Type I collagen and divalent cation shifts disrupt cell-cell adhesion, increase migration, and decrease PTHrP, IL-6, and IL-8 expression in pancreatic cancer cells

Background: We have shown in FG pancreatic cancer cells that α₂β₁ integrin-mediated type I collagen adhesion decreases parathyroid hormone-related protein (PTHrP), inerleukin-6 (IL-6), and interleukin-8 (IL-8) expression, decreases the localization of E-cadherin and β-catenin in cell-cell contacts, increases cell migration, and increases glycogen synthase kinase 3 (GSK3) and protein kinase B (PKB/Akt) phosphorylation states relative to α₅β₁ integrin-mediated fibronectin (Fn) adhesion. Aim of the Study: To extend our observations in FG cells to other pancreatic cancer cell lines, and to determine whether E-cadherin-mediated cell-cell adhesion and its downstream effectors were functionally involved in the ECM-mediated regulation of PTHrP, IL-6, and IL-8. Methods: We used standard biochemical techniques to determine ECM-specific differences in E-cadherin and β-catenin localization, GSK3 and PKB/Akt phosphorylation, haptokinetic cell migration, and cytokine expression in pancreatic cancer cells. We also conducted functional studies using pharmacological inhibitors for GSK3 and PKB/Akt, as well as elevated Mg²⁺/Ca²⁺ ratios similar to pancreatic juice, and examined their effects on cytokine expression. Results: Differences in E-cadherin and β-catenin localization along with GSK3 and PKB/Akt phosphorylation occur in multiple pancreatic cancer cell lines, resulting in differences in ECM-mediated haptokinesis and cytokine expression that are generally consistent with previous observations in FG cells. Our functional studies also suggest that E-cadherin-mediated cell-cell adhesion and downstream effectors are involved in PTHrP, IL-6, and IL-8 expression. Conclusions: These data indicate that α₂β₁ integrin-mediated type I collagen adhesion disrupts cell-cell adhesion architecture, resulting in increased migration and decreased PTHrP, IL-6, and IL-8 expression in pancreatic cancer cells.     

Higher CO2-insufflation pressure inhibits the expression of adhesion molecules and the invasion potential of colon cancer cells

AIM： To investigate the influence of CO2-insufflation pressure on adhesion, invasion and metastatic potential of colon cancer cells based on adhesion molecules expression. METHODS： With an/n vitro artificial pneumoperitoneum model, SW1116 human colon carcinoma cells were exposed to CO2-insufflation in 5 different pressure groups： 6 mmHg, 9 mmHg, 12 mmHg, 15 mmHg and control group, respectively for 1 h. Expression of E-cadherin, ICAM-I, CD44 and E-selectin was meas- ured at 0, 12, 24, 48 and 72 h after CO2-insufflation using flow cytometry. The adhesion and invasion capacity of SW1116 cells before and after exposure to CO2-insufflation was detected by cell adhesion/invasion assay in vitro. Each group of cells was injected intraperitoneally into 16 BALB/C mice. The number of visible abdominal cavity tumor nodules, visceral metas-tases and survival of the mice were recorded in each group. RESULTS： The expression of E-cadherin, ICAM-1, CD44 and E-selectin in SWl116 cells were changed significantly following exposure to CO2 insufflation at different pressures （P 〈 0.05）. The expression of E-cadherin, CD44 and ICAM-1 decreased with increasing CO2-insufflation pressure. The adhesive/ invasive cells also decreased gradually with increasing pressure as determined by the adhesion/invasion assay. In animal experiments, the number of abdominal cavity tumor nodules in the 15 mmHg group was also significantly lower than that in the 6 mmHg group （29.7± 9.91 vs 41.7±14.90, P = 0.046）. However, the survival in each group was not statistically different. CONCLUSION： CO2-insufflation induced a temporary change in the adhesion and invasion capacity of cancer cells in vitro. Higher CO2-insufflation pressure inhibited adhesion, invasion and metastatic potential in vitro and in vivo, which was associated with reduced expression of adhesion molecules.     

