Growth-inhibitory effects of selective cyclooxygenase-2 inhibitor on colon cancer cells and its possible mechanisms

AIM: To evaluate the growth-inhibitory effects of NS-398, a selective cyclooxygenase-2 inhibitor, in human colon cancer HT-29 cells and its possible mechanisms. METHODS: MTT assay was applied to detect the cell proliferation. Flow cytometry was performed to detect apoptosis rate and cell cycle. RT-PCR was used to detect the expression of bcl-2 mRNA and bax mRNA. Alteration of cytoskeleton component F-actin was observed by confocal laser scanning microscope. RESULTS: NS-398 could inhibit growth of HT-29 cells in dose-and time-dependent manners. Flow cytometry revealed that NS-398 could induce apoptosis and cause G0/G1 arrest of HT-29 cells in a dose-dependent manner. After 72 h incubation with NS-398 at different concentrations, the expression level of bcl-2 mRNA was lowered and the ratio of bcl-2 to bax was decreased in HT-29 cells. F-actin was mainly distributed around nuclei forming annular structure in HT-29 cells. After exposure to NS-398, the annular structure around nuclei disappeared and fluorescence intensity of F-actin decreased obviously. CONCLUSION: NS-398 can inhibit the growth effectively and induce apoptosis in HT-29 cells in vitro, which is associated with the down-regulation of bcl-2 to bax ratio and the disruption of cytoskeleton.     

Effects of NS-398 on the proliferation and apoptosis of HepG2 cells

AIM: To evaluate the effects of NS-398, a cyclooxygenase-2(COX-2) inhibitor, on the proliferation and apoptosis in HepG2 cells. METHODS: The effects of NS-398 on the proliferation of HepG2 cells was evaluated by MTT. DNA fragmentation gel analysis was used to analyze the apoptotic cells; DNA ploidy and apoptotic cell percentage were examined by flow cytometry. Furthermore, the expression of COX-2 and Bcl-2 mRNA was identified by competitive RT-PCR. RESULTS: NS-398 inhibited cell proliferation and induced apoptosis in HepG2 in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased with NS-398 concentration increasing. The quiescent G₀/G₁ phase was accumulated with decreasing of Bcl-2 mRNA. Whereas NS-398 had no effect on the expression of COX-2 mRNA, no correlations were found between COX-2 mRNA and the HepG2 cell proliferation and apoptosis induced by NS-398 (r=0.056 and r=0.119, respectively). CONCLUSION: NS-398 significantly inhibits the proliferation and induces apoptosis in HepG2. Mechanisms may be involved in accumulation of quiescent G₀/G₁ phase and decrease in Bcl-2 mRNA expression, but independent to COX-2 mRNA expression.     

BPH-1 induces aromatase expression in prostatic stromal cells through paracrine of PGE2

AIM: To characterize the effect of prostatic epithelial cell paracrine on aromatase expression in prostatic stromal cells.METHODS: Conditioned medium (CM) of prostatic epithelial cell lines (BPH-1, LNCap, DU-145 and PC3) were collected and used to treat prostatic stromal cells. Expression of aromatase was determined by real-time RT-PCR and Western blotting. Expression of cyclooxygenase-2 mRNA in prostatic epithelial cell lines and prostaglandin (PGE2) in CMs were examined by real-time RT-PCR and ELISA, respectively. The CM of BPH-1 cells cultured with NS-398, specific inhibitor of cyclooxygenase-2, were collected, and the effect of NS-398 and PGE2 on aromatase expression was analyzed.RESULTS: CM of human benign prostate hyperplasia epithelial cell line (BPH-1) stimulated expression of aromatase mRNA and protein in stromal cells. But CM of prostate cancer epithelial cell lines (LNCap, DU145, PC3) had no effect on aromatase expression. COX-2 mRNA level in BPH-1 was much higher than that of other cell lines and PGE2 concentration in BPH-1 CM was much higher than that of other CMs. PGE2 concentration of the CM from BPH-1 cultured with NS-398 significantly decreased. CM from BPH-1 cultured with NS-398 failed to stimulate aromatase expression, while PGE2 induced aromatase expression in prostatic stromal cells.CONCLUSION: BPH-1 could induce aromatase expression in prostatic stromal cells through paracrine of PGE2.     

