ObjectiveTo investigate the influence of hypoxia on pro-metastasis induced by epithelial-mesenchymal transition (EMT) of human lung adenocarcinoma. MethodsThe human lung cancer cell line H460 was cultured in hypoxic condition and the morphologic changes of the cells were observed under the microscope. The EMT-related markers including E-cadherin and vimentin were detected by Western blot. Transwell migration assay and transwell invasion assay were employed to detect the migratory and invasive activity of cancer cells. ResultsHypoxic induced morphological changes were consistent with the mesenchymal phenotype, such as an elongated fibroblastic morphology, and conversion from a tightly packed epithelial cobblestone pattern to a loosely packed scattered phenotype. Compared with the control group, hypoxic attenuated the quantity of E-cadhenrin, but increased vimentin, which resulted in promotion of migration and invasion of H460. ConclusionHypoxia induces EMT in H460 and enhances the invasive and migratory abilities of lung cancer cells.
Objective To examine the effects of newly designed LY52 on the expression of matrix metalloproteinases and invasive ability of hepatocellular carcinoma HepG2 cells. Methods The effects of LY52 on the proliferations of HepG2 cells were detected by MTT assay. Gelatin zymography and Western blot were used to detect the effects of LY52 on matrix metalloproteinase-2 expression in the cell line. Transwell chamber assay was used to detect the effects of LY52 on the invasion of the cells. Results No obvious inhibitory or cytotoxicity effects of LY52 was found in lower concentrations (lt;200 μg/ml) of LY52. Gelatin zymography and Western blot showed that matrix metalloproteinase-2 expression were inhibited by LY52 in a dose-dependent manner in HepG2 cells. Furthermore, transwell chamber assay showed that LY52 could significantly inhibit the invasion of the cell line in a dose-dependent manner.Conclusion The results suggest that LY52 may inhibit the invasion of hepatocellular carcinoma cells by suppressing the matrix metalloproteinase-2 activity.
Objective To detect the tissue factor (TF) mRNA expression in hepatocellular carcinoma and to elucidate its significance. Methods TF mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR） in 27 cases of human hepatocellular carcinoma tissue specimen with their adjacent tissues and in 27 non-tumorous process tissues. Then the relationship between mRNA expression and pathological data were analyzed. Results The expression and the relative expression intensity of TF in hepatocellular carcinoma tissues were 62.96%（17/27） and 0.567±0.268 respectively, which were significantly higher than those in their adjacent tissues 〔33.33%(9/27), 0.469±0.184〕 and in 27 non-tumorous process tissue 〔29.63%(8/27), 0.353±0.121〕, Plt;0.05. The relative expression intensity of TF were associated with tumor size, intrahepatic and extrahepatic metastasis and portal vein invasion, but unrelated to gender, AFP level, differentiation, HBsAg, cirrhosis, number of tumor lesions, and lymph node metastasis (Pgt;0.05). Conclusion Expression of TF mRNA were significantly higher in hepatocellular carcinoma and in the invasive and metastatic tissue, which indicated that TF may play an important role in carcinogenesis, invasion and metastasis of hepatocellular carcinoma.
ObjectiveTo investigate effect of small interfering RNA (siRNA) targeting inhibition of growth hormone receptor (GHR) on proliferation and invasion of human liver cancer line SMMC-7721. MethodsSMMC-7721 cells were transfected with siRNA targeting human GHR by GenMuteTM transfection regent.The cells were divided into three groups:blank control group (non-transfected siRNA),negative control group (transfected with non-specific siRNA),and specificity transfected group (transfected with expression specifically interfere by GHR siRNA).the relative expression of GHR mRNA was detected by using real-time PCR.the expression of GHR protein was detected by using Western blot.The cell proliferation was assessed by CCK-8 assay.And the ability of invasion was examined by Transwell assay. ResultsThe expressions of GHR mRNA and GHR protein in the specificity transfected group were significantly lower than those in the blank control group (P<0.05) and the negative control group (P<0.05).Compared with the blank control group and the negative control group,the absorbance value and the number of migrating cells of SMMC-7721 cells were decreased obviously (P<0.05) in the specificity transfected group. ConclusionsiRNA targeting human GHR could reduce capability of proliferation migration and invasion of SMMC-7721 cells.
