GSK3 and PKB/Akt are associated with integrin-mediated regulation of PTHrP, IL-6 and IL-8 expression in FG pancreatic cancer cells

We have demonstrated recently that PTHrP is upregulated in pancreatic adenocarcinoma and that the ECM exerts regulatory control, at least in part, over PTHrP expression. In our present study, we examined the potential signaling interactions between these 2 pathways. Our results demonstrate that, under serum-free conditions, adhesion of FG pancreatic adenocarcinoma cells on Fn is mediated by the alpha5beta1 integrin, whereas adhesion to Type I collagen is mediated by the alpha2beta1 integrin. alpha5beta1 integrin-mediated adhesion to Fn results in a phenotype that includes a reduction in cell proliferation, increased E-cadherin localization in cell-cell contacts, increased beta-catenin localization throughout the cell, inhibition of haptokinetic cell migration, and increased expression of PTHrP, IL-6 and IL-8 relative to alpha2beta1 integrin-mediated adhesion on Type I collagen. A phosphoprotein immunoblotting screen of FG pancreatic cancer cells grown on either Fn or Type I collagen indicates that GSK3 and PKB/Akt are differentially phosphorylated on these 2 substrates. These results implicate GSK3 and PKB/Akt in the integrin-mediated regulation of PTHrP, IL-6 and IL-8 in pancreatic cancer.     

Biological similarities and differences between pancreatic intraepithelial neoplasias and intraductal papillary mucinous neoplasms

Background: Ever since the classification of pancreatic intraepithelial neoplasia (PanIN) was published, studies on the precursor lesions of pancreatic cancer have been advancing along a new directions, using standardized terminology. There are few studies that have examined the biological differences between PanIN and intraductal papillary mucinous neoplasm (IPMN) in detail. Aims: PanIN and IPMN, which are similar in morphology, were compared using various indicators, with the aim of identifying the similarities and differences between the two. Methodology: A total of 46 PanINs and 37 ducts with IPMN were identified in 19 patients with invasive ductal carcinoma and 18 patients with IPMN. These PanINs and IPMNs were examined immunohistologically with respect to the expression patterns of HER2/neu, DPC4/Smad4, Akt/PKB, p53, cyclin A, Ki67, MUC1, and MUC2. Results: Significant differences in the expression of MUC1 and MUC2 were observed between IPMN-adenoma and PanIN-2 and between CIS and PanIN-3 (MUC1: p=0.001 and p=0.005, respectively; MUC2: p=0.002 and p<0.001, respectively). A significant difference in the p53 expression level was also observed between CIS and PanIN-3 (p=0.015). Conclusions: In both IPMN and PanIN, the grade of atypism increased with increasing expression of HER2/neu, DPC4/Smad4, and Akt/PKB, along with progression in the process of multistage carcinogenesis. Although the expression levels of these factors reflected the grade of atypism, they did not reflect any differences in the grade of biological malignancy between IPMN and PanIN. On the other hand, MUC1 and MUC2 may serve as indicators of the direction of differentiation, i.e., either progression to IDAC or IPMN. Positivity for MUC1 was believed to suggest differentiation into IDAC, and positivity for MUC2 appeared to be indicative of differentiation into IPMN. Such indication of the direction of differentiation seemed to appear in PanIN1-2, even before abnormalities of HER2/neu, Akt/PKB, DPC4/Smad4, p53, and cyclin A expression began to be detected.     

Capecitabine improves cancer cachexia and normalizes IL-6 and PTHrP levels in mouse cancer cachexia models

Purpose

To clarify the potential of parathyroid hormone-related protein (PTHrP) and interleukin-6 (IL-6) as cachectic factors in a colon 26 model and the effects of capecitabine on cancer cachexia as determined by plasma levels of IL-6 and PTHrP and body weight loss.

Methods

From two colon 26 sublines-cancer cachectic clone20 and non-cachectic clone5 plasma levels of PTHrP protein and mRNA expression levels in tumor tissues were compared. An IL-6 neutralizing antibody, a PTHrP neutralizing antibody, and capecitabine were administered into mice bearing clone20 and their anticachectic effects evaluated.

