Intracarotid transplantation of autologous adipose-derived mesenchymal stem cells significantly improves neurological deficits in rats after MCAo.

We aimed to evaluate whether adipose-derived mesenchymal stem cells (ADMSCs) that were transplanted via internal carotid can improve the neurological function after acute ischemic stroke and explore the underlying mechanisms. Total 40 adult Sprague-Dawley rats were subjected to transient (1.5 h) middle cerebral artery occlusion (MCAo) to induce ischemia/reperfusion injury. These rats were randomly divided into two groups with 20 ones in each group, which were intracarotid-injected with autologous ADMSCs (2.0 × 10(6)) and saline (control) at day 3 after MCAo, respectively. Behavioral tests (adhesive-removal and modified neurological severity score) were performed before and after MCAo. Histology was used to evaluate the ischemia lesion volume and pathological changes. The apoptosis and astroglial reactivity were determined by TUNEL and glial fibrillary acidic protein (GFAP) staining, respectively. Besides, we applied immunofluorescence to identify the distribution of ADMSCs and the neural makers (NeuN and GFAP) expressed by them under confocal microscope. Significant improvement of neurological deficits was observed in rats transplanted with ADMSCs when compared to controls. But there was no obvious difference on ischemia lesion volume between these two groups. The injected ADMSCs migrated to the brain infarct region and mainly localized in the ischemic core and boundary zone of the lesion, which can express NeuN and GFAP in the brain. In addition, autologous transplantation of ADMSCs significantly attenuated astroglial reactivity, inhibited cellular apoptosis and promoted cellular proliferation. Our data indicated that intracarotid transplantation of autologous ADMSCs had the potential therapeutic application for ischemic stroke.

Adenovirus mediated transfer of p53, GM-CSF and B7-1 suppresses growth and enhances immunogenicity of glioma cells.

OBJECTIVES: Malignant gliomas are good targets for gene therapy because they have been proven incurable with conventional treatments. However, malignant gliomas are genetically and physiologically highly heterogeneous, and current gene therapy interventions have been designed to target only a few variations of this kind of disease. Hence, we developed a combined gene therapy approach using a recombinant adenovirus carrying human wild-type p53 (WT-p53), granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7-1 genes (designated BB-102) to combat the disease. METHODS: Human malignant glioma cells U251 and U87 were transduced with BB-102. Expression of WT-p53, GM-CSF and B7-1 genes were determined by Western blot, enzyme linked immunosorbent assay and flow cytometric analysis, respectively. Growth rates were determined by serial cell counts. Apoptosis was detected by flow cytometric analysis. Proliferation of autologous peripheral blood lymphocytes (PBLs) and cytotoxicity against primary glioma cells were assessed by cell proliferation and cytotoxicity assay kits, respectively. RESULTS: By the transduction of BB-102, high expression levels of the three exogenesis genes were detected in glioma cells. Cell growth was inhibited and apoptosis was induced. Significant proliferation of autologous PBLs and specific cytotoxicity against primary glioma cells were also induced by the infection of BB-102 in vitro, with the effect being more evident than that of Ad-p53. CONCLUSION: These results suggest that glioma cell vaccination co-transferred with p53, GM-CSF and B7-1 genes may be a feasible and effective immunotherapeutic approach in glioma treatments.

KAI1 is a potential target for anti-metastasis in pancreatic cancer cells.

