Ecto-5′-nucleotidase promotes invasion, migration and adhesion of human breast cancer cells

Purpose

Associated with many molecules, metastasis includes cell adhesion to extracellular matrix, migration towards specific direction and invasion into local vessel of distant organs. The purpose of the present study was to evaluate the role of ecto-5′-nucleotidase (eN, ecto-5-NT, CD73) generated extracellular adenosine in biologically malignant behaviors of human breast cancer cell lines.

Materials and methods

Two human breast cancer cell lines, T-47D with lower expression of CD73 and MB-MDA-231 with higher expression of CD73, were used to investigate the functions of CD73. The effects of CD73 over-expression on invasion, migration and adhesion were observed in T-47D transfected with pcDNA-NT5E plasmid. The effects of specific CD73 inhibitor, α, ß-methylene ADP (APCP), were observed in MB-MDA-231 cells.

Results

The results showed CD-73 overexpression increased invasion, migration and adhesion to ECM of the pcDNA-NT5E transfected T-47D cells compared to the saline and mock vector controls. The increased cell mobility of CD-73-overexpressed T-47D cells was blocked by APCP. Adenosine increased the mobility of wild type T-47D cells. APCP inhibited the mobility of the MB-MDA-231 cells.

Conclusion

Taken together, our results indicated that CD73 may facilitate the adhesion, migration and invasion of human breast cancer cells through its enzyme activity of generating adenosine. This study provided a possibly molecular mechanism of metastasis of breast carcinoma.     

Enhancement of migration and invasion of hepatoma cells via a Rho GTPase signaling pathway

AIM:Intrahepatic extension is the main cause of liver failureand death in hepatocellular carcinoma patients.The smallGTPase Rho and one of its effector molecules ROCK regulatecytoskeleton and actomyosin contractility,and play a crucialrole in cell adhesion and motility.We investigated the roleof small GTPase Rho in biological behaviors of hepatocellularcarcinoma to demonstrate the importance of Rho in cancerinvasion and metastasis.METHODS:Using Western blotting,we quantitated Rhoprotein expression in SMMC-7721 cells induced byLysophosphatidic acid (LPA).Furthermore,we examined therole of Rho signaling in regulating the motile and invasiveproperties of tumor cells.RESULTS:Rho protein expression was stimulated by LPA.Using the Rhotekin binding assay to assess Rho activation,we observed that the level of GTP-bound Rho was elevatedtransiently alter the addition of LPA,and Y-27632 decreasedthe level of active Rho.LPA enhanced the motility of tumorcells and facilitated their invasion.Rho played an essentialrole in the migratory process,as evidenced by the inhibitionof migration and motility of cancer cells by a specific inhibitorof ROCK,Y-27632.CONCLUSION:The finding that invasiveness of hepatocellularcarcinoma is facilitated by the Rho/Rho-kinase pathway islikely to be relevant to tumor progression and Y-27632 maybe a new potential effective agent for the prevention ofintrahepatic extension of human liver cancer.     

Integrin β7-mediated regulation of multiple myeloma cell adhesion, migration, and invasion

Integrin-β7 (ITGB7) mRNA is detected in multiple myeloma (MM) cells and its presence is correlated with MAF gene activation. Although the involvement of several integrin family members in MM-stoma cell interaction is well documented, the specific biologic functions regulated by integrin-β7 in MM are largely unknown. Clinically, we have correlated integrin-β7 expression in MM with poor survival outcomes post autologous stem cell transplantation and postsalvage therapy with bortezomib. Functionally, we have found that shRNA-mediated silencing of ITGB7 reduces MM-cell adhesion to extra-cellular matrix elements (fibronectin, E-cadherin) and reverses cell-adhesion-mediated drug resistance (CAM-DR) sensitizing them to bortezomib and melphalan. In addition, ITGB7 silencing abrogated MM-cell transwell migration in response to SDF1α gradients, reduced vessel density in xenografted tumors, and altered MM cells in vivo homing into the BM. Mechanistically, ITGB7 knockdown inhibited focal adhesion kinase (FAK) and Src phosphorylation, Rac1 activation, and SUMOylation, reduced VEGF production in MM-BM stem cell cocultures and attenuated p65-NF-κB activity. Our findings support a role for integrin-β7 in MM-cell adhesion, migration, and BM homing, and pave the way for a novel therapeutic approach targeting this molecule.     

