Claudin 7 as a possible novel molecular target for the treatment of pancreatic cancer

Background/objectives

Pancreatic cancer consists of various subpopulations of cells, some of which have aggressive proliferative properties. The molecules responsible for the aggressive proliferation of pancreatic cancer may become molecular targets for the therapies against pancreatic cancer.

Proteinase-activated receptor-2-mediated signaling and inhibition of DNA synthesis in human pancreatic cancer cells

Summary Conclusion Proteinase-activated receptor-2 (PAR-2)-mediated effects contribute to the intracellular signaling network in pancreatic tumor cells. A role of PAR-2 as negative regulator in human pancreatic tumor growth might be implied. Background Using the human pancreatic tumor cell line MIA PaCa-2, we evaluated cellular effects of trypsin and the PAR-2-activating peptide SLIGRL on [Ca²⁺]_i mobilization, Ins(1,4,5)P₃ level, and protein kinase (PKC) activation. Furthermore, PAR-2 involvement in the regulation of cell proliferation has been estimated by measurement of [³H]thymidine incorporation in MIA PaCa-2 cells. Results Trypsin and the PAR-2 synthetic peptide agonist SLIGRL induced [Ca²⁺]_i mobilization, transient increase in inositol (1,4,5) triphosphate level, and PKC translocation in MIA PaCa-2 cells. In addition, SLIGRL induced a decrease in DNA synthesis in MIA PaCa-2 cells.     

Fascin promotes the motility and invasiveness of pancreatic cancer cells

AIM:To explore the role of actin-bundling protein, fascin during the progression of pancreatic cancer. METHODS:The plasmid expressing human fascin-1 was stably transfected into the pancreatic cancer cell line MIA PaCa-2. The proliferation, cell cycle, motility, scattering, invasiveness and organization of the actin filament system in fascin-transfected MIA PaCa-2 cells and control non-transfected cells were determined. RESULTS:Heterogeneous overexpression of fascin markedly enhanced the motility, scattering...     

Nintedanib inhibits epithelial-mesenchymal transition in A549 alveolar epithelial cells through regulation of the TGF-β/Smad pathway

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disorder. Recent studies have suggested that epithelial-mesenchymal transition (EMT) of alveolar epithelial cells influences development of pulmonary fibrosis, which is mediated by transforming growth factor β (TGF-β). Tumor necrosis factor α (TNF-α), an important proinflammatory cytokine in IPF, has been shown to enhance TGF-β-induced EMT. Nintedanib, a multiple tyrosine kinase inhibitor that is currently used to treat IPF, has been shown to suppress EMT in various cancer cell lines. However, the mechanism of EMT inhibition by nintedanib and its effect on TGF-β and TNF-α signaling pathways in alveolar epithelial cells have not been fully elucidated.MethodsA549 alveolar epithelial cells were stimulated with TGF-β2 and TNF-α, and the effects of nintedanib on global gene expression were evaluated using microarray analysis. Furthermore, Smad2/3 phosphorylation was assessed using western blotting.ResultsWe found that in A549 cells, TGF-β2 and TNF-α treatment induces EMT, which was inhibited by nintedanib. Gene ontology analysis showed that nintedanib significantly attenuates the gene expression of EMT-related cellular pathways and the TGF-β signaling pathway, but not in the TNF-α-mediated signaling pathway. Furthermore, hierarchical cluster analysis revealed that EMT-related genes were attenuated in nintedanib-treated cells. Additionally, nintedanib was found to markedly suppress phosphorylation of Smad2/3.ConclusionNintedanib inhibits EMT by mediating EMT-related gene expression and the TGF-β/Smad pathway in A549 alveolar epithelial cells.     

MiR-21 regulates epithelial–mesenchymal transition in intestinal fibrosis of Crohn's disease by targeting PTEN/mTOR

BackgroundPrevious studies suggested miR-21 regulated epithelial–mesenchymal transition (EMT) and fibrosis in organs. The aim of this study was to explore the role and mechanism of miR-21 in EMT process of CD(Crohn's disease)-associated intestinal fibrosis.MethodsTissue biopsies from fibrotic and nonfibrotic intestine of CD patients, and non-CD patients were obtained; chronic intestinal fibrosis model established by TNBS was treated with antagonist of miR-21; human intestinal epithelial cell, NCM460, were transfected with miR-21 mimics or inhibitor. The expressions of PTEN and mTOR, EMT-related markers and severity of colitis and fibrosis were examined.ResultsCompared to the controls, miR-21 was significantly upregulated in the intestinal tissues from CD patients with fibro stenosis, followed by decreased PTEN expression, increased EMT markers, and mTOR expression, and imbalanced ratio of MMP9(matrix metalloproteinase 9)/TIMP1(tissue inhibitor of metalloproteinase 1). MiR-21 downregulated the expression of PTEN and upregulated mTOR signal in NCM460 cell. Also, knocking miR-21 down reduced EMT in vitro. Inhibiting miR-21 with antagonists reversed TNBS-induced intestinal fibrosis in vivo, through suppressing EMT and balancing MMPs/TIMPs.ConclusionWe identified the involvement of miR-21 in EMT during intestinal fibrosis via targeting PTEN and mTOR, and miR-21 inhibition relieved intestinal fibrosis by regulating extracellular matrix (ECM) remodeling . Our results indicated miR-21 as a potential new target for the treatment of fibrosis complication in CD.     