Results

The plasma level of PTHrP protein in mice bearing clone20 was higher than that in mice bearing clone5. The expression level of PTHrP mRNA was 49-fold higher in tumor tissues of clone20 than of clone5, according to GeneChip^® analysis. PTHrP antibody as well as IL-6 antibody suppressed wasting of the body and gastrocnemius and adipose tissue weights. PTHrP antibody suppressed the induction of hypercalcemia but not hypoglycemia or elevation of IL-6, whereas IL-6 antibody suppressed the induction of hypoglycemia but not hypercalcemia or elevation of PTHrP. Capecitabine, a fluorinated pyrimidine anticancer agent, improved body wasting of mice bearing clone20 at a low dose with no reduction of tumor volume. Furthermore, capecitabine lowered the levels of PTHrP and IL-6 in plasma and suppressed hypoglycemia and hypercalcemia in this model. Capecitabine also showed anticachectic effects on cachexia in a cancer model induced by human cervical cancer cell line Y (also known as Yumoto).

Conclusions

PTHrP and IL-6 were found to be factors in the development of cachexia in a colon 26 cancer model, and capecitabine improved cancer cachexia by suppressing the plasma levels of IL-6 and PTHrP in colon 26 and Y cachectic models.     

Collagen type I induces disruption of E-cadherin-mediated cell-cell contacts and promotes proliferation of pancreatic carcinoma cells

Pancreatic cancer is characterized by its invasiveness, early metastasis, and the production of large amounts of extracellular matrix (ECM). We analyzed the influence of type I collagen and fibronectin on the regulation of cellular adhesion in pancreatic cancer cell lines to characterize the role of ECM proteins in the development of pancreatic cancer. We show that collagen type I is able to initiate a disruption of the E-cadherin adhesion complex in pancreatic carcinoma cells. This is due to the increased tyrosine phosphorylation of the complex protein beta-catenin, which correlates with collagen type I-dependent activation of the focal adhesion kinase and its association with the E-cadherin complex. The activation and recruitment of focal adhesion kinase to the E-cadherin complex depends on the interaction of type I collagen with beta1-containing integrins and an integrin-mediated activation of the cellular kinase Src. The disassembly of the E-cadherin adhesion complex correlates with the nuclear translocation of beta-catenin, which leads to an increasing expression of the beta-catenin-Lef/Tcf target genes, cyclin D1 and c-myc. In addition to that, cells grown on collagen type I show enhanced cell proliferation. We show that components of the ECM, produced by the tumor, contribute to invasiveness and metastasis by reducing E-cadherin-mediated cell-cell adhesion and enhance proliferation in pancreatic tumor cells.     

Expression of Cadherin-Catenin Cell Adhesion Molecules, Phosphorylated Tyrosine Residues and Growth Factor Receptor-tyrosine Kinases in Gastric Cancers

Tyrosine phosphorylation of β-catenin, an intracytoplasmic E-cadherin-binding protein, has been shown to disrupt the cadherin-mediated cell adhesion system in vitro . In order to investigate the relationships of expression and tyrosine phosphorylation of cadherin-catenin molecules and expression of growth factor receptor-tyrosine kinase with loose cell-to-cell adhesion, immunohistochemical staining for E-cadherin, α- and β-catenin, phosphorylated tyrosine residues and tyrosine kinase receptors, including c-erbB-2 , epidermal growth factor-receptor (EGF-R), c-met and K- sam , in 17 undifferentiated- and 10 differentiated-type human gastric cancers was performed. Loss or reduced expressions of E-cadherin and α- and β-catenin (11, 11, 10 cancers, respectively) were observed in the former, but not the latter. Diffuse cytoplasmic staining of E-cadherin, α- and β-catenin and phosphotyrosine residues was observed frequently in the undifferentiated-type cancers. The cytoplasmic localization of phosphotyrosine residues in undifferentiated-type cancers was correlated significantly with K- sam expression ( P <0.01) and diffuse cytoplasmic staining of E-cadherin ( P <0.05) and β-catenin ( P <0.05). Expression of K-sam protein was detected significantly more frequently in undifferentiated- (6/17; P <0.05) than differentiated-type adenocarcinomas whereas the converse applied to c-erbB-2 expression (8/10 of the latter, P <0.05). Tyrosine phosphorylation of β-catenin was directly confirmed in the protein extracts of one undifferentiated-type gastric cancer. These data indicate that alteration of tyrosine phosphorylation status associated with K- sam expression may cause the cytoplasmic distribution of cadherin-catenin molecules and loose cell-cell adhesion in undifferentiated-type gastric cancers.     