Integrin-linked kinase overexpression promotes epithelial-mesenchymal transition via nuclear factor-κB signaling in colorectal cancer cells

AIM: To investigate the effect of integrin-linked kinase(ILK) on proliferation, metastasis, and invasion of the colorectal cancer cell line SW480. METHODS: In this study, the colorectal cancer cell line SW480 was stably transfected with ILK plasmids, and small interfering RNA(si RNA) was used to knockdown expression of nuclear factor(NF)-κB/p65. Methylthiazole tetrazolium(MTT) assay was performed to measure proliferation, and the wound healing migration assay and matrigel invasion assay were used to test the metastasis and invasion ability of SW480 cells. To explore the epithelial-mesenchymal transition(EMT) process, embryonic development, and the invasion and metastasis of tumors, the protein level of E-cadherin, vimentin, snail, and slug was detected by western blot. Immunofluorescence was also used to detect E-cadherin expression. Western blot was used to determine the level of phosphorylated-inhibitor of kappa B(IκB)a, inhibitor of gamma B(IγB)a, and nuclear factor kappa B(NF-κB) expressions and toexplore the ILK signaling pathway. RESULTS: Western blot results revealed that ILK expression significantly increased when ILK was overexpressed in SW480 cells(P 0.05). Proliferation, metastasis, and invasion ability were improved in the vector-ILK group compared to the vector group(P 0.05). Immunofluorescence results revealed that E-cadherin fluorescence intensity decreased after ILK was overexpressed(P 0.05). Western blot results revealed that the protein expression of E-cadherin was reduced, while vimentin, snail, and slug were upregulated when ILK was overexpressed in SW480 cells(P 0.05). In order to determine the role of the NF-κB signaling pathway in ILK overexpression promoted EMT occurrence, we overexpressed ILK in SW480 cells and found that levels of NF-κB/p65 and cytoplasmic phosphorylated-IκBa were increased and that cytoplasmic IкBa levels were decreased compared to the control group(P 0.05). Furthermore, NF-κB/p65 knockout revealed that E-cadherin was increased in the overexpressed ILK group. CONCLUSION: ILK overexpression improved the proliferation, metastasis, and invasion ability of SW480 cells, and this effect may be mediated by the NF-κB signaling pathway.     

Expression of mucins and E-cadherin in gastric carcinoma and their clinical significance

AIM: To investigate the expression of three types of mucin(MUC1, MUC2, MUC5AC) and E-cadherin in human gastric carcinomas and their clinical significance. METHODS: Ninety-four gastric cancer specimens were classified according to WHO criteria and detected by immunohistochemical assay of expression of mucins and E-cadherin. RESULTS: The positive expression rates of MUC1, MUC2, MUC5AC and E-cadherin were 82% (77/94), 84% (79/94), 40% (38/94) and 56% (53/94) respectively. MUC1 expression was significantly correlated with the types of cancer (the positive rates of MUC1 in well and moderately differentiated tubular adenocardnoma, poorly differentiated adenocardnoma, signet-ring cell carcinoma and mucinous carcinoma were 91%, 87%, 71%, 71%, respectively, P<0.05), age of patients (the positive rates of it among the people who are younger than 40 years, between 40-60 years and over 60 yearwere 74%, 81%, 89%, P<0.05), lymph nodes involvement (the positive rates in the non-interfered group and the interfered group were 78%, 85%, P<0.05) and tumor size (the positive rates in the tumors with the size less than 3 cm, 3-6 cm and larger than 6 cm were 69%, 92%, 69%, P<0.05); MUC2 expression was significantly associated with types of cancers and had the strongest expression in mucinous carcinomas (the posrdve rates of MUC2 in well and moderately differentiated tubular adenocardnoma, poorly differentiated adenocardnoma,signet-ring cell carcinoma and mucinous carcinoma were 94%, 70%, 81%, 100%, P<0.05), but it had no obvious relation to age, gender, tumor location, lymph nodes involvement,depth of invasion and metastasis to extra-gastric organs (P>0.05); MUC5AC expression was not related to any of the characteristics investigated except that it had relation to gender, whereas MUC5AC showed the tendency to higher expression in less invasive lesions and lower expression in advanced stage cancers (P>0.05); No significant difference was found for E-cadherin expression. There were strong positive relationships between the expression of MUC1 and E-cadherin, MUC2 and E-cadherin, MUC1 and MUC2(R=0.33, R=0.22, R=0.32, respectively, P<0.05). According to the COX proportional hazards model, older patients, involvement of lymph nodes, different types of gastri ccancer and MUC2 expression were significantly associated with poorer outcome of gastric carcinoma patients (β=0.08,β=3.94, β=1.33, β=0.75, respectively, P<0.05). CONCLUSION: MUC1 and MUC2 are good markers of different types of gastric cancer. MUC2 is especially a good marker of mucinous carcinoma. MUCl, MUC2 may interfere with the function of E-cadherin in gastric carcinomas, and have synergic effect on progression of gastric cancers.     

