Inhibitory effect of interferon-α-2b on expression of cyclooxygenase-2 and vascular endothelial growth factor in human hepatocellular carcinoma inoculated in nude mice

AIM： To evaluate the effects of interferon-α-2b （IFN- α-2b） on expression of cyclooxygenase-2 （COX-2） and vascular endothelial growth factor （VEGF） in human hepatocellular carcinoma （HCC） inoculated in nude mice and to study the underlying mechanism of IFN-α- 2b against HCC growth. METHODS： Thirb/-two nude mice bearing human HCC were randomly divided into four groups （n = 8）. On the 10th day after implantation of HCC cells, the mice in test groups （groups A, B and C） received IFN-α- 2b at a serial dose （10000 IU for group A, 20000 IU for group B, 40000 IU for group C sc daily） for 35 d. The mice in control group received normal saline （NS）. The growth conditions of transplanted tumors were observed. Both genes and proteins of COX-2 and VEGF were detected by RT-PCR and Western blot. Apoptosis of tumor cells in nude mice was detected by TUNEL assay after treatment with IFN-α-2b. RESULTS： Tumors were significantly smaller and had a lower weight in the IFN-α-2b treatment groups than those in the control group （P 〈 0.01）, and the tumor growth inhibition rate in groups A, B and C was 27.78%, 65.22% and 49.64%, respectively. The expression levels of both genes and proteins of COX-2 and VEGF were much lower in the IFN-α-2b treatment groups than in the control group （P 〈 0.01）. The apoptosis index （AI） of tumor cells in the IFN-α-2b treatment groups was markedly higher than that in the control group （P 〈 0.01）. Group B had a higher inhibition rate of tumor growth, a lower expression level of COX-2 and VEGF and a higher AI than groups A and C （P 〈 0.05）, but there was no significant difference between groups A and C. CONCLUSION： The inhibitory effects of IFN-α-2b on implanted tumor growth and apoptosis may be associated with the down-regulation of COX-2 and VEGF expression. There is a dose-effect relationship. The medium dose of IFN-α-2b for inhibiting tumor growth is 20 000 IU/d.     

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.     

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.     

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.     

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.     

Correlation of expression of multidrug resistance protein and messenger RNA with ^99mTc-nethoxyisobutyl isonitrile（MIBI） imaging in poatients with hepatocellular carcinoma

AIM: To explore whether P-glycoprotein (Pgp) and other pumps, multidrug resistance-associated protein (MRP) and lung resistance protein (LRP), could affect tumor accumulation and efflux of 99mTc-MIBI in liver cancer. METHODS: Surgically treated 78 liver cancer patients were included in this study. Before surgery, 99mTc-MIBI SPECT was performed 15 min and 120 min after injection of 20 mCi 99mTc-MIBI, respectively. Early uptake, delayed uptake (L/Nd), and washout rate (L/Nwr) of 99mTc-MIBI were obtained. Expressions of Pgp, MRP and LRP were investigated with Western blotting and immunohistochemistry. Messenger RNA (mRNA) level of Pgp, MRP and LRP was determined by RT-PCR. RESULTS: No 99mTc-MIBI uptakes in tumor lesions of 68 of 78 (87.2%) patients with hepatocellular carcinoma were found on 99mTc-MIBI SPECT. P-gp expression was observed in tumor tissues of the patients with no uptake of 99mTc-MIBI (P<0.017). No appreciable correlation was found between liver cancer 99mTc-MIBI images and expression of MRP or LRP on the level of protein or mRNA. CONCLUSION: 99mTc-MIBI SPECT is noninvasive, and useful in predicting the presence of MDR1 gene-encoded Pgp in patients with hepatocellular carcinoma.     