Pancreatic cancer is associated with aberrant monocyte function and successive differentiation into macrophages with inferior anti-tumour characteristics

Background/objectivesInflammation is related to the development and progression of pancreatic cancer (PC). Locally, anti-inflammatory macrophages (M2), and systemically, high levels of certain inflammation-modulating cytokines associate with poor prognosis in PC. The detailed effects of systemic inflammation on circulating monocytes and macrophage polarisation remain unknown. We aimed to find out how intracellular signalling of peripheral blood monocytes is affected by the systemic inflammatory state in PC patients and how it affects their differentiation into macrophages.MethodsMonocytes were isolated from 50 consenting PC patients and 20 healthy controls (HC). The phosphorylation status of the signalling molecules was assessed by flow cytometry both from unstimulated and appropriately stimulated monocytes. Monocytes derived from HC and PC patients were co-cultured with cancer cells (MIA PaCa-2 and HPAF-II) in media supplemented with autologous serum, and the CD marker expression of the obtained macrophages was assessed by flow cytometry.ResultsPhosphorylation levels of unstimulated STAT2, STAT3 and STAT6 were higher (p < 0.05) and those of stimulated NF-kB (p = 0.004) and STAT5 (p = 0.006) were lower in patients than in controls. The expression of CD86, a proinflammatory (M1) marker, was higher in control- than patient-derived co-cultured macrophages (p = 0.029).ConclusionsCirculating monocytes from PC patients showed constitutive phosphorylation and weaker response to stimuli, indicating aberrant activation and immune suppression. When co-culturing the patient-derived monocytes with cancer cells, they differentiated into macrophages with reduced levels of M1 macrophage marker CD86, suggesting compromised anti-tumour features. The results highlight the need for global management of tumour-associated immune aberrations in PC treatment.     

Portal blood – A new source of dendritic cells for pancreatic cancer vaccine

IntroductionCohort study evaluated dendritic cells (DCs) subsets in portal and peripheral blood of patients with pancreatic cancer (PC) and chronic pancreatitis (CHP).Material and methodsMyeloid type 1 (mDCs1) and 2 (mDCs2), plasmocytoid (pDCs) and SLAN + DCs were assessed in PC (n = 20) and CHP (n = 6) patients.ResultsPercentage of mDCs1 was significantly lower in PC patients when compared to CHP (0.48 ± 0.26 vs 0.76 ± 0.3; p = 0.038) only in portal, but not peripheral blood.DiscussionFurther studies to assess the functional properties of portal blood DCs and their applicability in anticancer vaccination are needed.     

Cholecystokinin analog,JMV-180, stimulates growth of human pancreatic cancer

The gastrointestinal peptide CCK has been shown to stimulate growth of normal and malignant pancreatic tissue. The CCK receptor possesses several different binding sites for CCK. By using the CCK analog JMV-180, which is a functional agonist at CCK high-and low-affinity receptors and an antagonist at very low affinity receptors, and carbachol, which down-regulates binding to CCK high-affinity receptors, we evaluated which receptor is involved in growth of human pancreatic cancer cells. PANC-1 and MIA PaCa-2 human pancreatic cancer cell lines were grown for four to six days in the presence or absence of JMV-180 (10^–10–10^–6 M) alone or in combination with carbachol (10 mM). Growth was evaluated by counting cells and by [³H]thymidine incorporation. JMV-180 increased cell number in PANC-1 and MIA PaCa-2 cells 123% and 86%, respectively, over controls (P=0.004). DNA synthesis by [³H]thymidine uptake was increased 64% and 40% in PANC-1 and MIA PaCa-2 cells, respectively, over controls (P<0.001). The trophic effect of JMV-180 was not inhibited by the addition of carbachol. Since JMV-180 stimulated the growth and since the effect was not inhibited by carbachol, we suggest that the growth effects of CCK in pancreatic cancer cells are mediated by the low-affinity receptor.     