Inhibitory effects of nm23-H1 gene on proliferation and invasion of A549 cell line

AIM:To study the inhibitory effects of nm23-H1 gene on proliferation and invasion of human lung adenocarcinoma A549 cell line. METHODS:Recombinant eukaryotic expression vector pcDNA3.1-nm23-H1 containing full length of human nm23-H1 cDNA was constructed and transfected into a human lung adenocarcinoma A549 cell line by lipofectamine. Cell strain that expressed nm23-H1 stably was screened out by G418 and named pcDNA-nm23-A549. Expression of nm23-H1 was identified by RT-PCR and immunohistochemistry. Growth curves were drawn to detect the inhibitory effects on cell proliferation. Cell cycle of pcDNA-nm23-A549 was examined by flow cytometry. Atomic force microscopy was used to observe the filopodia on the surface of the cells. RESULTS:Introduction of nm23-H1 obviously inhibited the proliferation of A549. Expression of nm23-H1 did not induce apotosis in A549 cells but increased the percentage of phase G₁ cells and decreased phase S cells. Meanwhile, phase G₁ to phase S transition was restrained. Filopodia in the cell surface was much fewer and its structure changed in cells transformed. CONCLUSION:nm23-H1 is capable of inhibiting A549 proliferation and decreasing its metastatic ability, probably by interfering with cell cycle and cell surface structure.     

Inhibitory effects of a cyclooxygenase-2 inhibitor,NS398,on cancer cells

AIM:To study the mechanism of growth inhibitory effect of a selective cyclooxygenase-2 (COX-2) inhibitor,NS-398,on cancer cells.METHODS:The esophageal cancer cell line (EC9706),which expresses COX-2 constitutively,and hepatocellular carcinoma cell line (SMMC7721),which expresses no COX-2,were studied.The cell lines were incubated with NS-398 at doses of 10,20,50,100 μmol/L for 24 h,48 h and 72 h.Antiproliferation effect was measured by ［3H］-TdR incorporation.The cell apoptosis were determined by flow cytometry (FCM) and DNA fragmentation analysis.Survivin was detected by immunocytochemical technique.RESULTS:The growth inhibition was induced by NS398 in a dose- and time-dependent manners in both cell lines.FCM analysis revealed a high sub-G1 cell peak in EC9706 group and agarose electrophoresis showed marked apoptosis ladder pattern.However,no apoptosis was observed in SMMC7721 cells treated with NS-398.The difference of apoptosis percentage in EC9706 and SMMC7721 was (45.23±1.08)% and (3.05±0.15)% (P<0.01).After 24 h incubation with NS-398 at concentration of 100 μmol/L,the expression of survivin was markedly reduced in EC9706,no change was observed in SMMC7721.CONCLUSION:NS-398 suppresses cell growth in cancer cell lines by different mechanism.NS-398 suppresses cell growth and increases apoptosis in the cancer cells that expresses COX-2.     

Effects of propofol on invasion and migration of human gastric cancer SGC-7901 cells

AIM: To investigate the effects of propofol on invasion and migration of gastric cancer cell line SGC-7901. METHODS: Cultured gastric cancer cell line SGC-7901 was randomly divided into 4 groups, and then diffe-rent concentrations (1, 3, 5 and 7 mg/L) of propofol were added and incubated for 24 h. The cell viability was measured by MTT assay. The invasion and migration abilities of the SGC-7901 cells were detected by Transwell assay and wound-healing assay. The expression of cysteine-rich angiogenic inducer 61 (CYR61), CD44v6 and matrix metalloproteinase-7 (MMP-7) in the SGC-7901 cells were examined by immunocytochemistry and Western blot.RESULTS: Propofol at 5 mg/L does not affect the viability of SGC-7901 cells, whereas significantly suppresses the invasion and migration abilities, and down-regulates the expression of CD44v6 and MMP-7 (P<0.05). CONCLUSION: The decreased invasion and migration abilities of SGC-7901 cells were partly due to the inhibition of CD44v6 and MMP-7 expression.     