Clinicopathological and prognostic implications of endoglin （CD105） expression in hepatocellular carcinoma and its adjacent non-tumorous liver

AIM: The expression pattern of endoglin (CD105) in hepatocellular carcinoma (HCC) has not been reported so far. We hypothesized that CD105 could differentially highlight a subset of microvessels in HCC, and intratumoral microvessel density (IMVD) by CD105 immunostaining (IMVD-CD105) could provide better prognostic information than IMVD by CD34 immunostaining (IMVD-CD34). METHODS: Paraffin blocks of tumor and adjacent non-tumorous liver tissues from 86 patients who underwent curative resection of HCC were used for this study. Serial sections were stained for CD105 and CD34, respectively, to highlight the microvessels. IMVD was counted according to a standard protocol. RESULTS: In the HCC tissues, CD105 was either negatively or positively stained only in a subset of microvessels. In contrast, CD34 showed positive and more extensive microvessel staining in all cases examined. However, in the adjacent non-tumorous liver sections, CD105 showed a diffuse pattern of microvessel staining in 20 of 86 cases, while CD34 showed negative or only focal staining of the sinusoids around portal area. Correlation with clinicopathological data demonstrated that lower scores of IMVD-CD105 were found in larger sized tumors [mean 41.4/0.74 mm2 (>5 cm tumor) vs 65.9/0.74 mm2 (≤5 cm tumor), P= 0.043] and more aggressive tumors, as indicated by venous infiltration [36.8/0.74 mm2 (present) vs 64.2/0.74 mm2 (absent), P = 0.020], microsatellite nodules [35.1/0.74 mm2 (present) vs 65.9/0.74 mm2 (absent), P= 0.012], and advanced TNM tumor stage [38.8/0.74 mm2 (stage 3 or 4) vs 68.3/0.74 mm2 (stage 1 or 2), P= 0.014]. No prognostic significance was observed when median values were used as cut-off points using either IMVD-CD105 or IMVD-CD34. However, the presence of the diffuse pattern of CD105 expression in the adjacent non-tumorous liver tissues predicted a poorer disease-free survival (median 8.6 vs 21.5 mo, P= 0.026). CONCLUSION: Our data demonstrate that a lower IMVD-CD105 is associated with larger and more aggressive tumors. In this study, IMVD-CD105 did not provide significant prognostic information. However, active angiogenesis as highlighted by diffuse CD105 staining of the microvessels in the adjacent non-tumorous liver tissues is predictive of early recurrence.     

Correlation of integrin β3 mRNA and vascular endothelial growth factor protein expression profiles with the clinicopathological features and prognosis of gastric carcinoma

AIM： To investigate integrin β3 mRNA and vascular endothelial growth factor （VEGF） protein expression in gastric carcinoma, and its correlation with microvascular density, growth-pattern, invasion, metastasis and prognosis. METHODS： In situ hybridization（ISH） of integrin β3 mRNA and immunohistochemistry of VEGF and CD34 protein were performed on samples from 118 patients with gastric cancer. RESULTS： The positive rate of integrin β3 mRNA in non-tumor gastric mucosa （20%） was significantly lower than that of the gastric cancer tissue （52.5%, X^2 = 10.20, P 〈 0.01）. In patients of infiltrating type, stage T3-T4, vessel invasion, lymphatic metastasis, hepatic or peritoneal metastasis, the positive expression rates of integrin β3 mRNA were significantly higher than those in patients of expanding type （P 〈 0.01）, stage T1-T2 （P 〈 0.01）, non-vessel invasion （P 〈 0.01）, without lymphatic metastasis （P 〈 0.01）, without hepatic and peritoneal metastasis （P 〈 0.01）, respectively. In patients of infiltrating type, stage T3-T4, vessel invasion, lymphatic metastasis, hepatic or peritoneal metastasis, the positive expression rates of VEGF protein were significantly higher than those in patients of expanding type （P 〈 0.01）, stage T1-T2 （P 〈 0.01）, non-vessel invasion （P 〈 0.01）, without lymphatic metastasis （P 〈 0.01）, without hepatic and peritoneal metastasis （P 〈 0.01）, respectively. In patients of infiltrating type, stage T3-T4, vessel invasion, lymphatic metastasis, hepatic or peritoneal metastasis, the mean MVD were significantly higher than those in patients of expanding type （P 〈 0.01）, stage T1-T2 （P 〈 0.01）, non-vessel invasion （P 〈 0.01）, without lymphatic metastasis （P 〈 0.01）, without hepatic and peritoneal metastasis （P 〈 0.01）, respectively. It was found that the positive expression rate of integrin β3 mRNA was positively related to that of VEGF protein （P 〈 0.01） and MVD （P 〈 0.05）,