Silencing of LRRFIP1 enhances the sensitivity of gemcitabine in pancreatic cancer cells by activating JNK/c-Jun signaling

BackgroundThe epithelial-mesenchymal transition (EMT) in cancer cells has been shown to closely associate with the survival and drug resistance of cancer cells. We recently provided evidence that Wnt signal activator leucine-rich repeat in flightless-1-interacting protein 1 (LRRFIP1) regulates EMT in pancreatic cancer. LRRFIP1 silencing inhibits the translocation of β-catenin to the nucleus, which led to reverse EMT in cancer cells. It was suggested that LRRFIP1 was implicated in gemcitabine sensitivity by regulating EMT signaling.MethodsGemcitabine chemosensitivity was investigated in LRRFIP1-knockdown pancreatic cancer cells (PANC-1 and MIA Paca-2). In addition, the effects of LRRFIP1 knockdown on JNK/SAPK (stress activated-protein kinase) signaling and apoptosis were evaluated.ResultsLRRFIP1 silencing accelerates gemcitabine-induced caspase activity and cell death in pancreatic cancer cells. It was also revealed that gemcitabine-induced phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun were increased in LRRFIP1 knockdown cells. The activation of JNK/c-Jun in LRRFIP1-knockdown cells was significantly diminished by the inhibition of Rac activity. It was confirmed that the acquisition of gemcitabine sensitivity by LRRFIP1 silencing largely depends on the stimulation of JNK/SAPK (stress activated-protein kinase) signaling.ConclusionsOur findings suggest that reversing EMT and transient activation of JNK might be essential for the gemcitabine sensitivity in LRRFIP1 knockdown pancreatic cancer cells. Our discoveries highlight the potential role of LRRFIP1 in the chemosensitivity related to the regulation of EMT signaling.     

Autophagic tumor-associated macrophages promote the endothelial mesenchymal transition in lung adenocarcinomas through the FUT4/p-ezrin pathway

BackgroundLung adenocarcinoma is one of the most common malignant tumors with high morbidity and mortality, but the effect of Tumor-associated macrophages (TAMs) on lung adenocarcinoma has not been studied clearly now.MethodsIn this study, TAMs were stably transfected with Atg5 silence or overexpression lentiviral vectors to inhibit or induce autophagy of TAMs. In addition, the expression of fucosyltransferase IV (FUT4) or Ezrin were interfered in TAMs with autophagy. The above treated TAMs were then co-cultured with A549 or H1299 cells. The expressions of genes were detected by qPCR, western blotting, cell immunofluorescence, and enzyme-linked immunosorbent assay. Meanwhile, cell migration and invasion were analyzed by Transwell assay and wound healing assay. Furthermore, the effects of TAMs with autophagy were explored in lung adenocarcinoma xenograft model of mice.ResultsThe results showed that overexpression of autophagy-related gene 5 (ATG5) induced autophagy in TAMs, which increased the expression of FUT4, TGF-β1, and p-ezrin, and promoted epithelial-mesenchymal transition (EMT) in lung adenocarcinoma cells. However, FUT4 silencing partially reversed the effects of TAM autophagy, specifically, the expression of TGF-β1 and p-ezrin was inhibited and EMT in lung adenocarcinoma cells was suppressed. Notably, ezrin deletion in autophagic TAMs induced by rapamycin reduced TGF-β1 expression and suppressed EMT in lung adenocarcinoma cells. Consistently, in vivo experiments also revealed that autophagic TAMs increased the expression of FUT4, TGF-β1, and p-ezrin, and promoted EMT in lung adenocarcinomas. Similarly, FUT4 silencing partially reversed the effects of autophagic TAMs on EMT in lung adenocarcinomas.ConclusionsIn conclusion, autophagic TAMs promoted TGF-β1 secretion through the FUT4/p-ezrin pathway and induced EMT in co-cultured lung adenocarcinoma cells.     

KAI1 gene suppresses invasion and metastasis of hepatocellular carcinoma MHCC97‐H cells in vitro and in animal models

