patients was evaluated. RESULTS: The expression rate of Sp1 in normal gastric mucosa was 12.5% (5/40). The positively stained glandular cells were mainly limited to those in the neck region. Cells at the basal portion of the gland were essentially negative. In sharp contrast, Sp1 expression rate in gastric cancer lesions was 53.8% (35/65). The medium survival time in patients who had a tumor with negative, weak and strong Sp1 expression was 1700, 1560 and 1026 days, respectively (P = 0.036). Sp1 protein expression was closely related to the depth of tumor invasion and TNM stage (P = 0.001, P = 0.026), but not related to the number of metastatic lymph nodes and Lauren's classification (P = 0.306, P = 0.667). CONCLUSION: Normal and malignant gastric tissues have unique Sp1 expression patterns. Sp1 might be served as an independent prognostic factor.

[uPA expression of gastric cancer cell lines and its correlation with peritoneal seeding].

OBJECTIVE: To study the correlation between expression of urokinase-type plasminogen activator (uPA) and capability of tumor cell seeding to the peritoneal membrane by different gastric cancer lines. METHODS: Expression of uPA in 4 human gastric cancer cell lines was examined by semi-quantitative RT-PCR, ELISA and Western blot. uPA activity was determined by an assay kit. After ip inoculation of cancer cells to nude mice, tumors on peritoneal membrane was grossly examined for tumor cell seedings. RESULTS: SGC7901 was the highest in uPA expression among human gastric cancer cell lines AGS, SGC7901, MKN45, and MKN28. MKN45 had the strongest uPA activity, while AGS was lowest in both uPA expression and activity. Peritoneal seeding tumors of various sizes were observed in mice inoculated with SGC7901 and MKN45 cells. In addition to peritoneal seedings, bloody ascites was present in mice inoculated with MKN28. The MKN45-inoculated mice took the least time to develop tumors and had the shortest surviving period. No peritoneal seeding was seen in mice inoculated with AGS cells. CONCLUSION: Three of 4 human gastric cancer cell lines studied express uPA mRNA and activity, which correlate with their peritoneal seeding potentials.

[Inhibitory effect of dendritic cells pulsed with MAGE-3 peptide on transplanted murine gastric cancer in mice].

OBJECTIVE: To observe the inhibitory effect of MAGE-3 peptide pulsed DC on transplanted murine gastric cancer in 615 mice. METHODS: The CTL clones directed against MAGE-3 peptide were tested for the ability to lyse the gastric cancer cell line MFC which can express MAGE-3 antigen. In immunoprotection experiment, mice of the study group were immunized with MAGE-3 peptide pulsed DC (DC/MAGE-3) at the dosage of 1 x 10(6) on d0 and d7 by sc inoculation, mice of control groups were immunized with influenza virus peptide pulsed DC (DC/Nup) or unpulsed DC at the same dosage on days as the DC/MAGE-3 group. On d14, all the mice were challenged with sc injections of 5 x 10(5) MFC gastric cancer cells. In immunotherapy experiment, all the mice were sc injected 5 x 10(5) MFC gastric cancer cells on d0, and on d3, d10 mice of each groups were sc inoculated with DC/MAGE-3, DC/Nup or unpulsed DC at the dosage of 1 x 10(6) respectively. All mice were monitored closely with respect to tumor growth and survival times. RESULTS: The CTL clone induce by MAGE-3 peptide could lyse the MFC cells efficiently. Immunization of mice with DC pulsed with MAGE-3 generated partial protective immunity against MFC tumor, as well as significant inhibition of tumor growth in a 3-day tumor model. CONCLUSION: The tumor vaccine with DCs pulsed with MAGE-3 peptide possesses the ability to stimulate tumor specific CTL activity and to establish antitumor immunity when administered in vivo.

TK gene combined with mIL-2 and mGM-CSF genes in treatment of gastric cancer.

AIM: Cancer gene therapy has received more and more attentions in the recent decade. Various systems of gene therapy for cancer have been developed. One of the most promising choices is the suicide gene. The product of thymidine kinase (TK) gene can convert ganciclovir (GCV) to phosphorylated GCV, which inhibits the synthesis of cell DNA, and then induces the cells to death. Cytokines play an important role in anti-tumor immunity. This experiment was designed to combine the TK gene and mIL-2/mGM-CSF genes to treat gastric cancer, and was expected to produce a marked anti-tumor effect. METHODS: TK gene was constructed into the retroviral vector pLxSN, and the mIL-2 and mGM-CSF genes were inserted into the eukaryotic expressing vector pIRES. The gastric cancer cells were transfected by retroviral serum that was harvested from the package cells. In vitro study, the transfected gastric cancer cells were maintained in the GCV- contained medium, to assay the cell killing effect and bystander effect. In vivo experiment, retroviral serum and cytokines plasmid were transfected into tumor-bearing mice, to observe the changes of tumor volumes and survival of the mice. RESULTS: In vitro experiment, 20 % TK gene transduced cells could cause 70-80 % of total cells to death. In vivo results showed that there was no treatment effect in control group and TK/GCV could inhibit the tumor growth. The strongest anti-tumor effect was shown in TK+mIL-2+mGM-CSF group. The pathologic examination showed necrosis of the cancer in the treated groups. CONCLUSION: TK/GCV can kill tumor cells and inhibit the tumor growth in vivo. IL-2 and GM-CSF strongly enhance the anti-tumor effect. Through the retrovirus and liposome methods, the suicide gene and cytokine genes are all expressed in the tissues.

