Impacts of Bevacizumab on vascular endothelial growth factor and Sp1 expression in gastric cancer xenografts / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To evaluate the effects of Bevacizumab on the tumor growth, proliferation and apoptosis of gastric cancer xenograft, and the impacts on the VEGF and Sp1 expression.</p><p><b>METHODS</b>Gastric cancer xenografts in nude mice were established using SGC-7901 gastric cancer cell line. The nude mice were randomly divided into two groups, Bevacizumab treatment group and PBS group. The tumor sizes were measured for tumor growth curve. The proliferation and angiogenesis were evaluated by immunohistochemistry (IHC) staining of Ki67 and CD34. TUNEL assay was used for apoptosis evaluation. The expression of VEGF and Sp1 in tumor cells were detected by IHC and Western blot.</p><p><b>RESULTS</b>Compared to the PBS group, the tumor growth decreased significantly (P<0.05), the proliferation of tumor cells and angiogenesis decreased, and apoptosis index increased significantly [(5.3 ± 1.8)% vs. (16.7 ± 6.7)%, P<0.01] in Bevacizumab group. The results of IHC and Western blot demonstrated that the expression of VEGF and the microvessel density (MVD) was decreased (4.0 ± 1.0 vs. 16.3 ± 1.5, P<0.001) in Bevacizumab treatment group. No obvious changes of Sp1 expression were observed in Bevacizumab treatment group.</p><p><b>CONCLUSIONS</b>Bevacizumab can inhibit the growth of gastric cancer xenografts in nude mice, decrease the VEGF expression and MVD. However, the compensatory up-regulation of transcription factor Sp1 is not affected by Bevacizumab.</p>

Expression of metastasis associated 1 family member 2 (MTA2) in gastric cancer and its relationship with transcription factor Sp1 / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To determine the expression patterns of metastasis associated 1 family member 2 (MTA2) in gastric cancer and non-cancerous gastric mucosa, and analyze its relationship with nuclear transcription factor Sp1 expression.</p><p><b>METHODS</b>Tissue samples and clinicopathological information from 83 gastric cancer patients, who underwent surgery, were collected from Shanghai Rui Jin Hospital. All samples included cancer tissue and non-cancerous mucosa which was 5 cm away from the tumor lesion. The expression of MTA2 and Sp1 were detected by immunohistochemistry (IHC) staining. The mRNA of MTA2 was also detected by reverse transcription-polymerase chain reaction (RT-PCR). SPSS software was used for statistical analysis.</p><p><b>RESULTS</b>The expression of MTA2 protein was significantly higher in primary lesions of the gastric cancer than that in non-cancerous mucosa by IHC (31.3% vs 12.0%, P < 0.01). MTA2 expression was closely related with tumor invasion or T staging (χ(2) = 5.677, P < 0.05). Yet, no significant relationship was observed between MTA2 expression and other clinicopathological parameters, including the age, sex, tumor differentiation, Lauren classification, lymph node metastasis, distant metastasis, as well as pathological staging. Furthermore, MTA2 expression was concomitant with Sp1 expression (r = 0.320, P < 0.05). Elevated MTA2 expression was observed in Sp1 positive cancer tissues (χ(2) = 9.565, P < 0.01). RT-PCR results also demonstrated that MTA2 mRNA was also highly expressed in the tissue samples with Sp1 expression.</p><p><b>CONCLUSIONS</b>MTA2 is highly expressed in the primary lesions of gastric cancer than that in adjacent non-cancerous tissues, and is closely related with tumor invasion. MTA2 expression is elevated in Sp1 positive gastric cancer.</p>

Establishment of experimental angiogenic models with applications of quantitative digital image analysis / 中华病理学杂志