Expression of tissue microarray p53, p16 and cyclooxygenase-2 in gastric cancer

Objective To constructed tissue microarray containing specimens from gastric cancer and adjacent noncancer tissue to survey the expression of p53, pl6 and cyclooxygenase-2 (COX-2) , and to obtain a comprehensive survey on the expression of three proteins in gastric cancer progression and their clinical significance. Methods We constructed a tissue microarray containing 50     

Estrogen receptor β potentiates the antiproliferative effect of raloxifene and affects the cell migration and invasion in HCT-116 colon cancer cells

Background

Estrogen receptor β (ERβ) is the predominant ER in the colorectal epithelium, whose expression is greatly reduced in colorectal cancer compared with normal colon tissue. Recent in vitro studies suggested that ERβ may suppress tumor growth. No research was reported whether ERβ can be used as therapeutic agent for colon cancer.

Methods

In this study, ERβ gene constructed into adenoviral (Ad) vectors was used to treat colon cancer HCT-116 cells alone or in combination with raloxifene. In vitro and in vivo studies were conducted to investigate the therapeutic effects of ERβ and raloxifene in HCT-116 cells.

Results

Our results indicated that, although Ad-ERβ alone had no effect on the proliferation of HCT-116 cells, the combination of Ad-ERβ with raloxifene significantly inhibited the proliferation of HCT-116 cells. The apparently apoptotic induction effects may partly explain the cytotoxicity of the two agents. The results of the study of ERβ on migration and invasion of HCT-116 cells demonstrated that overexpression of ERβ significantly decreased cell migration and increased invasion of cells. The antitumor efficacies of ERβ as well as raloxifene were further investigated on HCT-116 tumor bearing mice. Results demonstrated that both Ad-ERβ and raloxifene individually inhibited tumor growth. The combination group showed the highest inhibitory efficiency compared with other three groups.

Conclusion

These findings demonstrated that combined administration of Ad-ERβ with raloxifene represents a promising colon cancer therapeutic strategy.     

Expression of IL-13Rα2 in liver cancer cells and its effect on targeted therapy of liver cancer

Purpose

Liver cancer is the third leading cause of cancer-related deaths globally. The number of liver cancers diagnosed in the world is increasing at an alarming rate. It is of great significance to find the new targets of the tumor cells and specific medicine. This research investigated the expression of interleukin-13 receptor α2 (IL-13Rα2) in different liver cancer cell lines and liver cancer tissues, and assessed the cytotoxin DT389-hIL13-13E13K (IL-13 and diphtheria toxin fusion protein) targeted killing effect on liver cancer cells. Based on study above, we further analyzed the function of IL-13Rα2 on the targeted liver cancer therapy. The results will provide a novel strategy and an alternative way for liver cancer therapy.

Methods

The expression of IL-13Rα2 in different liver cancer cell lines and tissues were analyzed by RT-PCR and immunohistochemistry. Cytotoxicity assay of DT389-hIL13-13E13K was performed in eight different concentrations in liver cancer cell lines in vitro. At the same time, siRNA-mediated knockdown was introduced to assess the role of IL-13Rα2 in liver cancer therapy.

Results

Two out of four tested liver cancer cell lines and 27 out of 33 (81.82%) liver tissues expressed the IL-13Rα2. The fusion protein DT₃₈₉-hIL13-13E13K showed a moderate cytotoxicity to the cancer cell line BEL-7402 in vitro, which 50% inhibition (IC₅₀) concentration occurred at 1.4 × 10^?5 M. Besides, the sensitivity to fusion protein DT₃₈₉-hIL13-13E13K was decreased in siRNA-transfected liver cells compared with control ones. These results suggest that IL-13Rα2 chain is a specific target for IL-13-directed fusion protein.

Conclusions

We reported the expression of IL-13Rα2 in liver cancer cell lines and tissues as well as investigated the cytotoxin (DT389-hIL13-13E13K) targeted killing efficiency of liver cancer cells and potential role of IL-13Rα2 in the cancer treatment.     