Effects of αvβ6 integrin-mediated cell adhesion on 5-fluorouracil induced apoptosis in human colon carcinoma cell lines

AIM: To investigate the effects of αvβ6 integrin-mediated cell adhesion on 5-fluorouracil (5-FU) induced apoptosis in colon carcinoma cell lines.METHODS: The expression of the αvβ6 integrin in colon carcinoma cell lines HT-29 and WiDr cells was analyzed by flow cytometry. The apoptosis induced by 5-FU and the effects of αvβ6 integrin-mediated cell adhesion on 5-FU induced cell apoptosis were measured by enzyme-linked immunosorbent assay (ELISA) method and acridine orange-ethidium bromide (AO-EB) double fluorescent dye staining.RESULTS: Both the colon carcinoma cell lines HT-29 and WiDr cells expressed the αvβ6 integrin. The percentages of HT-29 and WiDr cells expression were 80.82% and 82.96%. 5-FU induced the apoptosis of colon carcinoma cell lines HT-29 and WiDr. The result of ELISA method displayed that enrichment factor (EF) of HT-29 and WiDr cells planted on fibronectin (FN)-ligand of αvβ6 integrin was lower significantly than the EF of HT-29 and WiDr cells planted on non-integrin ligand polylisin (1.11±0.04 vs 3.68±0.03, 1.09±0.02 vs 3.72±0.02, P<0.01) after cultured in medium containing 20 mg/L 5-FU for 48 h. When HT-29 and WiDr cells preincubated with αvβ6 integrin blocking antibody were planted on FN again, the EF of HT-29 and WiDr cells was higher significantly than those directly planted on FN without being blocked by αvβ6 integrin antibody (2.12±0.04 vs 1.11±0.04, 2.14±0.03 vs 1.09±0.02, P<0.01). The AO-EB double fluorescent dye staining displayed that the apoptosis percents of HT-29 and WiDr cells planting on FN were (5.6±1.1)% and (5.3±0.7)%, which were lower significantly than those planting on polylisin (37.0±1.4)%, (38.5±0.9)%, P<0.01. When HT-29 and WiDr cells preincubated with αvβ6 integrin blocking antibody were planted on FN again the percents of HT-29 and WiDr cells apoptosis were (19.5±1.2)% and (20.0±0.7)%, which increased significantly compared with those directly planting on FN without being blocked by αvβ6 integrin antibody (P<0.01). CONCLUSION: Colon carcinoma cell lines HT-29 and WiDr cells expressed αvβ6 integrin. The cell adhesion with FN mediated by αvβ6 integrin inhibits 5-FU-induced colon carcinoma cell apoptosis. The results suggest that cell adhesion may enhance drug resistance in colon carcinoma cell lines through inhibiting the cell apoptosis.     

Experiment research of anti-PG cell metastasis and mechanism of a new Chinese herb Qcimum basilicum polysaccharide

AIM: In this paper, we studied the efficiencies and the mechanisms of a new Chinese herb Qcimum basilicum polysaccharide (BP) on PG cell metastasis in vitro. METHODS: The number of tumor cells going through matrigel was assayed and used to represent the ability of the invasion and migration of PG cells. Using Scrape-loading and dye transfer (SLDT) technique, the efficiencies of BP on recovering PG cell gap junction -mediated intercellular communication (GJIC) was measured. The expressions of c-myc, nm23-H1 and Tiam-1 genes mRNA in PG cells treated with BP were determined by RT-PCR. RESULTS: Compared with the control, the action of invasion and migration of PG cells were decreased after treated with BP (P<0.05). The GJIC of PG cells were recovered in different degrees after treated with BP for 8 h. The fluorescence reached some rows of cells from the scraped line. The expressions of c-myc and Tiam-1 genes in PG cells treated with BP were decreased, but the expression of nm23-H1 gene was increased compared with the control. CONCLUSIONS: BP is a new anti-pulmonary cancer cell metastasis reagent. Its antitumor metastasis action could be achieved by recovering the cell GJIC and changing the expression of some metastasis associated genes.     

αvβ6-ERK pathway participates in norcantharidin-induced apoptosis of HT-29 colon cancer cells