AIM: To investigate whether KAI1, as a metastasis suppressor gene, is associated with invasive and metastatic ability of pancreatic cancer cells. METHODS: KAI1 gene was transfected into pancreatic cancer cell line MiaPaCa II by liposomes selected with G418. Expression of transfected cells was measured by Western blotting, immunofluorescence and immunocytochemistry. Tumor cell invasion and metastatic ability were detected through gelatinase activity and reconstituted basement membrane (Matrigel) assay. pCMV-KAI1 was directly injected into the heterotopic human pancreatic adenocarcinoma successfully established in the groin of BALB/C nude mice, by subcutaneous injection of MiaPaCa II pancreatic cancer cells. The statistical analysis between groups was determined by Student's two tailed t test. RESULTS: By Western blotting, MiaPaCa II cells transfected by KAI1 gene indicated KAI1 expression at approximately 29.1 kDa. Cytoplasm staining was positive and uniformly spread in transfected cancer cells, using immunohistochemistry and immunofluorescence. The most obvious difference was present after 30 h (MiaPaca II 43.6 +/- 9.42, pCMV-MiaPaca II 44.8 +/- 8.56, pCMV-KAI1-MiaPaca II 22.0 +/- 4.69, P < 0.05). Gelatinolysis revealed a wider and clearer band of gelatinolytic activity in non-transfected than in transfected cells (MiaPaCa II cells 30.8 +/- 0.57, transfected cells 28.1 +/- 0.65, P < 0.05). In vivo tumor growth rates of KAI1 transfectants with KAI1-Lipofectamine 1.22 +/- 0.31 in A group were lower than control 4.61 +/- 1.98 and pCMV-KAI 11.67 +/- 0.81. Analyses of metastases with and without KAI1 transfection in mice were different in liver and lung between controls 1.62 +/- 0.39, 0.45 +/- 0.09, pCMV-KAI 1.01 +/- 0.27, 0.33 +/- 0.09 and KAI1-Lipofectamine 0.99 +/- 0.21, 0.30 +/- 0.09 respectively (P < 0.05). CONCLUSION: High expression of KAI1 gene was found in transfected MiaPaCa II human pancreatic cancer cells with lower metastatic ability. KAI1 gene plays an important role in inhibiting metastasis of pancreatic cancer after direct injection into pancreatic adenocarcinoma. These results show that the suppressed invasion and motor function of pancreatic cancer cells may be a key reason why the KAI1 gene controls pancreatic cancer cell metastasis.

KAI1 inhibits anchorage-dependent and -independent pancreatic cancer cell growth.

Decreased expression of the tumor suppressor gene, KAI1, is associated with metastasis formation in pancreatic cancer. The aim of the present study was to investigate whether KAI1 influences pancreatic cancer cell growth and colony formation. A full-length KAI1 cDNA expression vector was stably transfected into Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. Transfection was confirmed by Western blot analysis and immunohistochemistry. Tumor cell growth and cell cycle distribution were determined by MTT cell growth assays, colony formation assays, and flow cytometric analysis. KAI1-transfected, but not control-transfected pancreatic cancer cells displayed cytoplasmic KAI1 immunoreactivity. Cell proliferation decreased in the KAI1-transfected cells compared to parental and control cells together with a Go/G1-phase cell cycle arrest. Colony formation was reduced by 2.6- and 3.5-fold in the KAI1-transfected Panc-1 and MiaPaCa-2 pancreatic cancer cells, respectively, compared with parental cells. KAI1 blocks pancreatic cancer cell growth through cell cycle arrest and inhibits anchorage-independent cell growth. These findings support the premise that KAI1 functions as a tumor suppressor in this malignancy.

New tumor-associated antigen SC6 in pancreatic cancer.

AIM: To examine the concentration of a new antigen SC6 (SC6-Ag) recognized by monoclonal antibody (MAb) in patients with pancreatic cancer and other malignant or benign diseases and to understand whether SC6-Ag has any clinical significance in distinguishing pancreatic cancer from other gastrointestinal diseases. METHODS: Six hundred and ninety-five serum specimens obtained from 115 patients with pancreatic cancer, 154 patients with digestive cancer and 95 patients with non-digestive cancer were used and classified in this study. Serum specimens obtained from 140 patients with benign digestive disease and 89 patients with non-benign digestive disease served as controls. Ascites was tapped from 16 pancreatic cancer patients, 19 hepatic cancer patients, 16 colonic cancer patients, 10 gastric cancer and 6 severe necrotic pancreatitis patients. The samples were quantitated by solid-phase radioimmunoassay. The cut-off values (CV) of 41, 80, and 118 U/mL were used. RESULTS: The average intra- and interassay CV detected by immunoradiometric assay of SC6-Ag was 5.4% and 8.7%, respectively. The sensitivity and specificity were 73.0% and 90.9% respectively. The levels in most malignant and benign cases were within the normal upper limit. Among the 16 pancreatic cancer cases, the concentration of SC6-Ag in ascites was over the normal range in 93.8% patients. There was no significant difference in the concentration of SC6-Ag. Decreased expression of SC6-Ag in sera was significantly related to tumor differentiation. The concentration of SC6-Ag was higher in patients before surgery than after surgery. The specificity of SC6-Ag and CA19-9 was significantly higher than that of ultrasound and computer tomography (CT) in pancreatic cancer patients. Higher positive predictive values were indicated in 92.3% SC6-Ag and 88.5% CA19-9, but lower in 73.8% ultrasound and 76.2% CT. CONCLUSION: The combined test of SC6-Ag and CA19-9 may improve the diagnostic rate of primary cancer. The detection of SC6-Ag is valuable in the diagnosis of pancreatic cancer before and after surgery.