Background: Downregulation of KAI1 gene expression has been found in many types of cancer cells and is closely related to cancer invasion and metastasis. This study was aimed at investigating the effects and possible underlying mechanisms of KAI1 gene on invasion and metastasis of human hepatocellular carcinoma (HCC). Methods: The invasive ability, visco‐elastic properties and cell adhesion forces were analysed in different HCC cells originating from the MHCC97‐H cell line transfected with either the sense or the antisense KAI1 expression plasmid. Tumuorigenicity, metastatic abilities, extracellular matrix (ECM) and intercellular adhesion molecule‐1 (ICAM‐1) expression were also evaluated in the nude mouse models of the xenografted and orthotopic liver cancer cells. Results: Compared with their parental cells, in the HCC cells transfected with the sense KAI1 gene, the invasive ability in vitro was significantly decreased (P<0.01); the cellular elastic coefficients K₁, K₂ and μ were significantly higher (P<0.05); the cells adhesion forces to fibronectin were significantly lower (P<0.01). The sense KAI1 gene transfection into the cancer cells also inhibited their invasion and lung metastasis in the orthotopic liver cancer nude mice. However, the opposite changes were observed in the HCC cells transfected with the antisense KAI1 gene. KAI1 gene transfection also affected ECM and ICAM‐1 expression in the transplanted liver cancer. Conclusion: The KAI1 gene plays an important role in the invasion and metastasis of human HCC and its upregulation in HCC cells suppresses their invasive and metastatic abilities. KAI1 gene functioned as a metastasis inhibitor by regulating the HCC cell biophysical behaviours including aggregation, adhesion, motility and visco‐elastic properties.     

Overexpression of GP73 promotes cell invasion,migration and metastasis by inducing epithelial-mesenchymal transition in pancreatic cancer

Pancreatic cancer is one of the most difficult clinical cases to diagnose with a very low 5-year survival rate of 5%, regardless of the advances made in both the medical and surgical treatment of the disease. One of the contributing factors for the high mortality rate seen of pancreatic cancer patients is the lack of effective chemotherapies, which is believed to be due to drug-resistance. Based on recent evidence, epithelial-mesenchymal transition (ETM) of pancreatic cancer cells has been found to be associated with the development of drug resistance and an increase in cell invasion. Therefore, we conducted the present study in order to investigate the regulatory effects of Golgi protein-73 (GP73) on PC. GP73 and EMT-related gene expressions in PC, along with the adjacent and chronic pancreatitis tissues were determined by means of RT-qPCR and Western blot analysis. Cultured PC cells were treated with pAdTrack-CMV, si-NC, GP73 overexpression, Si-GP73, Snail-siRNA and GP73 + Snail-siRNA. Cell invasion, migration and metastasis were measured in vitro and in vivo. The results revealed that the PC tissues and chronic pancreatitis tissues exhibited diminished E-cadherin expression and amplified GP73, N-cadherin, Vimentin and Snail expression. In response to GP73 gene silencing, PC cells presented with increased E-cadherin expression and decreased N-cadherin, Vimentin, Snail expression in addition to the inhibition of the number of invasive cells, tumor volume and number of liver lesions. These findings highly indicated that the overexpression of GP73 promotes cell invasion, migration and metastasis by inducing EMT in PC.     

Role of adipocytokines and its correlation with endocrine pancreatic function in patients with pancreatic cancer

IntroductionSome authors suggest that adipocytokines contribute to the induction of pancreatic carcinogenesis as well as the development of endocrine insufficiency.AimsWe evaluate the circulating concentrations of leptin, resistin and visfatin in patients with newly diagnosed pancreatic cancer (PC) and relationship between serum adipocytokines level and clinicopathological features of PC. Moreover the usefulness of those adipocytokines as possible biomarkers of endocrine pancreatic function in PC has been assessed.MethodsThe pilot study group consisted of 45 individuals (mean age 65.6 ± 11.5 years, BMI 21.8 ± 3.4 kg/m²) with newly diagnosed PC (within last 1–3 months) and 13 healthy individuals with age, gender and BMI matched to the study group. Among PC patients 18 (40%) had recently diagnosed diabetes. Fasting plasma leptin, resistin, visfatin concentrations were determined with ELISA (R&D Systems, Phoenix Pharmaceuticals) and insulin by RIA (DakoCytomation).ResultsPatients with PC as compared to controls had significantly lower plasma leptin (40.6 ± 21.3 vs 63.2 ± 16.3 pg/mL; p < 0,0008). In contrast PC patients showed more than six fold higher level of resistin (126.2 ± 143.2 vs 18.9 ± 7.2 ng/mL; p < 0.009) than controls. The median plasma visfatin was 2.8 ± 1.8 ng/mL, which was not significantly different from the controls (3.8 ± 1.1 ng/mL). When PC patients with and without diabetes were considered separately, plasma leptin concentrations among nondiabetic patients were slightly, but not significantly higher (44.6 ± 21.0) as compared to diabetics (34.5 ± 20.7). Moreover there was no difference between visfatin and resistin level in PC, among patients with and without diabetes. No significant differences between serum level of leptin, visfatin and resistin and age, gender, BMI, smoking status, tumor localization, distant metastases and pain has been found.ConclusionThe results of this study confirm previous findings that patients with newly diagnosed pancreatic cancer are characterized with lower level of leptin. This pilot study showed significantly higher resistin concentrations in patients with PC in comparison to healthy controls, which may be helpful in PC early diagnosis. Changes in leptin and resistin level in PC are not likely related to endocrine disorders.     