AIM: To investigate the pharmacological mechanism of norcantharidin (NCTD)-induced apoptosis of HT-29 colon cancer cells. METHODS: Hoechst 33258 staining was used to analyze the apoptosis of HT-29 cells treated with NCTD. The effects of NCTD on the expression of integrin in HT-29 cells were determined by flow cytometry. The effects of several functional blocking antibodies on HT-29 cells were detected by MTT method. The expression and the phosphorylation of mitogen activated protein kinases (MAPKs) in HT-29 cells were measured by Western blotting. Co-immunoprecipitation assay was used to detect the activity of αvβ6-extracellular signal-regulated kinase (ERK) direct linkage in HT-29 cells.RESULTS: NCTD induced the apoptosis of HT-29 colon cancer cells. The expression of integrin αvβ6 in HT-29 cells treated with NCTD was reduced, but the expression of αvβ3 and αvβ5 was not changed. A function-blocking antibody to αvβ6,10D5,strengthened the growth inhibitory effect of NCTD on HT-29 cells ,but LM609 (a function-blocking antibody to αvβ3) and P1F6 (a function-blocking antibody to αvβ5) did not. The level of phosphorylated ERK (p-ERK) was decreased substantially after treated with NCTD in a dose-and time-dependent manner. NCTD also affected the association of αvβ6 and ERK. CONCLUSION: NCTD decreases the expression of integrin αvβ6 and interferes with the phosphorylation of ERK. As a result, the formation of αvβ6-ERK direct linkage is affected and the signal transduction mediated by αvβ6 is disturbed. The mechanism of NCTD-induced HT-29 cell apoptosis is involved in the αvβ6-ERK signaling pathway.     

In vitro enhancement of radiation-induced apoptosis in human hepatoma cell line HepG2 by a selective inhibitor of cyclooxygenase-2 enzyme NS-398

AIM:To investigate the possible role of NS-398, a selective inhibitor of cyclooxygenase-2 enzyme, in radiation-induced apoptosis of human hepatoma cell line HepG2 in vitro. METHODS:Hepatoma cell line HepG2 was treated with various concentrations (25, 50, 100, 200 μmol/L) of NS-398 before MTT assay was used to evaluate the cytotoxicity of NS-398. Transmission electron microscopy (TEM) was used to observe the changes of apoptosis in morphology. FCM was performed to quantify the apoptotic percentage. Real-time PCR was used to detect the expression of bcl-2, bax and caspase-3 mRNA, Western blotting was used to measure the expression of Bcl-2 and bax protein, and colorimetric method was provided to analyze the change of caspase-3 activity. RESULTS:The cytotoxicity of NS-398 increased in time-dependent and dose-dependent manners. NS-398 significantly enhanced radiation-induced apoptosis (P<0.01), increased the expression of bax mRNA, Bax protein, caspase-3 mRNA and enhanced caspase-3 activity, whereas no significant change in Bcl-2 expression was found (P>0.05). CONCLUSION:NS-398 enhances radiation-induced apoptosis in hepatoma cell line HepG2. The mechanism may be associated with the up-regulation of the expression of Bax, caspase-3 and enhancement of the activity of caspase-3, which ultimately induce apoptosis in HepG2.     

HAPLN1 induces resistance to MTX in human colorectal cancer HT-29 cells though IKK/p65 pathway

AIM:To investigate the changes of hyaluronan and proteoglycan link protein 1 (HAPLN1) expression before and after resistance to methotrexate (MTX) in human colorectal cancer HT-29 cells and its effect on this drug resistance, and to explore the molecular mechanism in the process. METHODS:The drug-resistant HT-29/MTX cells were established by stepwise exposure of the cells to MTX, and then the HT-29/MTX cells were stably transfected with specific shRNA interference plasmid vectors targeting HAPLN1 and multidrug resistance-associated protein 2 (MRP2). The mRNA expression levels of HAPLN1 and MRP2 were measured by RT-PCR. CCK-8 assay was used to detect the viability of HT-29/MTX cells. The apoptosis rate was analyzed by flow cytometry. The protein levels of HAPLN1, MRP2, IκB kinase (IKK) α/β, p-IKKα/β (Ser176/Ser177), p65 and p-p65 (Ser536) were determined by Western blot. RESULTS:The HT-29/MTX cells had significantly higher mRNA and protein levels of HAPLN1 and MRP2 than HT-29 cells (P<0.05) with resistant factor of 463.756. HAPLN1 and MRP2 gene silencing significantly increased the cytotoxicity and apoptosis of HT-29/MTX cells induced by MTX (P<0.05). The IC₅₀ value was decreased from 15.304 μmol/L to 6.119 μmol/L and 7.801 μmol/L, respectively, and their reversal folds were 2.501 and 1.962, respectively. Silencing of HAPLN1 and IKK inhibitor IKK16 inhibited the phosphorylation of IKKα/β and p65 (P<0.05), and down-regulated the protein level of MRP2 in the HT-29/MTX cells (P<0.05). However, IKK16 did not affect the protein level of HAPLN1 in the HT-29/MTX cells.CONCLUSION:Knock-down of HAPLN1 gene expression reverses the resistance to MTX in human colorectal cancer HT-29/MTX cells possibly by blocking the IKK/p65 signaling pathway and thus down-regulating the expression of MRP2.     