血清KL-6水平在胰腺癌诊断与鉴别诊断中的价值

目的 检测胰腺癌患者血清KL-6水平,探讨其临床诊断价值.方法 收集随访资料完整的53例胰腺癌(PC)、68例慢性胰腺炎(CP)、51例高危人群(high risk person,HR)的血清样本,以50例健康体检者作为对照.用ELISA方法检测血清CA50、MUCA、KL-6水平,放免法测定血清CA19-9水平.分析它们诊断胰腺癌的敏感性、特异性及与临床病理参数、患者预后的关系.结果 PC组、CP组、HR组及对照组的血清KL-6水平分别为(753±548)、(135 ±93)、(105±55)及(99±50)U/ml,PC组显著高于其他3组(P<0.01).以>232 U/ml为界,KL-6诊断胰腺癌的敏感性为96%,特异性为94%;以>244 U/ml为界,鉴别诊断PC与CP的敏感性为97%,特异性为91%.KL-6诊断胰腺癌的临床价值高于CA19-9、CA50及MUC4.胰腺癌患者血清KL-6水平与肿瘤的临床病理参数均无相关性.血清KL-6≤300 U/ml患者的平均生存期为(9.3±1.2)个月,较KL-6>300 U/ml患者平均生存期(4.6±0.7)个月显著延长(P=0.006).结论 KL-6可作为诊断胰腺癌的血清学指标,且对胰腺癌和慢性胰腺炎的鉴别诊断有一定意义.     

MUC2反义脱氧寡核苷酸对胃癌细胞体外黏附侵袭活性的影响

目的 探讨黏蛋白MUC2反义脱氧寡核苷酸（ASODN）对人胃癌细胞株SGC7901黏附侵袭活性的影响。方法 采用人工合成的MUC2 ASODN经阳离子脂质体包裹后转染入SGC7901细胞中,采用黏附试验、Boyden小室体外侵袭试验观察比较转染前后癌细胞黏附率,穿膜细胞相对百分率及组织蛋白酶D、钙黏蛋白表达的变化。结果 转染SGC7901细胞48h后,癌细胞黏附率在30、60、90、120min各时间段逐渐升高,但低于空白对照组（P均〈0．01）;转染后癌细胞穿膜细胞相对百分率明显下降;转染后SGC7901细胞的E-钙黏蛋白表达明显增高,组织蛋白酶D水平明显降低。结论 人工合成的MUC2 ASODN能有效抑制胃癌细胞株SGC7901黏附侵袭能力。     

Involvement of Ataxin-3 (ATXN3) in the malignant progression of pancreatic cancer via deubiquitinating HDAC6

BackgroundPancreatic cancer is a common digestive system cancer and one of the most lethal malignancies worldwide. Ataxin-3 (ATXN3) protein is a deubiquitinating enzyme implicated in the occurrence of diverse human cancers. The potential role of ATXN3 in pancreatic cancer still remains unclear.MethodsATXN3 was screened from differentially-upregulated genes of GSE71989, GSE27890 and GSE40098 datasets. The mRNA and protein levels of ATXN3 was evaluated in pancreatic cancer samples and cell lines. Through the gain- and loss-of-function experiments, the effects of ATXN3 on cell proliferation, migration and invasion were evaluated using cell counting kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU) staining, wound healing and Transwell assays. Subsequently, the interaction between ATXN3 and HDAC6 was confirmed using double immunofluorescence staining, co-immunoprecipitation (co-IP) and proximity ligation assay (PLA). The underlying mechanism of ATXN3 was determined by knockdown of HDAC6 in ATXN3-upregulated pancreatic cancer cells. The function of ATXN3 in vivo was verified through xenograft assay.ResultsHigh expression of ATXN3 was found in pancreatic cancer tissues. Increased ATXN3 expression dramatically promoted cell proliferation, migration, and invasion. The malignant phenotypes were suppressed in ATXN3-silenced pancreatic cancer cells. ATXN3 was proved to interact with HDAC6 and regulate its degradation through deubiquitination. Downregulation of HDAC6 inhibited ATXN3-induced development of pancreatic cancer cells through regulating the expression of PCNA, vimentin and E-cadherin. ATXN3 facilitated tumor growth of pancreatic cancer and increased HDAC6 expression in vivo.ConclusionsThis study confirmed that ATXN3 facilitated malignant phenotypes of pancreatic cancer via reducing the ubiquitination of HDAC6.     