[The mechanism of KAI1 gene in inhibition of metastasis of primary pancreatic cancer].

OBJECTIVE: To study the effect on invasive and motor ability of MiaPaCa II pancreatic cancer cell transfected by recombinant plasmid pCMV-KAI1, alteration of KAI1 in the transfecting cancer cells, and the metastatic mechanism in the malignancy. METHODS: MiaPaCa II pancreatic cancer cell was transfected with pCMV-KAI1 by liposome, the invasive and gelatinase ability of two pancreatic cancer cell lines were analyzed with transwell culture cell and polyacrylamided gel electrophoresis (PAGE) before and after KAI1 cDNA transfection. RESULTS: After KAI1 cDNA transfected into MiaPaCa II, the migrative ability of passing through the membrane filter decreased evidently (P < 0.05). The width of negative staining band narrowed, the intensity level of tape weakened through PAGE. These results showed that reduced the ability of degrading basement membrane and extra-cell base and metastasis were influenced after MiaPaCa II pancreatic cancer cells were transfected. CONCLUSION: The suppressed invasion and motor function of pancreatic cancer cells seems to influence their metastatic ability and thereby entrances the malignant potential of pancreatic cancer.

Effect of bax, bcl-2 and bcl-xL on regulating apoptosis in tissues of normal liver and hepatocellular carcinoma.

AIM: To investigate the expression of bax, bcl-2 and bcl-xL mRNA in the tissues of normal liver and hepatocellular carcinoma (HCC), and analyze the relationship between the expression of bax, bcl-2 and bcl-xL mRNA and clinical parameters of HCC patients. METHODS: The expression of bax, bcl-2 and bcl-xL mRNA of normal liver and HCC was measured by Northern blot. Statistical analyses were made by t test and correlation analysis. RESULTS: A very low mRNA level was indicated at bax, bcl-2 and bcl-xL in the HCC tissues in contrast to the tissues of normal liver by Northern blot analysis. The analyses of mRNA level revealed that HCC tissues exhibited a mean 7.6-fold decrease in bax, 4.2-fold in bcl-2 and 3.5-fold in bcl-xL in comparison with normal control tissues, respectively. Positive correlation was found between bax and bcl-xL (r=0.7061, P<0.01). There was no significance between the mRNA expression of these three genes and age, gender, tumor differentiation and tumor stage of HCC patients. CONCLUSION: The results are consistent with the fact that apoptosis rarely occurs in normal livers but increases in HCC, indicating that bcl-2 and bcl-xL may play a very important role in regulating the apoptosis of normal liver and HCC.

KAI1 gene is differently expressed in papillary and pancreatic cancer: influence on metastasis.

AIM:To compare KAI1 in cancer of papilla of Vater and pancreas to evaluate whether there are differences in biologic behavior which might account for prognosis.METHODS:We compared the expression in 24 papillay and 29 pancreatic cancers using Northern blot analysis, immunochemical assay and in situ hybridization, and investigated whether early diagnosis or molecular differences predict the outcome in these tumor entities.RESULTS:By Northern blot analysis there is no statistical difference of KAI1 levels in normal and cancerous papilla. No association between KAI1 mRNA expression and tumor stage or tumor differentiation was found in the tumors. By immunohistochemical assay, KAI1 staining in cytoplasm of papillary cancer cells was similar to that of normal papillary cells. By in situ hybridization, the results of KAI1 mRNA expression in normal and cancerous papilla were similar to those with immunohistochemical assay. The normal and cancerous pancreas tissues were also analyzed by the methods used in papillary samples.CONCLUSION:Although the biologic roles of KAI1 have not been clarified, our results suggest that KAI1 may restrict the progression of malignant papillary cancer, but its expression might not have any effect on the characteristics of papillary tumor, whereas by the analysis of KAI1 gene, its reduced expression is closely related to the progression and metastases of pancreatic cancer.