Inhibitory effect of antisense vascular endothelial growth factor 165 eukaryotic expression vector on proliferation of hepatocellular carcinoma cells

AIM:To construct antisense VEGF_(165) eukaryotic expressionvector PCDNA_3-as-VEGF_(165) and to study its expression and effecton the proliferation of hepatocarcinoma SMMC-7721 cells.METHODS:VEGF_(165) cDNA was inserted into polylinker sitesof eukaryotic expression vector PCDNA_3 to construct PCDNA_3-as-VEGF_(165).Then the vector was transferred into humanhepatocarcinoma cell strain SMMC-7721 with cationlipofectamine 2000 mediated methods to evaluate theexpression of VEGF protein and the inhibitory effect on theproliferation of hepatocarcinoma SMMC-7721 cells.RESULTS:The detection indicated the presence of VEGFcDNA in normally cultured SMMC-7721 cells by PCR.VEGFmRNA expression was notably decreased in SMMC-7721 cellsby RT-PCR after PCDNA_3-as-VEGF_(165) transfection.The expressionof VEGF protein was dramatically inhibited (142.01±7.95 vs1625.52±64.46 pg·ml~(-1),P<0.01) 2 days after transfection,which correlated with the dose of PCDNA_3-as-VEGF_(165) gene.VEGF protein was most expressed in PCDNA_3 transferredSMMC-7721 cells but few in PCDNA_3-as-VEGF_(165) transferredcells by immunohistochemical staining.The apoptotic rateof hepatocarcinoma SMMC-7721 cells was significantlypromoted (17.98±0.86% vs 4.86±0.27%,P<0.01) and thesurvival rate was notably decreased (80.99±3.20% vs93.52±3.93%,P<0.05) due to antisense VEGF_(165) by flowcytometry (FCM).The transfection of antisense VEGF_(165) generesulted in the inhibitory effect on the proliferation ofhepatocarcinoma cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the death of allhepatocarcinoma cells on day 6 after transfection.CONCLUSION:It is confirmed that antisense VEGF_(165) caninhibit the expression of VEGF protein,interfere with theproliferation and induce the apoptosis of hepatocarcinomacells in our study.Antisense VEGF_(165) gene therapy may playan important role in the treatment of human hepatocardnoma.     

Inhibitory effect of dimeric β peptide on the recurrence and metastasis of hepatocellular carcinoma in vitro and in mice

AIM：To block the adhesion of tumor cells to the extracellular matrix, and prevent tumor metastasis and recurrence, the dimer of the β peptide （DLYYLMDLSYSMKG- GDLYYLMDLSYSMK, β2） was designed and synthesized and its anti-adhesion and anti-invasion effects on hepa- tocellular carcinoma cells were assessed. Additionally, its influence on the metastasis and recurrence of mouse hepatocellular carcinoma was measured.
METHODS：The anti-adhesion effect of β2 on the highly metastatic hepatocellular carcinoma cell line HCCLM6 cells and fibronectin （FN） was assayed by the MTT as- say. The inhibition of invasion of HCCLM6 cells by β2 was observed using a Transwell （modified Boyden chamber） and matrigel. Using the hepatocellular carcinoma metas- tasis model and LCI-D20 nude mice, the influence of β2 on the metastasis and recurrence of hepatocellular carci- noma after early resection was investigated.
RESULTS：HCCLM6 cells co-incubated with 100 mmol/L, 50 mmol/L, 20 mmol/L or 10 mmol/L β2 for 3 h showed an obvious decrease in adhesion to FN. The adhesion inhibition ratios were 11.8%, 21.7%, 29.6% and 48.7%, respectively. Additionally, HCCLM6 cells cultured with 100 mmol/L β2 had a dramatic decrease in cell invasion. β2 was also observed to inhibit the incisal edge recur- rence and the distant metastasis of nude mice hepato- cellular carcinoma after early resection （P 〈 0.05）.
CONCLUSION：The β2 peptide can specifically block the adhesion and invasion of HCCLM6 cells, and can inhibit HCC recurrence and metastasis of LCI-D20 model pos-thepatectomy in vivo. Thus, β2 should be further studied as a new anti-tumor drug.     