Expression of p53, p16 and COX-2 in pancreatic cancer with tissue microarray

BACKGROUND: Pancreatic cancer development and progression is driven by the accumulation of genetic changes. In this study we constructed tissue microarray containing specimens from pancreatic cancer, adjacent non-cancer tissue and normal tissue to survey the expression of p53, p16 and cyclooxyganase-2 (COX-2). METHODS: Tissue microarray containing 337 specimens from different stages of pancreatic cancer, adjacent noncancer tissue and normal tissues was constructed, and the expression of p53, p16 and COX-2 was assayed by immunohistochemistry to consecutive formalin-fixed tissue microarray sections. RESULTS: The expression of p53, p16 and COX-2 was significantly higher in tumorous tissues than in non-tumorons ones. A significant relationship was observed between p53 and COX-2, or p16 and COX-2. But no obvious correlation was seen between p53 and p16 expressions. Logistic regression analysis showed p53 and COX-2 as dependent predictors in pancreatic carcinogenesis, and a reciprocal relationship to neoplastic progression between p53 and COX-2. CONCLUSION: Combination analysis of p53 and COX-2 may be useful in predicting pancreatic carcinogenesis.     

Arylsulfatase,β-galactosidase and lysozyme in gastric cancer cells and its relationship to invasion

Ａｒｙｌｓｕｌｆａｔａｓｅ，βｇａｌａｃｔｏｓｉｄａｓｅａｎｄｌｙｓｏｚｙｍｅｉｎｇａｓｔｒｉｃｃａｎｃｅｒｃｅｌｓａｎｄｉｔｓｒｅｌａｔｉｏｎｓｈｉｐｔｏｉｎｖａｓｉｏｎＹＩＹｏｎｇＦｅｎａｎｄＨＵＡＮＧＹｏｕＲｏｎｇＳｕｂｊｅｃｔｈｅａｄ...     

Inhibitory effects of synthetic β peptide on invasion and metastasis of liver cancer

Purpose: To study the inhibitory effects of synthetic β peptide on invasion and metastasis of liver cancer. Methods: Membrane-type intercellular adhesion molecule-1 (ICAM-1) expression of SMMC-7721 cultured hepatoma cells (7721 cells) was detected by immunofluorescence cell flowmeter. The adhesion of 7721 cells to fibronectin (FN) was assayed by the MTT method. The adhesion of 7721 cells to 7721 cells, 7721 cells to endothelial cells, and 7721 cells to lymphocyte cells was detected by adhesion assay. LCI-D20 human liver cancer metastasis model in nude mice was used in this experiment. One hundred micrograms of β peptide per mouse were injected subcutaneously after tumor was resected premetastatically or postmetastatically to observe its effect on liver cancer metastasis after hepatectomy. Results: Membrane-type ICAM-1 expression of SMMC-7721 cells treated by β peptide was lower than that of the untreated cells. The adhesion of 7721 cells to FN, 7721 cells to 7721 cells, 7721 cells to endothelial cells, and 7721 cells to lymphocyte cells was also lower in the β peptide group than in the untreated group. Conclusions:β Peptide can block the adhesion of 7721 cells to FN, 7721 cells to some host cells in vitro, and inhibit HCC metastasis of LCI-D20 model posthepatectomy in vivo, so it could potentially act as an anti-metastasis drug. Received: 23 November 1999 / Accepted: 10 March 2000     

Role of nucleostemin in growth regulation of gastric cancer,liver cancer and other malignancies

AIM:To examine the role of nucleostemin in the growthregulation of gastric cancer,liver cancer and other cancers.METHODS:RT-PCR was used to clone the fragment ofnucleostemin cDNA from HEK 293 cells.Eighteen kinds ofmalignant tumor tissues including gastric adenocarcinomaand liver cancer tissues,3 kinds of benign tumor tissues,3kinds of benign hyperplastic tissues and normal tissueswere employed to examine nucleostemin gene expressionby RT-PCR,Slot blot,Northern blot and in situ hybridization.RESULTS:We successfully cloned a 570 bp fragment ofnucleostemin-cDNA from HEK-293 cells.All detectedmalignant tumor tissues,benign tumor tissues,and benignhyperplastic tissues had high levels of nucleosteminexpression.Nucleostemin was also expressed in humanplacenta tissue at a high level.In terminally differentiatednormal human adult kidney and mammary gland tissues,no nucleostemin expression could be detected.CONCLUSION:Nucleostemin can help regulate theproliferation of both cancer cells and stem cells.It mightplay an important role in the growth regulation of gastriccancer,liver cancer and other cancers.     