A novel β-catenin signaling pathway activated by IL-1β leads to the onset of epithelial–mesenchymal transition in breast cancer cells

Interleukin 1β has been associated with tumor development, invasiveness and metastasis in various types of cancer. However, the molecular mechanisms underlying this association have not been clearly elucidated. The present study is the first to show, in breast cancer cells, that an IL-1β/IL-1RI/β-catenin signaling pathway induces β-catenin accumulation due to GSK3β inactivation by Akt phosphorylation. Translocation to the nucleus of accumulated β-catenin and formation of the TCF/Lef/β-catenin complex induce sequential expression of c-MYC, CCDN1, SNAIL1 and MMP2, leading to up-regulation of proliferation, migration and invasion; all of the processes shown to be required, in cancerous cells, to initiate transition from a non-invading to an invasive phenotype.     

Activation of phosphatidylinositol 3-kinase/Akt signaling by EGF downregulates membranous E-cadherin and β-catenin and enhances invasion in nasopharyngeal carcinoma cells

Dysregulation of E-cadherin and β-catenin function in cell-cell adhesion is common in nasopharyngeal carcinoma (NPC) and correlates with metastatic disease. In this study, we examined the role of EGF-activated phosphatidylinositol 3-kinase (PI3K)-Akt signaling in E-cadherin and β-catenin regulation. We found that reduced membranous E-cadherin and β-catenin expression was positively correlated with Akt phosphorylation in NPC tissues. EGF treatment disrupted cell-cell adhesion and resulted in mesenchymal morphological features in NPC cell lines (TW01, TW04, and TW06). Western blot analysis showed that the E-cadherin protein level was partially reduced in TW04 cells only and the β-catenin levels were not considerably affected upon EGF treatment. In contrast, quantitative real-time RT-PCR showed that the E-cadherin, but not β-catenin, mRNA levels were markedly reduced by EGF in all cell lines. Immunofluorescent staining revealed that E-cadherin and β-catenin appeared to be markedly reduced on the cell surface and more localized in the cytoplasm. Inhibition of PI3K by LY294002 did not abolish the EGF-induced downregulation of E-cadherin protein or mRNA in TW04 cells but moderately increased the β-catenin protein level in TW01 cells and mRNA level in TW06 cells. However, LY294002 substantially restored or increased cell surface E-cadherin and β-catenin in all EGF-treated cell lines, in concordance with the inhibition of cell morphological changes. Moreover, LY294002 significantly blocked EGF-driven cell invasion, correlating with the elevation of membranous E-cadherin and β-catenin levels. In conclusion, EGF-induced epithelial-to-mesenchymal transition may not be only dependent on downregulation of E-cadherin protein/mRNA but also on mislocalization of E-cadherin and β-catenin. The mechanisms involved may be related, at least in part, to the PI3K-Akt pathway.     