Effects of endostatin on expression of vascular endothelial growth factor and its receptors and neovascularization in colonic carcinoma implanted in nude mice

AIM:To investigate the antiangiogenic effects of endostatin on colonic carcinoma cell line implanted in nude mice and its mechanism. METHODS:Nude mice underwent subcutaneous injection with LS-174t colonic carcinoma cell line to generate carcinoma and were randomly separated into two groups.Mice received injection of vehicle or endostatin every day for two weeks. After the tumor was harvested,the tumor volumes were determined,and the expressions of CD34,VEGF and FIk-1 were examined by immunohistochemical method. RESULTS:Tumor volume was significantly inhibited in the endostatin group(84.17%)and tumor weight was significantly inhibited in the endostatin group(0.197±0.049) compared to the control group(1.198±0.105)(F=22.56, P=0.001),microvessel density(MVD)was significantly decreased in the treated group(31.857±3.515)compared to the control group(100.143±4.290)(F=151.62,P<0.001). Furthermore,the expression of FIk-1 was significantly inhibited in the treated group(34.29%) ompared to the control group(8.57%)(X~2=13.745,P=0.001).However no significant decrease was observed in the expression of vascular endothelial growth factor(VEGF)between these two groups(X~2=0.119,P=0.730). CONCLUSION:Endostatin can inhibit tumor growth and angiogenesis by blocking Vegf/FIk-1 pathway.This experiment provides the theory basis for developing a new anti-carcinoma drug through studying the properties of anti-angiogenesis inhibitors.     

Expression of plasma vascular endothelial growth factor in patients with hepatocellular carcinoma and effect of transcatheter arterial chemoembolization therapy on plasma vascular endothelial growth factor level

AIM:To investigate the expression level of plasma vascularendothelial growth factor(P-VEGF)in patients withhepatocellular carcinoma(HCC)and its relationship withthe clinicopathologic characteristics,and to examine thechanges of P-VEGF in the course of transcatheter arterialchemoembolization(TACE).METHODS:Peripheral blood samples were taken from 45HCC patients before and 1,3,7 d,and 1 mo after TACE.Plasma VEGF level was measured with the quantitativesandwich enzyme-linked immunosorbent assay(ELISA).Twenty patients with benign liver lesions and 17 healthycontrol subjects were also included in this study.RESULTS:Plasma VEGF levels in HCC patients weresignificantly elevated as compared to those in patients withbenign liver lesions(P=0.006)and in the normal controls(P=0.003).Significant differences were observed whenP-VEGF was categorized by tumor size(P=0.006),portalvein thrombosis(P=0.011),distant metastasis(P=0.017),arterial-portal vein shunting(P=0.026),and InternationalUnion Against Cancer(UICC)TNM stage(P=0.044).Therewas no correlation between plasma level of VEGF and thelevel of alpha fetoprotein(α-FP) (r=0.068,P=0.658)andweakly correlated with the number of platelets(r=0.312,P=0.038).P-VEGF levels increased significantly andreached the peak value on the first day after TACE,and thendecreased gradually.The change rate of P-VEGF concentration(one month post-TACE/pre-TACEx100%)was correlatedwith the retention rate of lipiodol oil(rs=0.494,P=0.001)and the tumor volume change(rs=0.340,P=0.034).The patients who achieved a partial or complete responseto TACE therapy showed significantly less pre-treatmentP-VEGF than those nonresponders(P=0.025).A high pre-therapeutic P-VEGF level was associated with poor responseto treatment(P=0.018).CONCLUSION:A high pre-treatment P-VEGF level is auseful marker for tumor progression,especially for vascular invasion.TACE increases the level of P-VEGF onlytemporarily which may be associated with tumor ischemia.P-VEGF may be useful in predicting treatment response,monitoring disease course after TACE and judging the effectof different TACE regimens.     