Discrepancy between E-cadherin protein expression and morphology in human gastric carcinoma cells

BACKGROUND/AIMS: The E-cadherin-mediated cell adhesion system is now considered to be an "invasion suppressor system" in cancer cells. The purpose of this study is to examine the effect of E-cadherin on morphogenesis of MKN28 human gastric carcinoma cell line in the course of E-cadherin antisense S-oligodeoxynucleotide (ODN) treatment. METHODOLOGY: The effect of E-cadherin antisense or random S-ODN treatment on cell growth, morphology in monolayer culture, and E-cadherin protein expression of MKN28 cells were evaluated. Further, immunohistochemical examination was performed. RESULTS: Cell growth under 3-microM and 6-microM E-cadherin antisense S-ODN treatment did not differ from that under random S-ODN treatment. Although the expression of E-cadherin was decreased assuredly at the time of 6 days after 3-microM E-cadherin antisense S-ODN treatment by immunohistochemical examination, cell-cell adhesion was still observed until Day 10 after the treatment. On Day 15, the cells lost the cell-cell adhesion and showed the detachment and intercellular slits at least. Expression of the insoluble fraction of E-cadherin protein decreased in E-cadherin antisense S-ODN treatment cells at 6 days. CONCLUSIONS: In this study, we demonstrated that discrepancy between E-cadherin protein expression and morphology exists in MKN28 cells treated with E-cadherin antisense S-ODN treatment.     

活化血管内皮生长因子受体-1诱导肝癌细胞MHCC97-H上皮-间叶表型转化的实验研究

目的 探讨血管内皮生长因子受体1(VEGFR-1)促肝细胞癌(HCC)侵袭转移的作用机制.方法 用VEGFR-1的特异性配体血管内皮生长因子-B(VEGF-B)诱导活化肝癌细胞MHCC97-H,观察MHCC97-H细胞形态学改变,RT-PCR和Western blot检测MHCC97 H细胞上皮标志物钙黏蛋白(E-cad)、连环蛋白-a(a-cat)和间叶标志物波形蛋白、神经钙黏蛋白(N-cad)的mRNA和蛋白质表达改变,细胞荧光免疫组织化学法检测E-cad、a-cat和波形蛋白、N-cad表达部位改变,细胞侵袭和迁移试验检测MHCC97-H细胞侵袭和运动能力的改变.组间比较采用单因素方差分析.结果 VEGFR-1活化后MHCC97-H细胞变成梭形、纺锤状,细胞间隙增宽,有的伸出伪足;活化前上皮标志物E-cad、a-cat的mRNA吸光度值(A值)分别为12.55±2.98、14.23±1.36,活化后E-cad、a-cat的A值分别为6.78±3.66、6.18±0.92,活化后上皮标志物的mRNA表达显著下调,F=17.21,P＜0.05.活化前上皮标志物E cad、a-cat蛋白的A值分别为20 878±11.54、7520.45±8.66,活化后E cad、a-cat的A值分别为8031.23±10.44、5425.15±7.37,活化后上皮标志物的蛋白表达显著下调,F=30.49,P＜0.05.波形蛋白、Ncad mRNA的A值分别为0.72±1.77、4.46±6.50,活化后的分别为26.58±7.97、26.98±10.79,活化后间叶标志物的mRNA表达显著上调,F=26.24,P＜0.05.活化前波形蛋白、Ncad蛋白的A值分别为6100.22±12.73、1244.64±10.27,活化后的分别为12836.99±9.67、4586.70±8.58,活化后间叶标志物的蛋白表达显著上调,F=19.16,P＜0.05.上皮标志物E-cad和a-cat在胞膜表达减少,胞质中的表达增加,波形蛋白和N-cad在胞质中表达显著增加;MHCC97-H细胞运动和侵袭能力显著增强,与活化前相比,F=20.13,P＜0.05,差异有统计学意义.结论 VEGFR-1促进肝细胞癌侵袭和转移是通过诱导肝癌细胞发生上皮-间叶表型转化实现的.     

Expression of E-cadherin,alpha-and beta-catenins in patients with pancreatic adenocarcinoma

Background/Aims: E-Cadherin and its associated cytoplasmic proteins, catenins, are important mediators of epithelial cell-cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor invasion suppressor role for E-cadherin and catenins and loss of expression, as well as mutations, has been described in a number of epithelial cancers. The aim of this study was to evaluate the expression of E-cadherin and catenins in pancreatic adenocarcinoma tissue, and to examine the relationship between these expression and various clinicopathological parameters. Methods: In this study, we conducted an immunohistochemical investigation of expression of E-cadherin, α- and β-catenins in 30 tissue samples obtained from pancreatic ductal adenocarcino-ma patients undergoing surgical treatment. Results: In the pancreatic mucosa of noncancerous areas, epithelial cells showed equally strong membranous expression of E-cadherin, α- and β-catenin proteins at the cell-cell boundaries. Reduced expression of E-cadherin, α- and β-catenins was demonstrated in 60.0, 40.0, and 56.7% of cancer tissues, respectively. Reduced expression of E-cadherin, α- and β-catenins correlated with tumor dedifferentiation (p = 0.012, 0.013, and 0.033, respectively). Reduced expression of E-cadherin correlated with stage and lymph node involvement (p = 0.031, 0.009, respec-tively). α-Catenin and β-catenin expression did not correlate with the patient's age and sex, with the tumor size, location, stage and depth of invasion, or lymph node involvement and distant metastasis. Conclusion: These results suggest that the E-cadherin and catenins may be a useful marker of differentiation and prognosis in pancreatic adenocarcinoma, although the mechanisms underlying changes in E-cadherin and catenin expression are not fully known.     