Antitumor effects of vaccine consisting of dendritic cells pulsed with tumor RNA from gastric cancer.

AIM: To investigate the immunotherapeutic potential of vaccine consisting of dendritic cells (DCs) pulsed with total RNA from MFC gastric cancer cells. METHODS: DCs were prepared from the spleens of strain 615 mice by magnetic cell sorting (MACS). After culture for 24 h, DCs were pulsed with total RNA from MFC gastric cancer cells. Mice of one group were immunized with tumor RNA pulsed DC (RNA/DC) at the dosage of 1X10(6) on d 14 and 7 by s c inoculation before tumor implantation. Mice of another group were immunized with unpulsed DC (UDC) at the same dosage on days as the RNA/DC group. The third group of control mice was untreated. On d 0, all the mice were challenged with s c injections of 5X10(5) MFC gastric cancer cells. After inoculation, the mice were monitored closely with respect to tumor growth. Activities of NK cells in PBL and splenocytes and CTL were tested. RESULTS: On d 21 after tumor cell inoculation, the mice of control group manifested the largest tumors with volume at a mean of 2.6323+/-1.1435 cm(3), followed by the UDC and RNA/DC groups with mean volumes at 0.7536+/-0.3659 cm(3) and 0.3688+/-0.6571 cm(3), respectively. The activities of NK cells in PBL and splenocytes in RNA/DC group were 66.2% and 65.4%, respectively, higher than that in the control group. The tumor specific CTL activity in RNA/DC group was 49.5%, higher than that in the control group. CONCLUSION: The tumor vaccine with DCs pulsed with total RNA from gastric cancer cells possesses the ability to stimulate tumor specific CTL activity and to establish anti-tumor immunity when administered in vivo.

Inhibition of human telomerase in MKN-45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest.

AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.

Methionine-dependence and combination chemotherapy on human gastric cancer cells in vitro.

AIM: To elucidate whether human primary gastric cancer and gastric mucosa epithelial cells in vitro can grow normally in a methionine (Met) depleted environment, i.e. Met-dependence, and whether Met-depleting status can enhance the killing effect of chemotherapy on gastric cancer cells. METHODS: Fresh human gastric cancer and mucosal tissues were managed to form monocellular suspensions, which were then cultured in the Met-free but homocysteine-containing (Met(-)Hcy(+)) medium, with different chemotherapeutic drugs. The proliferation of the cells was examined by cell counter, flow cytometry (FCM) and microcytotoxicity assay (MTT). RESULTS: The growth of human primary gastric cancer cells in Met(-)Hcy(+) was suppressed, manifested by the decrease of total cell counts [1.46 +/- 0.42 (x 10(9).L(-1)) in Met(-)Hcy(+) vs 1.64 +/-0.44(x 10(9).L(-1)) in Met(+)Hcy(-), P<0.01], the decline in the percentage of G(0)G(1) phase cells (0.69 +/- 0.24 in Met(-)Hcy(+) vs 0.80 +/- 0.18 in Met(+)Hcy(-), P<0.01) and the increase of S cells (0.24 +/- 0.20 in Met(-)Hcy(+) vs 0.17 +/- 0.16 in Met(+)Hcy(-), P<0.01); however, gastric mucosal cells grew normally. If Met(-)Hcy(+) medium was used in combination with chemotherapeutic drugs, the number of surviving gastric cancer cells dropped significantly. CONCLUSION: Human primary gastric cancer cells in vitro are Met-dependent; however, gastric mucosal cells have not shown the same characteristics. Met(-)Hcy(+) environment may strengthen the killing effect of chemotherapy on human primary gastric cancer cells.

Correlation of thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase with sensitivity of gastrointestinal cancer cells to 5-fluorouracil and 5-fluoro-2'-deoxyuridine.