Correlation between expression of cyclooxygenase-2 and the presence of inflammatory cells in human primary hepatocellular carcinoma： Possible role in tumor promotion and angiogenesis

AIM: To investigate the association of cyclooxygenase-2 (COX-2) expression with angiogenesis and the number and type of inflammatory cells (macrophages/Kupffer cells; mast cells) within primary hepatocellular carcinoma (HCC) tissues and adjacent non-tumorous (NT) tissues. METHODS: Immunohistochemistry for COX-2, CD34, CD68 and mast cell tryptase (MCT) was performed on 14 well-characterized series of liver-cirrhosis-associated HCC patients. COX-2 expression and the number of inflammatory cells in tumor lesions and surrounding liver tissues of each specimen were compared. Moreover, COX-2, CD34 staining and the number of inflammatory cells in areas with different histological degrees within each tumor sample were comparatively analyzed. RESULTS: The percentage of COX-2 positive cells was significantly higher in NT tissues than in tumors. COX-2 expression was higher in well-differentiated HCC than in poorly-differentiated tissues. Few mast cells were observed within the tumor mass, whereas a higher number was observed in the surrounding tissue, especially in peri-portal spaces of NT tissues. Abundant macrophages/ Kupffer cells were observed in NT tissues, whereas the number of cells was significantly lower in the tumor mass. However, a higher cell number was observed in the well-differentiated tumor and progressively decreased in relation to the differentiation grade. Within the tumor, a positive correlation was found between COX-2 expression and the number of macrophages/Kupffer cells and mast cells. Moreover, there was a positive correlation between CD34 and COX-2 expression in tumor tissues. Comparison between well- and poorly-differentiated HCC showed that the number of CD34-positive cells decreased with dedifferentiation. However, COX-2 was the only independent variable showing a positive correlation with CD34 in a multivariate analysis. CONCLUSION: The presence of inflammatory cells and COX-2 expression in liver tumor suggests a possible relationship with tumor angiogenesis. COX-2 expressing cells and the number of macrophages/Kupffer cells and mast cells decrease with progression of the disease.     

Reactivation of the insulin-like growth factor-Ⅱ signaling pathway in human hepatocellular carcinoma

Constitutive activation of the insulin-like growth factor （IGF）-signaling axis is frequently observed in human hepatocellular carcinoma （HCC）. Especially the overexpression of the fetal growth factor IGF-Ⅱ, IGF-Ⅰ receptor （IGF-IR）, and cytoplasmic downstream effectors such as insulin-receptor substrates （IRS） contribute to proliferation, anti-apoptosis, and invasive behavior. This review focuses on the relevant alterations in this signaling pathway and independent in vivo models that support the central role IGF-Ⅱ signaling during HCC development and progression. Since this pathway has become the center of interest as a target for potential anti-cancer therapy in many types of malignancies, various experimental strategies have been developed, including neutralizing antibodies and selective receptor kinase inhibitors, with respect to the specific and efficient reduction of oncogenic IGF- Ⅱ/IGF-IR-signaling.     

Pathobiological significance of vascular endothelial growth factor and Maspin expressions in human gastric carcinoma

AIM:To investigate the correlation between expressionof vascular endothelial growth factor (VEGF) and celldifferentiation,invasion,metastasis and Naspin expressionin gastric carcinoma.METHODS:Formalin-fixed paraffin-embedded tissuespecimens from 73 cases of gastric carcinoma were studiedwith SP immunohistochemistry,using anti-VEGF monoclonalantibody,and thirty-nine of them were studied using anti-Maspin monoclonal antibody.VEGF expression was comparedwith the dinical stage,lymph node metastasis,and Borrrnann'sand WHO's classification of gastric carcinoma.RESULTS:The positive rate of VEGF expression wassignificantly higher in adjacent non-carcinoma epithelia(ANCE) than in non-metaplastic,non-carcinoma gastricepithelia (NMNCE),which were at least 4 cm distant fromthe primary tumor (P=0.000,X~2=73.03).The positiverate of VEGF expression was significantly higher in advancedgastric carcinoma (AGC) than in early gastric carcinoma(EGC) (P=0.032,X~2=4.62).The positive rate of VEGFexpression in gastric carcinomas with lymph node metastaseswas significantly higher than that in those without metastasis(P=0.006,X~2=7.47).Maspin was weakly expressed in 16out of 39 cases of NINCE,and the positive immunoreactionwas limited to gland cells of the stomach body.There wasno significant correlation between the expression of VEGFand histological or gross classifications,and correlationbetween the expressions of VEGF and laspin in gastriccarcinoma (P=0.648,X~2=0.21).CONCLUSION:Expression of VEGF is significantly correlatedto the malignant biological behaviors of gastric carcinoma,but there is no significant correlation between the expressionof VEGF and Maspin.     