<p><b>OBJECTIVE</b>To establish experimental models for tumor neovascularization and to apply quantitative digital imaging analysis in the study.</p><p><b>METHODS</b>An endothelial tube formation model was established by human umbilical vein endothelial cells (HUVECs). A vasculogenic mimicry model was established by SGC-7901 gastric cancer cell line. Fertilized eggs were used to establish a chorioallantoic membrane angiogenesis model. Using gene transfection experiment, IRX1 tumor suppressor gene was chosen as a therapeutic target. Image Pro Plus (IPP) analysis software was used for digital vascular images analysis with parameters including points, lines, angles and integral absorbance (IA) for the tubular formation or vasculogenic mimicry.</p><p><b>RESULTS</b>Digital image analysis by IPP showed that HUVEC tubular formation was significantly inhibited in IRX1 transfectant, compared with controls. The tubular numbers in three groups were 12.80 +/- 3.83, 29.00 +/- 5.34 and 28.20 +/- 4.32 (P<0.01). The connection points of tubules in three groups were 13.20 +/- 2.59, 25.00 +/- 2.24 and 24.60 +/- 3.21 (P<0.01). The tubular lengths of three groups were (821.5 +/- 12.5), (930.9 +/- 13.5) and (948.4 +/- 18.1) microm (P=0.022). The IA values of PAS stain in three groups were 3606 +/- 363, 14 200 +/- 1251 and 15 043 +/- 1220 (P<0.01). In chick chorioallantoic membrane model, the angular numbers of tubules in three groups were 6.41 +/- 2.60, 10.27 +/- 2.65 and 9.18 +/- 1.99 (P<0.01).</p><p><b>CONCLUSIONS</b>The endothelial tube formation model, vasculogenic mimicry model and chorioallantoic membrane angiogenesis model are useful for gene therapy and drug screening with targeting neoplastic vascularization. Professional image analysis software may greatly facilitate the quantitative analysis of tumor neovascularization.</p>

Discovery and verification of matrix gla protein, a TNM staging and prognosis-related biomarker for gastric cancer / 中华病理学杂志

<p><b>OBJECTIVE</b>To analyze microarray datasets deposited in the public database and to identify TNM associated genes in gastric cancers.</p><p><b>METHODS</b>Microarray datasets of gastric cancer were selected from GEO database. Differentially expressed genes related to TNM staging were evaluated by significant analysis of the microarray using MultiExperiment Viewer (MEV) platform. Candidate gene expressions in gastric cancer tissues and cell lines were verified by reverse transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR, Western blot and immunohistochemistry.</p><p><b>RESULTS</b>GES4007 dataset was re-analyzed leading to the identification of 14 genes associated with TNM staging. Over-expression of matrix gla protein (MGP) was confirmed in gastric cancer cell lines and tumor tissues by quantitative RT-PCR, Western blot and immunohistochemistry. Increased MGP expression was found in 22 of 54 cases of (40.7%) gastric cancer specimens compared to the normal gastric tissues. The up-regulation of MGP mRNA expression closely correlated with TNM stage (P = 0.001) and prognosis (P = 0.006).</p><p><b>CONCLUSIONS</b>Public databases of microarray studies are the valuable resources for data mining. MGP has been identified and confirmed as a novel biomarker for the TNM stage and prognosis of gastric cancer.</p>

Significance of plasmic L-plastin levels in the diagnosis of colorectal cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the clinical significance of plasmic L-plastin level in patients with colorectal cancer.</p><p><b>METHODS</b>From March 2008 to March 2009, plasma samples were collected from 40 patients and 40 healthy controls. Plasmic L-plastin level was measured by ELISA kit and was compared to TIMP-1.</p><p><b>RESULTS</b>Plasmic L-plastin level in patients with colorectal cancer was higher than that in healthy adults (1.662±0.386 vs. 0.485±0.085 μg/L, P<0.01). The sensitivity of L-plastin in the diagnosis of colorectal cancer was 67.5%, and the specificity was 80.6%. The Youden index was 0.481 and AUC was 0.772 (P<0.01). Plasmic L-plastin levels were associated with the tumor size (P=0.006), serosal penetration (F=4.687, P<0.05) and lymphatic metastasis (P<0.01). Compared to plasmic TIMP-1 level, L-plastin showed the same capability in indicating the depth of tumor. The specificity of L-plastin was better in indicating lymphatic metastasis (86% vs. 58%, χ2=4.2, P<0.05).</p><p><b>CONCLUSIONS</b>Plasmic L-plastin level may serve as a potential marker in colorectal cancer.</p>