Cell adhesion system and human cancer morphogenesis

Cell-cell adhesion determines the polarity of cells and participates in the maintenance of the cell societies called tissues. Cell-cell adhesiveness is generally reduced in human cancers. Reduced intercellular adhesiveness allows cancer cells to disobey the social order, resulting in destruction of histological structure, which is the morphological hallmark of malignant tumors. Reduced intercellular adhesiveness is also indispensable for cancer invasion and metastasis. A tumor-suppressor gene product, E-cadherin, and its undercoat proteins, catenins, which connect cadherins to actin filaments, are located at lateral borders, concentrating on adherens junctions, of epithelial cells and establish firm cell-cell adhesion. The E-cadherin cell adhesion system in cancer cells is inactivated by various mechanisms that reflect the morphological and biological characteristics of the tumor. Silencing of the E-cadherin gene by DNA hypermethylation around the promoter region occurs frequently, even in precancerous conditions. In diffuse infiltrating cancers, mutations are found in the genes for E-cadherin and α-and β-catenins. At the invading front of cancers, the E-cadherin cell adhesion system is inactivated by tyrosine phosphorylation of β-catenin; an oncogene product, c- erb B-2 protein, is found to associate directly with β-catenin. The E-cadherin cell adhesion system cross-talks with the Wingless/Wnt signaling pathway through β-catenin, and expression of genes, which participate in cancer morphogenesis, may be regulated in conjunction with the Wingless/Wnt signaling pathway. Dysadherin, a newly identified cancer-associated cell membrane glycoprotein, down-regulates E-cadherin and promotes cancer metastasis. In conclusion, inactivation of the E-cadherin cell adhesion system by both genetic and epigenetic mechanisms plays a significant role during multistage human carcinogenesis.     

E-cadherin、β-catenin 表达与胰腺癌预后的关系

目的：探讨胰腺癌患者中E-cadherin和β-catenin的表达与其预后的关系。方法选取胰腺癌患者50例,检测E-cadherin及β-catenin在胰腺癌组织及癌旁组织的表达,并分析其与肿瘤临床分期、分化程度、淋巴结转移及血管侵犯的关系。结果胰腺癌组织中E-cadherin及β-catenin的阳性表达率均明显低于癌旁组织,差异具有统计学意义（ P＜0．05）;E-cadherin和β-catenin的表达与肿瘤分化程度、淋巴结转移及血管侵犯密切相关（P＜0．05）。结论联合检测胰腺癌组织中E-cadherin和β-catenin的表达情况,可在一定程度上分析胰腺癌患者的恶性程度,从而判断患者的预后情况。     

Antitumor effect of a combination of lysine, proline, arginine, ascorbic acid, and green tea extract on pancreatic cancer cell line MIA PaCa-2

Background: Current treatment of pancreatic cancer is generally associated with poor prognosis, even if diagnosed early, owing to its aggressive rate of metastasis and non-responsiveness to chemotherapy and radiotherapy. Matrix metalloproteinases (MMPs) have received much attention in recent years for their role in various malignancies, and have been implicated in tumor invasion, metastasis, and angiogenesis. Aim of Study: Reported antitum or properties of ascorbic acid, lysine, proline, and green tea extract prompted us to investigate the effect of a combination of lysine, proline, arginine, ascorbic acid, and green tea extract on pancreatic cancer cell line MIA PaCa-2 for viability, MMP expression, invasion, and morphology. Methods: Viability was evaluated based on cell proliferation by MTT assay and MMP expression in condition media by gelatinase zymography. Invasion through Matrigel? was assayed and morphology was observed by hematoxylin and eosin (H+E)staining. Data was analyzed by independent sample “t” test. Results: The nutrient mixture (NM) did not inhibit cell proliferation at 10 µg/mL and exhibited a dosedependent antiproliferative effect with maximum inhibition of 38% over the control at demonstrated production of only MMP-9, which showed a dose-dependent decreased expression that was abolished at 100 µg/mL of NM. Invasion through Matrigel was inhibited at 10, 50, 100, and 500 µg/mL by 66%, 66%, 87% and 100%, respectively. H&E staining did not indicate changes even at the highest concentration of NM. Conclusion: Our results suggest that the formulation of green tea extract, lysine, proline, and ascorbic acid, tested as a promising adjunct to standard treatment of pancreatic cancer, by inhibiting MMP expression and invasion without toxic effects—important parameters in cancer metastasis.     

In vitro treatment of carcinoma cell lines with pancreatic (pro)enzymes suppresses the EMT programme and promotes cell differentiation

Background

Previous research has suggested a putative utility of pancreatic (pro)enzymes in cancer treatment. The aim of the present study was to investigate the in vitro effects of a mixture of two pancreatic pro-enzymes, i.e., Chymotrypsinogen and Trypsinogen, and the enzyme Amylase on three human cancer cell lines, i.e., OE33 (derived from an oesophageal carcinoma), Panc1 (derived from a pancreatic carcinoma) and Caco-2 (derived from a colon carcinoma).