Effect of blocking IGF-Ⅰ receptor on growth of human hepatocellular carcinoma cells

AIM: To study the expression level and localization of insulin-like growth factor -I receptor (IGF-IR) in HepG2 cells and Chang liver cells, and to observe the effect of anti-IGF-IR monoclonal antibody (αIR3) on the growth of HepG2 cells. METHODS: The expression of IGF-IR in HepG2 cells and Chang liver cells was detected by immunohistochemistry. The influences of aIR3 on proliferation and apoptosis were examined by the 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and electron microscopy, respectively. Flow cytometry (FCM) was applied for the analysis of cell cycle and apoptosis was observed under electron microscope. RESULTS: IGF-IR was located in the membranes of both HepG2 and Chang liver cell lines, and the expression level of IGF-IR was higher in HepG2 cells than in Chang liver cells. Treated with 0.1μg/mLαIR3 for 48 h in vitro, the cell growth index (GI) of HepG2 cells was significantly higher than that of control (103.41% vs 100%, P<0.01). However, the aIR3 for 24 h at final concentration of 4.0μg/mL made the GI of HepG2 cells lower than that of control (93.37% vs 100%, P < 0.01). Compared with control, treated with aIR3 for 48 h at final concentrations ranging from 1.0μg/mL to 4.0μg/mL markedly reduced the GIs of HepG2 cells (97.63%, 97.16%, 95.13%, 92.53% vs 100%, P < 0.05 or P < 0.01), treated withαIR3 for 72 h at final concentrations ranging from 0.2μg/mL to 4.0μg/mL decreased the GIs of HepG2 cells obviously (95%, 91.63%, 90.77%, 89.84%, 88.51% vs 100%, P<0.01), and treated with aIR3 for 96 h at final concentrations ranging from 0.5μg/mL to 4.0μg/mL made GIs of HepG2 cells lower significantly (88.86%, 83.97%, 79.81%, 77.24%, 70.51% vs 100%, P<0.05 or P<0.01). Moreover, treated withαIR3 from 24 h to 96 h at final concentrations ranging from 0.2μg/mL to 4.0μg/mL reduced the GI of HepG2 cells from 97.63% to 70.51% in a dose- and time-dependent manner. Also,αIR3 treatment for 72 h at final concentration from 0.5μg/mL to 2.0μg/mL increased the proportion of G0/G1 phase cells(61.73%, 67.1%, 83.7%,76.87% vs 44.47%, P<0.01) and significantly decreased that of S phase cells(28.63%, 25.13%, 15.63%, 23.13% vs 53.17%, P<0.01), in contrast to the proportion of G2/M phase cells. The apoptotic rates of HepG2 cells were increased more than that of control (7.83%, 16.13%, 21.1%, 37.73% vs 4.13%, P< 0.01). CONCLUSION: The malignant cell phenotype of human hepatocarcinoma cell is related to overexpression of IGF-IR. The blockage of IGF-IR with aaaaaIR3 may contribute to the inhibition of proliferation and induction of apoptosis in HepG2 cells.     