Adipose-derived stem cells mediate immunosuppression of Th17 in multiple sclerosis

AIM: To investigate how human adipose-derived stem cells (hASCs) regulates the differentiation of Th17 cells in multiple sclerosis. METHODS: hASCs were isolated from the adipose tissues. Magnetic-activated cell sorting (MACS) kit was used to isolate CD4⁺ T cells from peripheral blood mononuclear cells (PBMCs) which were isolated by density gradient centrifugation. The percentage of CD4⁺ T cells was detected by flow cytometry. The activated CD4⁺ T cells were co-cultured with hASCs for about 4 d at different ratios of hASCs to CD4⁺ T cells (1:4 and 1:10) in a Th17 polarised condition. Another group adding anti-leukemia inhibitory factor (LIF) antibody was set up. Th17 cell proportion of the CD4⁺ T cells was determined by flow cytometry. The level of LIF in the supernatant of co-cultured system was measured by ELISA. The mRNA expression of retinoid-related orphan receptor γt (RORγt), interleukin-6 receptor (IL-6R), interleukin-23 receptor (IL-23R), LIF and leukemia inhibitory factor receptor (LIFR) was detected by real-time PCR. RESULTS: The result of flow cytometry suggested there were mainly hASCs, and the percentage of CD4⁺ T cells in the PBMCs were above 90% after MACS. The Th17 cell proportion decreased in 1:4 and 1:10 co-cultured groups in a dose-dependent manner. The mRNA expression of IL-6R, IL-23R and RORγt was downregulated and the expression of LIFR and LIF was up-regulated. When the anti-LIF was added into the co-cultured system, the ratio of Th17 cells increased and reached to the control level. The protein level of LIF obviously increased after co-cultured. After anti-LIF added, the mRNA expression of RORγt and IL-6R was up-regulated. CONCLUSION: hASCs inhibits the differentiation of Th17 cells from multiple sclerosis patients through the competitive inhibition of LIF/IL-6 by secreting LIF.     

Nanoparticle for siRNA delivery and its pancreatic cancer targeting ability

AIM: To synthesize a safe, efficient and targeted nanoparticulate carrier for siRNA delivery to pancreatic cancer cells. METHODS: Iron oxide nanocrystal with carboxylic acid group-polyethyleneimine (IONP-PEI) was synthesized and investigated as a nonviral carrier of siRNA to the pancreatic cells. The size, surface and charge using zeta potential were characterized. The perfect charge ratio between amino groups of IONP-PEI and phosphate groups of siRNA (N/P) was determined by the transfection efficiency detection, gel retardation assay and MTS assay. An antibody-directed nonviral vector, scFvCD44v6-IONP-PEI nanoparticle attaching to the cancer-associated CD44v6 single-chain variable fragment, was constructed as a cancer-targeting nanocarrier for siRNA delivery. Prussian blue staining and immunofluorescent staining were performed to detect the distribution of scFvCD44v6-IONP-PEI/siRNA complexes in the cells. The transfection efficiency, fluorescence intensity and the expression of KRAS at mRNA and protein levels in the cells transfected by IONP-PEI/siRNA and scFvCD44v6-IONP-PEI/siRNA were detected by flow cytometry, fluorescence microscopy, real-time PCR and Western blotting, respectively. RESULTS：The mass ratio of IONP to PEI was 0.75. The suitable ratio of N/P was 20. The averaged size and surface zeta potential of IONP-PEI/siRNA in deionized water were (51.3±2.2)nm (diameter) and (21.73±8.07)mV,respectively. Red fluorescence was seen in both targeting and nontargeting groups, which clearly revealed the intracellular distribution of siRNA and delivery agents. Transfection efficiencies in targeting and nontargeting groups were (89.75±1.81)% and (59.87±4.52)%, respectively. Down-regulation of the KRAS mRNA in Panc-1 cells transfected with siKRAS by scFvCD44v6-IONP-PEI and IONP-PEI was up to (34.02± 6.15)% and (51.09±6.70)%, respectively. The protein level of KRAS was lower in targeting group than that in nontargeting group. CONCLUSION：scFvCD44v6-IONP-PEI is a safe and efficient nanoparticulate carrier for gene delivery. It is more effective to transfer siRNA into the cells and mediate gene silencing effect in vitro than the nontargeting group.     