基质细胞衍生因子/趋化因子受体4轴参与胰腺癌细胞侵袭的体外实验研究

目的 探讨胰腺癌细胞株趋化因子受体(CXCR)4的表达及其与增殖、侵袭和粘附的关系.方法 采用RT-PCR检测3个胰腺癌细胞株中CXCR4 mRNA的表达;Western印迹法检测胰腺癌细胞株中CXCR4的蛋白表达;共聚焦显微镜检测基质细胞衍生因子(SDF)-1α作用下AsPC-1细胞内钙荧光强度变化;MTT法检测细胞增殖;细胞体外粘附试验及Transwell体外侵袭实验观察胰腺癌细胞的粘附及侵袭能力.结果 3个胰腺癌细胞株均不同程度地存在CXCR4 mRNA及蛋白表达,AsPC-1细胞表达最强,而SW1990表达最弱;CXCR4功能性表达于AsPC-1细胞;SDF-1α不同程度的促进3种胰腺癌细胞的增殖、侵袭及粘附,尤以AsPC-1细胞最为明显,而SW1990细胞最弱;AMD3100可抑制SDF-1α所致的促增殖、侵袭及粘附作用.结论 胰腺癌细胞表达CXCR4mRNA及蛋白,SDF-1α通过与CXCR4作用影响胰腺癌细胞的侵袭转移,该效应与CXCR4表达程度密切相关.     

Clobenpropit enhances anti-tumor effect of gemcitabine in pancreatic cancer

AIM: To evaluate the anti-tumor effect of clobenpropit, which is a specific H₃ antagonist and H₄ agonist, in combination with gemcitabine in a pancreatic cancer cell line.METHODS: Three kinds of human pancreatic cancer cell lines (Panc-1, MiaPaCa-2, and AsPC-1) were used in this study. Expression of H₃ and H₄ receptors in pancreatic cancer cells was identified with Western blotting. Effects of clobenpropit on cell proliferation, migration and apoptosis were evaluated. Alteration of epithelial and mesenchymal markers after administration of clobenpropit was analyzed. An in vivo study with a Panc-1 xenograft mouse model was also performed.RESULTS: H₄ receptors were present as 2 subunits in human pancreatic cancer cells, while there was no expression of H₃ receptor. Clobenpropit inhibited cell migration and increased apoptosis of pancreatic cancer cells in combination with gemcitabine. Clobenpropit up-regulated E-cadherin, but down-regulated vimentin and matrix metalloproteinase 9 in real-time polymerase chain reaction. Also, clobenpropit inhibited tumor growth (gemcitabine 294 ± 46 mg vs combination 154 ± 54 mg, P = 0.02) and enhanced apoptosis in combination with gemcitabine (control 2.5%, gemcitabine 25.8%, clobenpropit 9.7% and combination 40.9%, P = 0.001) by up-regulation of E-cadherin and down-regulation of Zeb1 in Panc-1 xenograft mouse.CONCLUSION: Clobenpropit enhanced the anti-tumor effect of gemcitabine in pancreatic cancer cells through inhibition of the epithelial-mesenchymal transition process.     

尼美舒利抑制结肠癌侵袭和转移的作用机制研究

目的观察选择性环氧合酶-2(COX-2)抑制剂尼美舒利对结肠癌细胞Caco-2同质性黏附能力,E-钙黏蛋白和CD44v6表达的影响.探讨其在抑制肿瘤侵袭和转移中的作用机制.方法培养结肠癌细胞细胞株Caco-2,用不同浓度的尼美舒利进行干预,观察细胞同质性黏附能力的变化.用Western blot检测E-钙黏蛋白表达的变化,用免疫细胞化学法检测CD44v6的变化.结果尼美舒利能增加Caco-2细胞的同质性黏附能力,增加E-钙黏蛋的表达,并呈剂量依赖性.结论尼美舒利可以通过增强结肠癌细胞的同质性黏附能力,增加E-钙黏蛋白的表达,减少CD44v6的表达来抑制肿瘤细胞的侵袭和转移.