Evaluation of VEGF and VEGF-C expression in gastric cancer cells producing α-fetoprotein

Background. Gastric cancers producing α-fetoprotein (AFP) are known to have a poor prognosis and to show a high incidence of liver metastasis. Vascular endothelial growth factor (VEGF) and its isoform VEGF-C are reported to be associated with tumor progression through an angiogenic or lymphangiogenic function. In the present study, to clarify the cellular characteristics of AFP-producing gastric cancers, the expression of VEGF and that of VEGF-C in AFP-producing gastric cancer was compared with their expression in gastric cancers that do not produce AFP. Methods. Twenty-six patients with AFP-producing gastric cancers [AFP(+)] and 26 patients with stage-matched gastric cancers that did not produce AFP [AFP(−)] were evaluated for VEGF and VEGF-C expression and vessel density, using immunohistochemical analysis. Results. The survival rate of the AFP(+) group was significantly worse than that of the stage-matched AFP(−) group (P < 0.05). The frequency of VEGF-C expression was significantly higher in the AFP(+) group than in the AFP(−) group (P < 0.01). There was no significant difference in VEGF expression between the AFP(+) and AFP(+) groups. The microvessel density was higher in the AFP(+) group than in the AFP(−) group (P < 0.05). Conclusions. A higher expression of VEGF-C might be one explanation for the poorer prognosis of AFP-producing gastric cancers. Received: June 7, 2002 / Accepted: October 25, 2002 RID="*" ID="*" Reprint requests to: K. Kono     

Expression of intercellular adhesive molecule-1 in liver cancer tissues and liver cancer metastasis

Ｅｘｐｒｅｓｓｉｏｎｏｆｉｎｔｅｒｃｅｌｕｌａｒａｄｈｅｓｉｖｅｍｏｌｅｃｕｌｅ１ｉｎｌｉｖｅｒｃａｎｃｅｒｔｉｓｓｕｅｓａｎｄｌｉｖｅｒｃａｎｃｅｒｍｅｔａｓｔａｓｉｓＳＵＮＪｉｎｇＪｉｎｇ，ＺＨＯＵＸｉｎＤａ，ＺＨＯＵＧｅａｎｄＬＩＵＹｉｎ...     

Expression of intercellular adhesive molecule1 in liver cancer tissues and liver cancer metastasis

AIM: To study the relationship between intercellular adhesive molecule-1 (ICAM-1) and liver cancer metastasis and to search for factors to predict metastasis of liver cancer.METHODS: ICAM-1 expression in fresh tissues of normal liver and hepatocellular cancer (HCC) was examined by immunoperoxidase staining. The expression of ICAM-1 in human hepatoma, tumor surrounding tissues and normal livers were semiquantitatively analyzed by Dot immuno blot. Tissue ICAM-1 expression at mRNA level was detected by Northern blot.RESULTS: All 6 cases of normal liver samples were negative in anti-ICAM-1 immunohistochemical staining, 80.0% (36/45) of HCC presented various ICAM-1 expression. The number of positive cells was a little higher in large tumors, tumors with intact capsule and metastasis, but there was no significant difference. Two cases with cancer embolus also had high ICAM-1 expression. ICAM-1 concentration in HCC (13.43 ± 0.09) was higher than that in tumor surrounding tissues (5.89 ± 0.17, P < 0.01) and normal livers (4.27 ± 0.21, P < 0.01). It was also higher in metastasis group (20.24 ± 0.30) than in nonmetastasis group (10.23 ± 0.12, P < 0.05). Northern blot analysis revealed that ICAM-1 expression at mRNA level was also higher in HCC and cancer embolus than that in tumor surrounding tissues and normal livers.CONCLUSION: Tissue ICAM-1 could indicate the growth and metastasis of HCC, and may be an index that can predict liver cancer metastasis.     