Synergistic effect of decitabine and valproic acid on apoptosis and cell cycle arrest at G0/G1 phase in gastric cancer MGC-803 cells

AIM: To investigate the synergistic effect of decitabine (DCA) and valproic acid (VPA) on apoptosis and cell cycle arrest at G₀/G₁ phase in gastric cancer MGC-803 cells. METHODS: Gastric cancer MGC-803 cells were used in the study and divided into the following groups according to the treatment with different drugs for 72 h: DCA 1.5 μmol/L,DCA 3.0 μmol/L, VPA 1.5 mmol/L, DCA 1.5 μmol/L+VPA 1.5 mmol/L and DCA 3.0 μmol/L+VPA 1.5 mmol/L. The early and late apoptotic rates were detected by annexin V and PI staining. The cell cycle was also determined by flow cytometry. The relative nm23-H1 mRNA expression level was measured by real-time quantitative PCR. RESULTS: The apoptotic rates in VPA 1.5 mmol/L+DCA 1.5 μmol/L group (early: 33.58%±3.88%; late: 31.52%±4.20%) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (early: 42.61%±4.23%; late: 38.01%±3.86%), the percentages of the cells in G₀/G₁ phase in VPA 1.5 mmol/L+DCA 1.5 μmol/L group (61.55%±2.38%) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (66.75%±2.48%), and the relative nm23-H1 mRNA expression levels in VPA 1.5 mmol/L +DCA 1.5 μmol/L group (1.84±0.46) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (3.02±0.36) were all significantly higher than those in the corresponding concentrations of single drug treatment groups (P<0.01). CONCLUSION: Synergistic effect of VPA and DCA on apoptosis and cell cycle arrest in gastric cancer MGC-803 cells is possibly via inactivation of nm23-H1 gene expression.     

Effects of cyclooxygenase-2 inhibitors on gastric epithelial cell proliferating and gastric healing following hydrochloric acid-induced injury in rats

AIM: To clarify the effects of specific and non-specific cyclooxygenase-2 (COX-2) inhibitors on gastric epithelial cell proliferating and gastric healing following acid-induced damage. METHODS: Male Sprague-Dawley rats were given 1 mL of 0.6 mol/L hydrochloric acid (HCl) into the stomach. Ten minutes after the administration of the acid, the animals were given NS-398 (COX-2 inhibitor) or indomethacin. Levels of COX-1 and COX-2 in the gastric mucosa before and after HCl-administration were analyzed using western blotting and immunohistochemical staining. Proliferating cell nuclear antigen (PCNA) was detected using immunohistochemistry for epithelial cell proliferation. Gastric lesion index (LI) was assessed using planimetry. RESULTS: Expression of COX-2 was enhanced mainly in surface epithelial cells and neck cells following HCl-administration. At 24 h following acid administration, PCNA labeling index (PCNA-LI) was (22.72±4.33) % and (21.98±5.18) % in the groups treated with 40 mg/kg of NS-398 and indomethacin respectively, which was significantly lower than that in the control group [ (34.46±3.61) %, P< 0.05 ]; LI was (1.28±0.58) % and (1.16±0.56) % in the groups treated with 4 mg/kg and 40 mg/kg of NS-398, respectively, which was significantly higher than that in the control group [ (0.58±0.24) %, P< 0.05 ]. CONCLUSIONS: The present study demonstrated that cyclooxygenase-2 inhibitors delayed gastric mucosal healing by suppressing expansion of the mucosal proliferative zone. These results provide evidence that cyclooxygenase-2 plays an important role in gastric mucosal regeneration.     

Metastasis-related function of osteopontin between two different colorectal cancer cell lines

AIM:To analyze the metastasis-related function of osteopontin(OPN) in colorectal cancer cell lines. METHODS:The sense- and antisense-osteopontin eukaryotic expression plasmids were transfected into Colo205 and SW480 cell lines. The metastatic function was detected by flow cytometry, immunohistochemistry, homogeneous and heterogeneous adhesion in different cell lines. RESULTS:High expression of OPN reduced E-cadherin expression and enhanced CD44v6 expression in colorectal cancer cells. Homogeneous adhesion was weakened, but heterogeneous adhesion was enhanced among these cells. CD44v6 expression was intensified to accelerate colorectal cancer cell adhering with ECM and invading into blood vessels and liver. CONCLUSION:OPN is one of potential and important factors in the process of colorectal cancer liver metastasis.     