Expression of angiostatin cDNA in human hepatocellular carcinoma cell line SMMC-7721 and its effect on implanted carcinoma in nude mice

AIM:To transfect murine angiostatin cDNA into humanhepatocellular carcinoma cell line SMMC-7721 and toinvestigate its effects on implanted carcinoma in nude mice.METHODS:A eukaryotic expression vector of pcDNA3.1-mAST containing murine angiostatin was constructed.ThenpcDNA3.1-mAST plasmid was transfected into cell line SMMC-7721 by Lipofectamine.The resistant clone was screenedby G418 filtration and identified by RT-PCR and Westernblotting.Nude mice were divided into three groups of 10each.Mice in blank control group were only injected withSMMC-7721 cells.Mice in vector control group were injectedwith SMMC-7721 cells transfected with pcDNA3.1 ( ) vector,whereas mice in angiostatin group were injected with SMMC-7721 cells transfected with pcDNA3.1-mAST plasmid.Volume,mass and microvessel density (MVD) of the tumorsin different groups were measured and compared.RESULTS:Murine angiostatin cDNA was successfullycloned into the eukaryotic expression vector pcDNA3.1 ( ).pcDNA3.1-mAST was successfully transfected into SMMC-7721 cell line and showed stable expression in this cell line.No significant difference was observed in the growth speedof SMMC-7721 cells between groups transfected with andwithout angiostatin cDNA.Tumor volume,mass and MVD inthe angiostatin group were significantly lower than those inthe blank control group and vector control group (P<0.01).The inhibitory rate of tumor reached 78.6%.Mass and MVDof the tumors only accounted for 34.6% and 48.9% respectivelyof those in the blank control group.CONCLUSION:Angiostatin cDNA could be stably expressedin human hepatocellular carcinoma cell line SMMC-7721without obvious inhibitory effects on the growth of SMMC-7721 cells.When implanted into nude mice,SMMC-7721cells transfected with angiostatin cDNA show a decreasedtumorigenic capability.It suggests that angiostatin caninhibit tumor growth through its inhibition on angiogenesisin tumors.     

Successful treatment of advanced hepatocellular carcinoma by combined administration of 5-fluorouracil and pegylated interferon-α

We report a case of hepatocellular carcinoma (HCC) treated successfully by transarterial chemoembolization (TACE) followed by combination therapy of 5-fluorouracil (5-FU) and pegylated interferon-alpha (PEG-IFN-alpha). In the present case, the patient had massive and advanced HCC with a diameter of over 8 cm located in segment 7 (S7) of the liver. Furthermore, the tumor invaded into the major branch of the portal vein (Vp3). After TACE, combined administration of 5-FU and PEG-IFN-alpha was performed for 5 mo. HCC was totally eradicated and the serum levels of tumor markers were markedly decreased by the treatment. Although it has been reported that the combined use of conventional IFN-alpha and 5-FU showed striking effects on HCC in some cases, this case may suggest the more promising effect of PEG-IFN-alpha with a long-lasting effect, in the combined use with 5-FU for the treatment of massive advanced HCC.     

Spider angiomas in patients with liver cirrhosis： Role of vascular endothelial growth factor and basic fibroblast growth factor

AIM: To investigate whether vascular endothelial growth factor (VEGF) and basic fibroblastic growth factor (bFGF) are associated with spider angiomas in patients with liver cirrhosis. METHODS: Eighty-six patients with liver cirrhosis were enrolled and the number and size of the spider angiomas were recorded. Fifty-three healthy subjects were selected as controls. Plasma levels of VEGF and bFGF were measured in both the cirrhotics and the controls. RESULTS: Plasma VEGF and bFGF were increased in cirrhotics compared with controls (122 +/- 13 vs. 71 +/- 11 pg/mL, P=0.003 for VEGF; 5.1 +/- 0.5 vs. 3.4 +/- 0.5 pg/mL, P=0.022 for bFGF). In cirrhotics, plasma VEGF and bFGF were also higher in patients with spider angiomas compared with patients without spider angiomas (185 +/- 28 vs. 90 +/- 10 pg/mL, P=0.003 for VEGF; 6.8 +/- 1.0 vs. 4.1 +/- 0.5 pg/mL, P=0.017 for bFGF). Multivariate logistic regression showed that young age and increased plasma levels of VEGF and bFGF were the most significant predictors for the presence of spider angiomas in cirrhotic patients (odds ratio [OR]=6.64, 95 % confidence interval [CI]=2.02-21.79, P=0.002; OR=4.35, 95% CI=1.35-14.01, P=0.014; OR=5.66, 95% CI=1.72-18.63, P=0.004, respectively). CONCLUSION: Plasma VEGF and bFGF are elevated in patients with liver cirrhosis. Age as well as plasma levels of VEGF and bFGF are significant predictors for spider angiomas in cirrhotic patients.     