Human mesenchymal stem cells are recruited to injured liver in a β1-integrin and CD44 dependent manner

Human bone marrow mesenchymal stem cells (hMSCs) have shown benefit in clinical trials of patients with liver disease. Efficient delivery of cells to target organs is critical to improving their effectiveness. This requires an understanding of the mechanisms governing cellular engraftment into the liver. Binding of hMSCs to normal/injured liver tissue, purified extracellular matrices, and human hepatic sinusoidal endothelial cells (HSECs) were quantified in static and flow conditions. To define the mechanisms underpinning hMSC interactions, neutralizing adhesion molecule antibodies were used. Fluorescently labelled hMSCs were infused intraportally into CCl(4) -injured mice with and without neutralizing antibodies. hMSCs expressed high levels of CD29/β1-integrin and CD44. Using liver tissue binding assays, hMSC adhesion was greatest in diseased human liver versus normal liver (32.2 cells/field versus 20.5 cells/field [P = 0.048]). Neutralizing antibodies against CD29 and CD44 reduced hMSC binding to diseased liver by 34% and 35%, respectively (P = 0.05). hMSCs rolled at 528 μm/second on HSECs in flow assays. This rolling was abolished by CD29 blockade on hMSCs and vascular cell adhesion molecule-1 (VCAM-1) blockade on HSECs. Firm adhesion to HSECs was reduced by CD29 (55% [P = 0.002]) and CD44 (51% [P = 0.04]) blockade. Neutralizing antibodies to CD29 and CD44 reduced hepatic engraftment of hMSCs in murine liver from 4.45 cells/field to 2.88 cells/field (P = 0.025) and 2.35 cells/field (P = 0.03), respectively. hMSCs expressed modest levels of chemokine receptors including CCR4, CCR5, and CXCR3, but these made little contribution to hMSC adhesion in this setting. Conclusion: hMSCs bind preferentially to injured liver. Rolling of hMSCs is regulated by CD29/VCAM-1, whereas CD29/CD44 interactions with VCAM-1, fibronectin, and hyaluronan on HSECs determine firm adhesion both in vitro and in vivo as demonstrated using a murine model of liver injury. (HEPATOLOGY 2012;56:1063-1073).     

Troglitazone, a peroxisome proliferator-activated receptor γ ligand, induces growth inhibition and apoptosis of HepG2 human liver cancer cells

AIM： To examine the effect of troglitazone, a peroxisome proliferator-activated receptor γ （PPARγ） ligand, on the proliferation and apoptosis of human liver cancer cells. METHODS： Liver cancer cell line HepG2 was cultured and treated with troglitazone. Cell proliferation was detected by 3-（4-,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide （MTT） assay; apoptosis was detected by flow cytometry and terminal deoxynucleotidyl transferase- mediated nick end labeling of DNA fragmentation sites （TUNEL） assay; and apoptosis-related protein was detected by immunocytochemistry and Western blotting. RESULTS： Troglitazone inhibited growth and induced apoptosis of HepG2 cells in a dose-dependent manner, and induced activation of caspase-3 expression. Troglitazone not only drove apoptosis-inhibiting factor survivin to translocate incompletely from the nucleus to the cytoplasm, but also inhibited expression of survivin, while it did not affect expression of apoptosis-promoting factor Bax. CONCLUSION： PPARγ ligands inhibit growth and induce apoptosis of liver cancer cells, and may have applications for the prevention and treatment of liver cancer.     

Early apoptosis and cell death induced by ATX-S10Na （ Ⅱ ）-mediated photodynamic therapy are Bax- and p53-dependent in human colon cancer cells

AIM： To investigate the roles of Bax and p53 proteins in photosensitivity of human colon cancer cells by using lysosome-localizing photosensitizer, ATX-S10Na （Ⅱ）. METHODS： HCT116 human colon cancer cells and Bax-null or p53-null isogenic derivatives were irradiated with a diode laser. Early apoptosis and cell death in response to photodynamic therapy were determined by MTT assays, annexin Ⅴ assays, transmission electron microscopy assays, caspase assays and western blotting. RESULTS： Induction of early apoptosis and cell death was Bax- and p53-dependent. Bax and p53 were required for caspase-dependent apoptosis. The levels of anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-xL, were decreased in Bax- and p53-independent manner. CONCLUSION： Our results indicate that eady apoptosis and cell death of human colon cancer cells induced by photodynamic therapy with lysosome-localizing photosensitizer ATX-S10Na （Ⅱ） are mediated by p53- Bax network and low levels of Bcl-2 and Bcl-xL proteins. Our results might help in formulating new therapeutic approaches in photodynamic therapy.