Objective To observe the effect of epidermal growth factor (EGF) on the proliferation, adhesion, invasiveness and the activation of nuclear factor-κB (NF-κB), matrix metalloproteinases (MMPs) expression and explore related mechanisms in pancreatic cancer cells. Methods Cell invasion assay, proliferation assay and adhesion assay were used to examine the proliferation, adhesion and invasiveness of pancreatic cancer cells, respectively. NF-κB activity was detected by electrophoretic mobility shift assay (EMSA), and MMPs protein and mRNA expressions were investigated by gelatin zymography, Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Results EGF increased the invasiveness of pancreatic cancer cell in a dose-dependent manner (P＜0.05), but did not affect cell proliferation or adhesion. The expressions of MMP-9 mRNA and protein significantly increased after induction by EGF and were highest when EGF concentration was 50 ng/ml, while there was no effect on the expressions of MMP-2 mRNA and protein. Furthermore, NF-κB activity increased with increased concentration of EGF in a concentration-dependent manner (P＜0.05). In addition, NF-κB activity and the expressions of MMP-9 mRNA and protein by pretreatment with both pyrrolidine dithiocarbamate (PDTC) and EGF decreased when compared that by pretreatment with EGF alone. The invasiveness of pancreatic cancer cell by pretreatment with both PDTC and EGF decreased when compared that by pretreatment with EGF alone and nothing (P＜0.05).Conclusion The findings indicate that the NF-κB-mediated MMP-9 induction is essential for EGF-induced invasiveness in pancreatic cancer cells, which can be inhibited by PDTC.
【 Abstract 】 Objective Overexpressions of epidermal growth factor (EGF) and EGF receptor have been associated with progression and invasive phenotype of pancreatic cancer. However, the underlying molecular mechanism by which EGF worked in pancreatic cancer cells has not been completely understood. In this study, effect of EGF on the invasion and metastasis of pancreatic cancer cells and its regulatory mechanism were investigated. Methods The effects of EGF on the proliferation, adhesion and invasion of pancreatic cancer cells were detected by WST-1 proliferation assay, adhesion assay and invasive assay, respectively. The activity and expression of MMP-2 and MMP-9 were examined by zymography, Western blot and RT-PCR, respectively. The activity of NF- κ B was examined by EMSA. Results EGF could significantly promote the invasiveness of pancreatic cancer cells but did not affect cell proliferation or adhesion. The expressions of NF- κ B and MMP-9 were significantly increased by EGF, but EGF did not affect the activity and expression of MMP-2. Furthermore, EGF stimulated the NF- κ B binding activity. Pretreatment with NF- κ B inhibitors, pyrrolidine dithiocarbamate (PDTC), could significantly inhibit the activity of NF- κ B induced by EGF. Meanwhile, the EGF-induced expression and activity of MMP-9, as well as cell invasiveness were also inhibited by NF- κ B inhibitor. Conclusion EGF could increase the expression and promote the invasiveness of MMP-9 via the activation of NF- κ B in pancreatic cancer cells, which implies that NF- κ B inhibitant, such as PDTC, may diminish the invasiveness of pancreatic cancer cells.
ObjectiveTo study the expression of matrilysin in gastric cancer and to evaluate the correlation between its expression and invasion, metastasis, and prognosis. MethodsA total of 52 patients with gastric cancer were selected and followed up. The expressions of matrilysin in gastric primary focus, normal gastric mucosa, and metastatic lymph nodes were examined by reverse transcriptionpolymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry, respectively. The correlations between matrilysin expression and tumor invasion, metastasis, and prognosis were assessed. ResultsThe expressions of matrilysin in gastric primary focus and metastatic lymph nodes significantly increased, while decreased or loss in normal gastric mucosa (Plt;0.001). The higher concordance was seen between the levels of mRNA and protein (Plt;0.001). Among patients with infiltrating type, penetrated serosa, area of serosa involved more than 20 cm2, and metastatic lymph nodes more than 7, the expression of matrilysin was significantly higher (Plt;0.01). The survival rate of patients with matrilysin higher expression (34.1%) was significantly lower than that with matrilysin lower expression (55.6%), χ2=9.778， P=0.002. Conclusions Up-regulated expression of matrilysin plays an important role in tumor invasion, metastasis, and poor prognosis, and it is a good molecular marker to reflect the biological behaviors of gastric cancer.