AIM: To determine the expression levels of three metabolic enzymes of fluoropyrimidines: thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in seven human gastrointestinal cancer cell lines, and to compare the enzyme levels with the sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd). METHODS: TS, TP and DPD mRNA levels were assessed by semi-quantitative RT-PCR, TP and DPD protein contents were measured by ELISA. Fifty percent inhibitory concentrations of growth (IC50), representing the sensitivity to drugs, were determined by MTT assay. RESULTS: IC50 values ranged from 1.28 to 12.26 microM for 5-FU, and from 5.02 to 24.21 microM for FdUrd, respectively. Cell lines with lower DPD mRNA and protein levels tended to be more sensitive to 5-FU (P<0.05), but neither TS nor TP correlated with 5-FU IC50 (P>0.05). Only TS mRNA level was sharply related with FdUrd sensitivity (P<0.05), but TP and DPD were not (P>0.05). A correlation was found between mRNA and protein levels of DPD (P<0.05), but not TP (P<0.05). CONCLUSION: DPD and TS enzyme levels may be useful indicators in predicting the antitumor activity of 5-FU or FdUrd, respectively.

Expression of vascular endothelial growth factor C and chemokine receptor CCR7 in gastric carcinoma and their values in predicting lymph node metastasis.

AIM: To study the expression of vascular endothelial growth factor C (VEGF-C) and chemokine receptor CCR7 in gastric carcinoma and to investigate their associations with lymph node metastasis of gastric carcinoma and their values in predicting lymph node metastasis. METHODS: The expression of VEGF-C and CCR7 in gastric carcinoma tissues obtained from 118 patients who underwent curative gastrectomy was examined by immunohistochemistry. Among these patients, 39 patients underwent multi-slice spiral CT (MSCT) examination. RESULTS: VEGF-C and CCR7 were positively expressed in 52.5 and 53.4% of patients. VEGF-C expression was more frequently found in tumors with lymph node metastasis than those without it (P<0.001). VEGF-C expression was also closely related to lymphatic invasion (P<0.001), vascular invasion (P<0.01), and TNM stage (P<0.001). However, there was no significant correlation between VEGF-C expression and age at surgery, gender, tumor size, tumor location, Lauren classification, and depth of invasion. CCR7 expression was significantly higher in patients with lymph node metastasis compared with those without lymph node metastasis (P<0.001) and was also associated with tumor size (P<0.01), depth of invasion (P<0.001), lymphatic invasion (P<0.001), and TNM stage (P<0.001). However, the presence of CCR7 had no correlation to age at surgery, gender, tumor location, Lauren classification, and vascular invasion. Among the 39 patients who underwent MSCT examination, only CCR7 expression was related to lymph node metastasis determined by MSCT (P<0.05). In the current retrospective study, the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of VEGF-C and CCR7 expression in the diagnosis of lymph node metastasis for patients with gastric carcinoma were 73.8%, 70.2%, 72.6%, 71.4% and 72.0%, and 82.0%, 77.2%, 79.4%, 80.0% and 79.7%, respectively. After subdivision according to the combination of VEGF-C and CCR7 expression, receiver operating characteristic (ROC) analysis showed that the accuracy of the combined examination of VEGF-C and CCR7 expression in predicting lymph node metastasis was relatively high (area under ROC curve [Az]=0.83). CONCLUSION: The expression of VEGF-C and CCR7 is related to lymph node metastasis of gastric carcinoma and both of them may become new targets for the treatment of gastric carcinoma. Furthermore, the combined examination of VEGF-C and CCR7 expression in endoscopic biopsy specimens may be useful in predicting lymph node metastasis of gastric carcinoma and deciding the extent of surgical lymph node resection.

[A preliminary study of endoscopic ultrasonography in the preoperative staging of early gastric carcinoma].

OBJECTIVE: To study the clinical value of endoscopic ultrasonography (EUS) in the preoperative staging of early gastric carcinoma. METHODS: EUS was performed in 149 gastric carcinoma patients proved by biopsy (including 33 patients with early gastric cancer), of which the results were compared with postoperative pathologic findings. RESULTS: The accuracy of EUS in determining the T stage of gastric carcinoma was 80.3% (T1 81.8%, T2 70.4%, T3 88.9%, T4 71.4%). The accuracy of EUS in differentiating early gastric carcinoma from advanced ones was 95.1%, and the accuracy of EUS in differentiating mucosal cancer from submucosal cancer was only 63.6%. The diagnostic accuracy of EUS for mucosal and submucosal cancer was 52.9% and 75%, with positive predictive value of 90% and 70.6%, respectively. The accuracy of invasion depth of EUS for the bulging and flat type of early gastric carcinoma was 100%, whereas the accuracy was only 58.6% for the depressed type. The accuracy of invasion depth of the differentiated and undifferentiated early cancer was 71.4%and 57.9%, without any significant difference (P > 0.05). The accuracy of invasion depth of EUS for early gastric carcinoma decreased as tumor size increased. The diagnostic accuracy of lymph node status of early gastric carcinoma by EUS was 90.9%, and the sensitivity and specificity of lymph node metastasis was 66.7% and 96.3%, respectively. CONCLUSION: The clinical value of endoscopic ultrasonography in the preoperative staging of early gastric carcinoma is relatively high.