Promoter cloning and activity analysis for iroquois homeobox gene 1 in gastric mucosa cell line / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To clone core promoter regions of iroquois homeobox gene 1 (IRX1) gene and evaluate the regulatory mechanism of IRX1 transcription.</p><p><b>METHODS</b>Upstream sequence from transcriptional start site was predicted using bioinformatics methods. Serial deleted fragments from IRX1 promoter sequences were amplified by PCR and luciferase reporter plasmids were constructed. The luciferase intensity was analyzed after transferring reporters into GES-1 gastric mucosa cell line.</p><p><b>RESULTS</b>The promoter of IRX1 was predicted to be within 700 bp from the 5'-flanking region of IRX1 gene. Eight serial deleted luciferase reporter plasmids were constructed. The transcriptional activity of different fragments was expressed as following: p-416>p-584>p-715>p-350>p-687>p-320>p-188>p-92. Except p-320 and p-188, the transcriptional activity of other 6 fragments was higher than that of PGL3-basic plasmid. The transcriptional activity was the highest in p-416 and decreased sharply from p-320 to p-188.</p><p><b>CONCLUSIONS</b>The fragment p-416 shows the strongest promoter activity. The sequence from -320 bp to -188 bp is identified as core promoter region, which is focused as key sequence for further regulatory analysis, since some binding sites for important transcriptional factors such as Sp1 and TFII D are predicted.</p>

EGFR-blockade by antibody Cetuximab inhibits the growth of human gastric cancer xenograft in nude mice and its possible mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>EGFR-mediated tumor proliferation plays an important role in the development of cancer, and is a key candidate for targeted therapy. The aim of this study is to evaluate the impact of EGFR monoclonal antibody Cetuximab (C225) on the growth, proliferation and apoptsis of gastric cancer xenograft in nude mice, and its possible mechanisms.</p><p><b>METHODS</b>A gastric cancer cell line SGC-7901 with high EGFR expression level was screened from 7 gastric cancer cell lines. Gastric cancer xenografts in nude mice were established, and randomly divided into C225 treatment group and PBS control group. Tumor growth curves were calculated, the impact of C225 on the tumor growth, proliferation and angiogenesis was evaluated by immunohistochemical (IHC) staining Ki67 and CD34, respectively. The effect of C225 on apoptosis in the gastric cancer cells was evaluated by TUNEL assay. The expression levels of EGFR and its transcription factor Sp1 were detected by IHC staining and Western blot.</p><p><b>RESULTS</b>After C225 treatment, the proliferation and growth of gastric cancer xenograft in nude mice were significantly decreased. In the contrast, the apopotic indexes in C225 treatment group and PBS control group were (16.4% +/- 0.3%) and (3.1% +/- 0.9%), respectively, with a significant difference (P < 0.001). There was no significant difference of the densities of CD34-positive microvessels between C225 treatment group and control group. Elevated expression of EGFR and Sp1 after C225 treatment was observed by IHC staining and Western blot assay.</p><p><b>CONCLUSION</b>EGFR monoclonal antibody cetuximab (C225) can effectively inhibit the growth of gastric cancer xenografts in nude mice, and trigger its apoptosis. Yet, C225 treatment may upregulate the expression of EGFR and its transcription factor Sp1. A "block-transcription activation-compensation" mechanism may exist to explain the molecular mechanism of acquired resistance of a single target blockade treatment.</p>

Correlation between liver stem cell activation and histopathologic changes in liver with hepatic failure / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the correlation between liver stem cell activation and histopathologic changes in liver transplant recipients with hepatic failure.</p><p><b>METHODS</b>A total of 33 cases of hepatic failure were enrolled into the study. Donor liver tissues were used as normal controls. Histopathologic changes, presence of hepatotropic virus antigens, history of artificial liver therapy and number of c-kit positive cells were analyzed.</p><p><b>RESULTS</b>There were a total of 25 males and 8 females. The age of patients ranged from 21 to 64 years. Among the 33 patients studied, 6 suffered from acute liver failure, 5 from subacute liver failure and the remaining from acute-on-chronic liver failure (associated with cirrhosis). Thirteen patients had a history of artificial liver therapy. Activated liver stem cells expressed c-kit monoclonal antibody but were negative for toluidine blue stain. The number of c-kit-positive cells in acute liver failure, subacute liver failure and acute-on-chronic liver failure were 3.50 +/- 2.66 (0 to 8) per mm(2), 11.47 +/- 8.85 (3 to 30) per mm(2) and 15.50 +/- 10.95 (5 to 45) per mm(2), respectively (P < 0.05). The number of c-kit-positive cells in cases with or without artificial liver therapy showed no statistically significant difference.</p><p><b>CONCLUSIONS</b>The poor prognosis of acute liver failure is mainly due to massive liver necrosis and insufficient stem cell activation. Liver stem cell level is increased whenever there is progression into subacute liver failure and chronic liver failure. Actively treating acute liver failure with stimulation of the self-regeneration system in liver is thus useful.</p>