ObjectiveTo investigate the expressions of Snail and VEGF gene in invasion ductal carcinoma tissues and analyze their clinicopathologic relationship. MethodsThe expressions of Snail and VEGF gene were detected on mammary gland hyperplasia (30 cases), intraductal breast cancer (30 cases), and invasion ductal carcinoma (70 cases) by in situ hybridization， to compare with the expression difference of the two genes in the different pathological changed tissues of mammary gland and among the clinicopathological facters of invasion ductal carcinoma as well as the relationship. ResultsThe expression rate of Snai mRNA in mammary gland hyperplasia， intraductal breast cancer, and invasion ductal carcinoma was 23.3% (7/30)， 46.7% (14/30), and 81.4% (57/70), respectively, there was statistical difference among them (χ 2=32.4, Plt;0.05)； The expression rate of VEGF mRNA in mammary gland hyperplasia， intraductal breast cancer, and invasion ductal carcinoma was 33.3% (10/30)， 50.0% (15/30), and 71.4% (50/70), respectively, there was statistical difference among them (χ 2=13.4, Plt;0.05). The expression rates of Snail mRNA and VEGF mRNA in lymphatic metatasis group were significantly higher than those in no lymphatic metatasis group 〔92.7% (38/41) vs. 65.5% (19/29)， χ 2=8.29, Plt;0.05； 85.4% (35/41) vs. 51.7% (15/29), χ 2=9.42, Plt;0.05， respectively 〕. The expression rates of Snail mRNA and VEGF mRNA in Ⅲ-Ⅳ stage of TNM clinical stage were significantly higher than those in Ⅰ-Ⅱ stage 〔939% (46/49) vs. 52.4% (11/21), χ 2=14.14， Plt;0.05； 81.6% (40/49) vs. 47.6% (10/21), χ 2=8.32， Plt;0.05〕. The expressions of Snail mRNA and VEGF mRNA were related to the expressions of ER, PR, HER-2, and vessel cancer embolus （Plt;0.01）. The expressions of Snail mRNA and VEGF mRNA were not related to age, tumor size, and histological grade (Pgt;0.05). There was a positive correlation between the expressions of Snail mRNA and VEGF mRNA （r=0.67， Plt;0.05）. ConclusionsThe overexpressions of Snail mRNA and VEGF mRNA in invasion ductal carcinoma has a synergetic effect on occurrence and development, therefore, combined detecting the expressions of Snail mRNA and VEGF mRNA are some significance to predict infiltration and metastasis of the invasion ductal carcinoma.
ObjectiveTo explore the expression of collagen Ⅳ in breast cancer and its clinical significance. We analyzed the correlation of the results with other prognostic parameters which included tumor size, status of estrogen receptor, axillary nodal status, TNM grade, and 5 years survival. The expression of collagen Ⅳ in 93 cases of human primary breast cancer as well as 5 cases of benign breast masses were examined.MethodsUsing monoclonal antibodies of collagen Ⅳ, the expression of collagen Ⅳ in breast masses were detected with immunohistochemical technique (LSAB).ResultsThe absent expression of collagen Ⅳ in the tumor masses was correlated with axillary lymph node involvement, tumor size and poor prognosis (5 years survival). The patients who had no expression of collagen Ⅳ in tumor masses had a shorter survival. ConclusionThe expression of collagen Ⅳ in tumor samples are correlated with axillary node involvement and prognosis. Collagen Ⅳ would be helpful for evaluation of invasion and treatment in breast carcinoma.
For an advanced elucidation of mechanisms of nm23-H1 suppressive effects on metastasis of primary hepatocellular carcinoma (HCC), it is necessary to investigate the correlation between nm23-H1 expression and relative factors involved in the HCC invasion. In present report, full-length cDNA of nm23-H1 was subcloned into pBKCMV vector and transfected into HCC cell line to observe its effects on invasion, cytosolic free Ca2+ and Nras mRNA expression. The results showed that lower expression of N-ras and higher cytosolic free Ca2+ in transfected cell line were detected, while the potential of invasion was depressed. It suggests that the suppressive effects on HCC metastasis might interact with intracellular signal transduction which is essential for stimulating cell invasion.