Results

After treatment of the three cancer cell lines with different doses of the (pro)enzymes for up to 7 days, we observed (i) growth inhibition in a dose-dependent manner, (ii) enhanced expression of β-catenin and E-cadherin and decreased expression of several epithelial-mesenchymal transition (EMT)-associated genes, such as Vimentin, Snail and Slug, (iii) differentiation of Caco-2 cells, including the appearance of cell-specific differentiated structures such as microvilli and tight junctions, the acquisition of a more regular polygonal morphology, and an increased expression of the intestinal differentiation markers alkaline phosphatase and cytokeratin 8, and (iv) differentiation of Panc1 cells, including the formation of cell aggregates, an increment on lamellar bodies and an increased expression of the pancreatic differentiation markers glucagon and insulin.

Conclusions

Our results show that the treatment of three different human cancer cell lines with pancreatic (pro)enzymes results in an enhancement of cell adhesion, an attenuation of several EMT-associated markers, and an increase in the expression of several differentiation-associated markers, suggesting the acquisition of a less malignant phenotype and a decrease in proliferative capacity due to lineage-specific cellular differentiation.     

Claudin-1 enhances tumor proliferation and metastasis by regulating cell anoikis in gastric cancer

Claudin-1 (CLDN1) is overexpressed in gastric cancer and correlated with tumor invasion, metastasis and poor outcome. Here, we both down and up regulated CLDN1 expression in gastric cancer cells to elucidate its role in gastric carcinogenesis and tumor progression. We found that deficiency of CLDN1 inhibited cells migration, invasion, and colony formation in vitro and tumorigenicity, metastasis in vivo. Also, CLDN1 promoted cell aggregation and increased anoikis resistance. Down or up regulation of CLDN1 was accompanied with changes of membrane β-catenin expression as well as Akt and Src activities. When β-catenin was up-regulated in CLDN1-KD cells, cell aggregation and anoikis resistance were restored, and Akt and Src signal pathways were re-activated. Taken together, these findings suggest that CLDN1 is oncogenic in gastric cancer and its malignant potential may be attributed in part to regulation of anoikis, by mediating membrane β-catenin-regulated cell-cell adhesion and cell survival.     

The metastatic potential of human pancreatic cell lines in the liver of nude mice correlates well with cathepsin B activity

Background. Cathepsin B, a lysosomal cysteine protease, has a major role in the mechanisms of tumor metastasis. The aim of the present work was to examine the correlation between cathepsin B activity and the metastatic potential of human pancreatic cancer. Methods. The primary cell line COLO 357 and the derivative tumor cell lines FG, L3.1, L3.2, L3.3, L3.4, and L3.5, which are characterized by progressively increasing metastatic potential, were injected intrasplenically in the athymic mice. Cathepsin B activity, metastasis, and ultrastructural characteristics were assessed. Results. An increased number of liver tumor nodules was observed with each subsequent intrasplenic inoculation (p=0.0001), associated with lymph node, splenic, and pancreatic involvement. Cathepsin B activity progressively increased (p=0.001) and was strongly positively correlated with the metastic potential. However, no correlation was found between the metastatic potential and ultrastructural characteristics. Conclusions. These findings further support the central role of cathepsin B in metastasis in a combined in vitro/in vivo model.     

Relation between aberrant α-catenin expression and loss of E-cadherin function in prostate cancer