Clinical Experience of Liver Retransplantation in 8 Patients / 上海第二医科大学学报

Objective To summarize the clinical experience in liver retransplantation. Methods From June 2002 to December 2005,a total of 185 cases of liver transplantation were performed in our hospital,including 8 cases of retransplantation.Those clinical data were analyzed retrospectively. Results The rate of liver retransplantation was 4.32%.The average MELD scores before primary transplantation and retransplantation were 15.6 and 23.9,respectively.The average interval between primary transplantation and retransplantation was 316 days(78~725 days).Causes of retransplantation included 3 cases of biliary complications,2 cases of chronic rejection,1 case of hepatic artery thrombosis,1 case of acute rejection and 1 case of recurrence of hepatitis B.The former 3 cases died of severe infection combined with multiple organ failure 101,16 and 28 days after retransplantation.The latter 5 cases recovered smoothly,and have survived 27,12,8,4 and 3 months up to now. Conclusion Liver retransplantation is an effective way to save the patient with hepatic allograft failure.Good knowledge of the indications of retransplantation,careful selection of the operation time,excellent surgical skills and meticulous postoperative management will contribute to the success of liver retransplantation.

High-resolution analysis of chromosome 5 and identification of candidate genes in gastric cancer / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the relationship between loss of heterozygosity (LOH) of 5p with the histological phenotype in gastric cancer.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Transcription factor Sp1 expression in gastric cancer and its prognostic value / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression of transcription factor Sp1 in human gastric cancer tissues and normal gastric mucosa, and its prognostic significance.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>Normal and malignant gastric tissues have unique Sp1 expression patterns. Sp1 might be served as an independent prognostic factor.</p>

COX- 2 expression in gastric cancer and its relationship with lymphangiogenesis and lymph node metastasis / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor-C (VEGF-C) in human gastric cancer, the relationship between their expression and the clinicopathological features,as well as the relationship between these two parameter expression and lymphangiogenesis and lymph node metastasis.</p><p><b>METHODS</b>COX-2 and VEGF-C expressions were detected in 63 gastric cancer samples by immunostaining. Lymphangiogenesis was evaluated by immunostaining with the specific antibody LYVE-1.</p><p><b>RESULTS</b>The expression rates of COX-2 and VEGF-C were 66.7% (42/63), 52.4% (33/63), respectively in 63 gastric cancer specimens. LYVE-1 was positive in 35 cases (35/63), which indicated lymphangiogenesis in the tumors. The expression of COX-2 was significantly correlated with the expression of VEGF-C, tumor lymphangiogenesis and lymphatic metastasis (P< 0.05), however not gender, tumor size, tumor location, Lauren classification and serosa invasion (P< 0.05).</p><p><b>CONCLUSIONS</b>In gastric cancer, the expression of COX-2 is significantly associated with VEGF-C expression, lymphangiogenesis and lymphatic metastasis. COX-2 may up-regulate the expression of VEGF-C, which induces lymphangiogenesis and accordingly contributes to lymphatic metastasis.</p>

uPA expression of gastric cancer cell lines and its correlation with peritoneal seeding / 中华肿瘤杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>Three of 4 human gastric cancer cell lines studied express uPA mRNA and activity, which correlate with their peritoneal seeding potentials.</p>