Experimental study on ultrasound-guided intratumoral injection of“Star-99”in treatment of hepatocellular carcinoma of nude mice

AIM: To investigate the anti-cancer effect and the immunological mechanism of ultrasound-guided intratumoral injection of Chinese medicine "Star-99" in hepatocellular carcinoma (HCC) of nude mice.METHODS: Twenty-eight human hepatocellular carcinoma SMMC-7721 transplanted nude mice, 14 of hypodermically implanted and 14 of orthotopic liver transplanted, were randomly divided into three groups of which 14 mice with Star-99, and 7 with ethanol and saline respectively. Ten days after the transplantation the medicines were injected into the tumors of all the nude mice once every 5 days.After 4 injections the nude mice were killed. The diameters of three dimension of the tumors were measured by high frequency ultrasound before and after the treatment and the tumor growth indexes* (TGI) were calculated.Radioimmunoassay was used to detect the serum levels of interleukin-2 (IL-2) and tumor necrosis factor (TNF)-alpha.The tumor tissues were sent for flow cytometry (FCM) DNA analysis. Apoptotic cells were visualized by TUNEL assay.All the experiments were carried out by double blind method. zRESULTS: The TGI of Star-99 group (0.076±0.024) was markedly lower than that of the saline group (4.654±1.283)(P＜0.01). It also seemed to be lower than that of the ethanol group (0.082±0.028), but not significantly different (P＞0.05).Serum levels of IL-2 and TNF-α were markedly higher than those of ethanol group and saline groups (P＜0.05). The mean apoptotic index (AI: percentage of TUNEL signal positive cells)in Star-99 group (48.98±5.09 %) was significantly higher than that of the ethanol group (11.95±2.24 %) and the saline group (10.48±3.85 %) (P＜0.01). FCM DNA analysis showed that the appearance rate of the apoptosis peak in Srar-99group was 92.9 %, markedly higher than that of the ethanol group (14.3 %) and the saline group (0.0 %) (P＜0.01).Correlation (r=0.499, P＜0.05) was found between AI and serum level of TNF-α.CONCLUSION: Star-99 has an effect on the elevation of the serum levels of IL-2 and TNF-α. ft indicates that Star99 has the function of enhancing the cellular immunity and inducing cancer cell apoptosis. The correlation between AI and serum level of TNF-α indicates that the elevation of the serum of TNF-α induced by Star-99 may be an important factor in the promotion of the hepatic cancer cell apoptosis.Star-99 has strong effects on the inhibition and destruction of cancer cells. Its curative effect is as good as ethanol. Its major mechanisms can be as follows: (1) it increases the serum levels of IL-2 and TNF-α and triggers cellular immunity. (2) It can induce cancer cells apoptosis, the effective mechanism of the Star-99 is different from that of the ethanol. The mechanisms of triggering the immunologic function of the organism and inducing cell apoptosis are, of particular significance. This study will provide a new pathway of drug administration and an experimental basis for the treatment of HCC with Chinese herbal, and the study of Star-99 in the treatment of tumor is of profound significance with good prospects.     

Antiangiogenic effect of somatostatin receptor subtype 2 on pancreatic cancer cell line： Inhibition of vascular endothelial growth factor and matrix metalloproteinase-2 expression in vitro