It is now well documented that E-cadherin expression correlates inversely with tumor grade in various carcinomas including prostate cancer. We also demonstrated a statistically significant correlation between decreased E-cadherin expression and progression-free period in early stage patients treated by radical prostatectomy and decreased survival in patients with advanced stage disease. We now study the relationship between E cadherin and α-catenin expression, because in prostate cancer cell lines, mutational inactivation of the α-catenin gene can be the cause of the impaired E-cadherin function. Twenty patients treated by radical prostatectomy and 32 advanced stage patients were evaluated immunohistochemically for E-cadherin and α-catenin expression. The results were related to tumor grade and disease progression. Four patients in the radical prostatectomy group had aberrant E-cadherin and α-catenin expression and showed disease progression. The other 16 patients were free of progression and had normal E-cadherin and α-catenin expression. In the advanced stage group, 4 of 13 patients with normal E-cadherin staining showed aberrant α-catenin expression and 2 patients (50%) progressed, compared with only 22% progression in patients with both normal E-cadherin and α-catenin expression. The other 19 patients with aberrant E-cadherin and α-catenin staining had the poorest prognosis. Our results suggest that loss of α-catenin expression could be one of the mechanisms responsible for the loss of E-cadherin–mediated cell-cell adhesion in human prostate cancer and might in some cases provide prognostic information. Int. J. Cancer 74:374–377, 1997. © 1997 Wiley-Liss, Inc.     

Phase II clinical trial of 5-fluorouracil, trimetrexate, and leucovorin (NFL) in patients with advanced pancreatic cancer

Background. Advanced pancreatic cancer has limited treatment options. 5-fluorouracil (5-FU) is frequently used in the treatment of pancreatic cancer. Preclinical studies suggest synergism between trimetrexate (TMTX), 5-FU, and leucovorin (NFL). Aim. We conducted a phase II trial to evaluate the activity and safety of NFL in pancreatic cancer. Method. Eligible patients (n=21) with untreated advanced pancreatic cancer were treated with 110 mg/m² intravenous (IV) THTX on day 1 and 200 mg/m² IV leucovorin prior to 500 mg/m² IV 5-FU on day 2. Oral leucovorin (15 mg every 6 h for seven doses) started intravenous 24 h later. Results. Treatment was administered for 6 wk followed by a 2-wk rest period. Response was evaluated every 8 wk. All patients were evaluable for response and toxicity. Most patients (80%) had distant metastases. Forty-five cycles of chemotherapy were administered. The most common serious toxicities were Grade 3 diarrhea (23.8%) and nausea and vomiting (14.2%). The response rate was 4.1% (95% CI, 0–23%), median survival was 6.8 mo, and 1-yr survival was 19%. Conclusion. Treatment with NFL is well-tolerated in patients with advanced pancreatic cancer. The median survival and 1-yr survival in these patients with poor prognosis compares favorably with other treatment options.     

Activation of conventional PKC isoforms increases expression of the pro-apoptotic protein bad and trail receptors

Background. Pancreatic cancer is a leading cause of cancer death worldwide; current treatment options have been ineffective in prolonging survival. Agents that target specific signaling pathways (e.g., protein kinase C [PKC]) may regulate apoptotic gene expression rendering resistant cancers sensitive to the effects of other chemotherapeutic drugs. The purpose of our study was to assess the effect of PKC stimulation on apoptotic gene expression in pancreatic cancer cells. Methods. The human pancreatic cancer cell line, PANC-1, was treated with PKC-stimulating agents, phorbol 12-myristate 13-acetate (PMA) or bryostatin-1, and analyzed for expression of apoptosis-related genes. Results. Both PMA and bryostatin-1 induced expression of the pro-apoptotic gene Bad in a dosedependent fashion. The expression of Bad was blocked by the PKC inhibitors GF109203x, Gö6983, and Ro-31-8220, suggesting a role for the conventional isoforms of PKC. In addition, treatment with the MEK inhibitors PD98059 or UO126 reduced PMA-mediated induction of Bad gene expression. PMA also increased the expression of TRAIL receptors DR4 and DR5; this expression was inhibited by the PKC inhibitors GF109203x, Gö6983, and Ro-31-8220 and the MEK inhibitor UO126, suggesting a role for conventional PKC isoforms and MEK in the regulation of TRAIL receptor expression. Conclusions. PKC stimulation in PANC-1 cells increases expression of the pro-apoptotic gene Bad and the TRAIL receptors, DR4 and DR5, through both conventional PKC- and MEK-dependent pathways. Agents that stimulate PKC may sensitize pancreatic cancer cells to apoptosis and provide a potential adjuvant therapy for the treatment of chemoresistant pancreatic cancers.