Sensitivity and specificity of granzyme B and perforin in diagnosing acute rejection after liver transplantation / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the roles of granzyme B and perforin in diagnosing acute rejection after liver transplantation, and the relationship between their activity index (AI) and Banff's histological grading criteria.</p><p><b>METHODS</b>Liver biopsies were processed as for routine surgical specimens and labeled with granzyme B and perforin monoclonal antibodies. The number of positive cells/mm(2) was determined as activity index (AI) by IPP image analysis software. Histologic findings were used as the "gold standard" in diagnosing acute rejection.</p><p><b>RESULTS</b>Of 41 liver biopsy samples studied, acute rejection was noted in 21 cases, the remaining 20 cases showed no evidence of rejection. The AI of granzyme B and perforin in the acute rejection group was significantly higher than that in the non-acute rejection group (< 0.001). In the acute rejection group, the AI in moderate to severe acute rejection was higher than that in mild to indeterminate acute rejection (< 0.001). Compared with the "golden" histologic criteria, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of granzyme B in diagnosing acute rejection were 90.0%, 95.2%, 94.7%, 90.9% and 92.7% respectively. The values of these parameters for perforin were also above 80%.</p><p><b>CONCLUSIONS</b>Granzyme B and perforin are key markers of activated immune cells in acute rejection and highly expressed during acute liver rejection episodes. As ancillary investigations, these parameters demonstrated high sensitivity and specificity in diagnosing acute rejection in allograft post-transplant liver biopsies.</p>

Value of transabdominal ultrasonography in preoperative assessment of gastric carcinoma / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the value of transabdominal ultrasonography (TAUS) in preoperative assessment of TNM stage and tumor angiogenesis for patients with gastric carcinoma.</p><p><b>METHODS</b>Sixty- four patients with gastric carcinoma preoperatively underwent TAUS, in whom transabdominal color Doppler ultrasonography was used for measuring color Doppler vascularity index (CDVI) of each tumor in 37 cases and microvessel density (MVD) was evaluated by using immunohistochemical staining of surgical specimens with anti- CD34 antibody.</p><p><b>RESULTS</b>The overall accuracy rate was 56.0% for T staging of gastric carcinoma (T (1) 2/3 cases, T (2) 28.6% , T (3) 73.1% , T (4) 50.0% , respectively) by TAUS. The diagnostic accuracy rate was 63.3% for lymph node status of gastric carcinoma. The diagnostic sensitivity and specificity for lymph node metastasis was 37.9% and 100% respectively. The overall accuracy for N staging of gastric carcinoma was 57.1% (N (0) 100% , N (1) 16.7% , N (2) 35.3% , respectively). The diagnostic sensitivity and specificity for determining distant metastases was 58.3% and 100% respectively. The CDVI of gastric carcinoma determined by color Doppler ultrasonography was significantly correlated to vascular invasion (P=0.0418), a linear correlation between CDVI and MVD was determined by logistic regression analysis (r=0.5628, P< 0.01).</p><p><b>CONCLUSION</b>TAUS can be a routine diagnostic approach for preoperative gastric carcinoma patients.</p>

Perforin and granzyme B expression in liver allograft rejection / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the expression of perforin and granzyme B in rejection response following liver transplantation, and evaluate their roles to be used as predictive markers of rejection.</p><p><b>METHODS</b>The expression of perforin and granzyme B in liver biopsies obtained from liver allograft recipients was determined by immunohistochemistry. Biopsies were classified into two groups-no evidence of rejection and rejection-according to Histopathologic criteria. The relationship between the perforin/granzume B expression and acute rejection was analyzed.</p><p><b>RESULTS</b>From 19 patients, thirty-five liver biopsies were obtained after liver transplantation. Among them, nineteen samples were diagnosed as rejection response. The frequencies of perforin and granzyme B expression in rejection group were 100% (19/19) and 94.7% (18/19), respectively. While those in no rejection group were 25.0% (4/16) and 12.5 (2/16), respectively. In most rejected samples, perforin and granzyme B were expressed simultaneously. Only three samples showed perforin expression alone, while no samples demonstrated granzyme B expression alone. There was a close relationship between perforin/granzyme B expression and liver allograft rejection.</p><p><b>CONCLUSION</b>Perforin and granzyme B expression seemed to be related to the development of acute rejection following liver transplantation, and might be served as sensitive and reliable markers in diagnosing acute rejection in early stage.</p>