Therapeutic effect of MCP-1 mediated macrophages on ovarian epithelial carcinoma

AIM: To investigate the therapeutic effect of MCP-1 mediated macrophages on ovarian epithelial carcinoma and its mechanisms.METHODS: Retorviral expression vectors pLXSN/MCP-1 was transfected into the packaging cell line PA317 by lipofectin-mediated gene transfer system. The virus particles containing MCP-1 gene were collected to infect NuTu-19. RT-PCR and Boyden Chamber were used to confirm the expression of MCP-1. Rat Fischer344 spleen macrophages were isolated. MTT method was applied to investigate the tumoricidal effect of macrophages. The survival time of the intraperitoneal disseminating ovarian cancer animal model was observed, and flow cytometry method was applied to analyze the expression of CD25 or CD44v6, and then the anti-tumor mechanisms of gene modified tumor cell lines were discussed. RESULTS: Stable MCP-1 expression in the cell line NuTu-19/MCP-1 possessed the chemotatic activity. The maximum killing ratio of macrophages on NuTu-19/MCP-1 cells was 28%. In the animal models immunized by MCP-1 expressing cells, prolonged survival time was showed which had statistical significance compared with that in the control group (P<0.05). The expression rate of CD25 (25.82%) in the NuTu-19/MCP-1 cells was higher than that in NuTu-19/neo cells (8.73%). The expression of CD44v6 in NuTu-19/MCP-1 cells was significantly lower than that in control NuTu-19/neo cells. CONCLUSION: MCP-1 mediates macrophages and suppresses the growth of NuTu-19. MCP-1 gene modified tumor cells can induce anti-tumor immunity. This strategy would be used as a promising approach for the treatment of ovarian cancer.     

Effect of PTEN gene overexpression on cell cycle and tumor invasion in a human ovarian carcinoma SKOV3 cell line

AIM:To investigate the role of phosphatase and tensin homology deleted on chromosome(PTEN) gene in the cell cycle and invasion ability of human ovarian carcinoma SKOV3 cell line in vitro. METHODS:Human ovarian carcinoma SKOV3 cells were transfected with a eukaryotic expression plasmid vector containing PTEN gene in vitro,and then the positive cell clones were selected and amplified. MTT method was used to observe the inhibitory rate,flow cytometry was used to detect the cycle of transfected PTEN cells and apoptosis level. Western blotting analysis was used to determine PTEN gene expression. The invasiveness of transfected cells were measured quantitatively by Matrigel invasion assays (Transwell chamber). RESULTS:The expression of PTEN mRNA in SKOV3 cells increased after transfection with PTEN gene. Flow cytometry showed that the percentage of cells in S phase increased,but that in G₂/M phase decreased. Invasiveness of SKOV3 was significantly decreased. CONCLUSION:The transfection of PTEN gene into SKOV3 cells can inhibit human ovarian carcinoma SKOV3 cell proliferation,invasion and induce SKOV3 cell apoptosis.     

Effects of USP9X down-regulation on apoptosis and invasion ability of gastric carcinoma AGS cells

AIM: To investigate the effects of ubiquitin-specific peptidase 9, X-linked (USP9X) down-regulation on apoptosis and invasion ability in gastric carcinoma cells, and to explore its possible molecular mechanisms. METHODS: USP9X small interfering RNA (siRNA) and control siRNA were used to be transfected into gastric carcinoma AGS cells. The cells were divided into 3 groups, including untreated AGS group, control siRNA group and USP9X siRNA group. The expression of USP9X at mRNA and protein levels in the AGS cells with different treatments was determined by real-time PCR and Western blot. The cell viability was analyzed by CCK-8 assay. Flow cytometry and Boyden chamber were employed to examine the apoptosis and invasion ability of the AGS cells. RESULTS: USP9X siRNA significantly down-regulated the expression of USP9X at mRNA and protein levels in the AGS cells. Down-regulation of USP9X markedly induced apoptosis and reduced invasion ability of the gastric carcinoma AGS cells. Notably, down-regulation of USP9X significantly reduced the protein expression of Mcl-1 and MMP-2, but markedly increased the protein level of Bax. CONCLUSION: USP9X may be a key regulator for apoptosis and invasion in gastric carcinoma.