AIM: To investigate the anti-angiogenic effect of somatostatin receptor subtype 2 (SSTR2) gene transfer into pancreatic cancer cell line PC-3, and the mechanisms involved in this effect. METHODS: The full length human SSTR2 cDNA was introduced into pancreatic cancer cell line PC-3 by lipofectamine-mediated transfection. Positive clones were screened by G418 and stable expression of SSTR2 was detected by immunohistochemistry SABC methods and RT-PCR. Enzyme-linked immunosorbent assay (ELISA) was used to detect vascular endothelial growth factor (VEGF) levels in the cell culture supernatants of SSTR2-expressing cells, vector control and mock control cells. Furthermore, the expressions of VEGF and matrix metalloproteinase-2 (MMP-2) were detected by immunohistochemistry SABC methods and RT-PCR in these cells. RESULTS: VEGF levels in the cell culture supernatants were significantly reduced in the SSTR2-expressing cells (first week, 172.63+/-21.2 ng/L and after two months, 198.85+/-26.44 ng/L) compared with the vector control (first week, 790.39+/-86.52 ng/L and after two months, 795.69+/-72.35 ng/L) and mock control (first week, 786.42+/-90.62 ng/L and after two months, 805.32+/-84.36 ng/L) (P<0.05). The immunohistochemical assay showed a significant reduction of the integral optical density of VEGF and MMP-2 in the SSTR2-expressing cells (42.25+/-8.6 and 70.5+/-6.25, respectively) compared with the vector control (85.75+/-12.9 and 110.52+/-13.5, respectively) and mock control (82.6+/-9.28 and 113.56+/-9.62, respectively) (P<0.05). Conversely, the average gray value of VEGF and MMP-2 was significantly increased in the SSTR2-expressing cells (121.56+/-8.43 and 134.46+/-19.95, respectively) compared with the vector control (55.72+/-5.6 and 62.26+/-12.68, respectively) and mock control cells (58.48+/-6.2 and 65.49+/-9.16, respectively) (P<0.05). Moreover, the expressions of VEGF mRNA and MMP-2 mRNA were significantly reduced in the SSTR2-expressing cells (0.1384+/-0.017 and 0.2343+/-0.070, respectively) compared with the vector control (1.024+/-0.117 and 0.806+/-0.119, respectively) and mock control (1.085+/-0.105 and 0.714+/-0.079, respectively) (P<0.05). CONCLUSION: The expression of reintroduced human SSTR2 gene exerts its antiangiogenic effects by down-regulating the expressions of the factors involved in tumor angiogenesis and metastasis, suggesting SSTR2 gene transfer as a new strategy of gene therapy for pancreatic cancer.     

Influence of transarterial chemoembolization on angiogenesis and expression of vascular endothelial growth factor and basicfibroblast growth factor in rat with Walker-256 transplanted hepatoma： An experimental study

AIM: After transarterial chemoembolization (TACE), the residual cancer cells are under extensive hypoxic or even anoxic environment. Hypoxia can lead to adaptive responses. For example, angiogenesis will help these cells survive. In this study, we examined the effect of TACE on angiogenesis and expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) and to assess their relevance to Walker-256 transplanted hepatoma. METHODS: Male Wistar rats were inoculated with Walker-256 tumor in the left lobe of liver. Angiography and transarterial chemoembolization were performed at d14 after transplantation. Sixty rats bearing walker-256 transplanted hepatoma were randomly divided into control group, arterial infusion group and TACE group. Each group consisted of twenty rats. Normal saline, 5-Fu, 5-Fu and lipiodol were infused through hepatic artery respectively. Two weeks after the infusion, staining of factor VIII, VEGF and b-FGF was performed by immunohistochemistry method in routine paraffin-embedded sections. Microvessel density (MVD) was counted in endothelial cells with positive factor VIII. Their expression levels were analyzed in conjunction with the pathologic features. RESULTS: While a smaller tumor volume was found in TACE group (F=37.818, P<0.001), no statistical differences between MVD and expression of VEGF and b-FGF were found among the 3 groups. MVD of the control group, chemotherapy group and chemoemoblization group was 80.84+/-24.24, 83.05+/-20.29 and 85.20+/-23.91 (F=0.193, P=0.873), respectively. The positive expression of VEGF and b-FGF was 75%, 75%, 85% (chi2=0.449, P=0.799) and 30%, 25%, 30% (chi2=0.141, P=0.922), respectively. Statistical analysis revealed a positive correlation between the expression of VEGF and MVD (r=0.552, P<0.001). CONCLUSION: There has been little influence of lipiodol chemoembolization on the formation of tumor angiogenesis, but the development of neovascularization and expression of VEGF play important roles in establishment of collateral circulation and reconstruction of blood supply of residual cancer tissue.