Identification of dietary patterns in a general population of North Italian adults and their association with arterial stiffness. The RoCAV study

Background and aimsThe aims of this study were to identify dietary patterns in a general population of North Italian adults and to investigate the cross-sectional association between prevalent dietary patterns and arterial stiffness.Methods and resultsParticipants to the RoCAV study without chronic diseases at recruitment and with reliable dietary data were included. The food-frequency EPIC questionnaire was used to evaluate dietary habits. Dietary patterns were estimated using principal components analysis and Mediterranean diet adherence score (MedS). Carotid–femoral pulse wave velocity (cfPWV) was used as proxy of arterial stiffness. Basing on data from 2640 subjects (1608 men and 1032 women, mean ± SD 65.5 ± 6.7 years), four principal components (PC) were retained, explaining 24% of the overall variance. Considering 1284 subjects with cfPWV (mean ± SD 10.7 ± 2.5 m/s) data available, adherence to PC1 (Western-like dietary pattern) was associated with higher stiffness values (+0.29 m/s cfPWV for 1 SD increase of PC1, 95% CI:0.08,0.50; p = 0.007) in a multivariate model. Conversely, adherence to PC2 (Mediterranean-like) was not related to cfPWV values (?0.18, 95% CI: ?0.36, 0.004; p = 0.06). Likewise, MedS and other PC patterns did not show any significant association with cfPWV. Mediation analysis showed that the association between Western-like dietary pattern and cfPWV is mediated by higher levels of leucocytes (9.2% of the effect, p = 0.047).ConclusionsOur study in a Southern European population identified a Western-like dietary pattern associated with an increased cfPWV, a proxy of arterial stiffness. The association with cfPWV was in part mediated by inflammatory status.     

Comparative analysis of a human pancreatic undifferentiated cell line (MIA PaCa-2) to acinar and ductal cells

Pancreatic ductal cell secretion has not been well characterized due to the difficulty in obtaining sufficient quantities of purified ductal cells. To determine if the MIA PaCa-2 cell line would provide a useful model for in vitro studies of pancreatic ductal cell secretion, the present study was designed to characterize these cells in greater detail. In this investigation, the human pancreatic undifferentiated cell line, MIA PaCa-2, was compared with PANC-1 cells (a human ductal cell line previously characterized), isolated rat and human ducts, acinar cells, and nonpancreatic cell lines. The results indicate that while the morphology of the MIA PaCa-2 cell line is nonpolarized and generally atypical of either ductal or acinar cells, the cell line has retained certain biochemical similarities to ductal cells. Additional morphological studies indicated (a) the presence of intermediate filaments characteristic of epithelial cells, (b) the absence of zymogen granules, and (c) an apparent basolateral plasma membrane localization of Na+, K+-ATPase. Similar to ductal cells, biochemical analyses indicated (a) the presence of Na+, K+-ATPase based on [3H]-ouabain binding assays, (b) high levels of carbonic anhydrase, (c) low levels of gamma-glutamyl transpeptidase, (d) nondetectable levels of amylase, and (e) protein composition and protein synthetic patterns comparable to PANC-1 cells. Finally, as with PANC-1 cells and isolated rat and human ducts, the major sulfated secretory product of MIA PaCa-2 cells was a protein with a molecular weight of approximately 660,000 to 1 million.(ABSTRACT TRUNCATED AT 250 WORDS)     

KAI1 is a potential target for anti-metastasis in pancreatic cancer cells

AIM： To investigate whether KAI1, as a metastasis suppressor gene, is associated with invasive and metastatic ability of pancreatic cancer cells.METHODS： KAI1 gene was transfected into pancreatic cancer cell line MiaPaCa Ⅱ by liposomes selected with G418. Expression of transfected cells was measured by Western blotting, immunofluorescence and immunocytochemistry. Tumor cell invasion and metastatic ability were detected through gelatinase activity and reconstituted basement membrane （Matrigel） assay. pCMV-KAI1 was directly injected into the heterotopic human pancreatic adenocarcinoma successfully established in the groin of BALB/C nude mice, by subcutaneous injection of MiaPaCa Ⅱ pancreatic cancer cells. The statistical analysis between groups was determined by Student＇s two tailed t test.RESULTS： By Western blotting, MiaPaCa Ⅱ cells transfected by KAI1 gene indicated KAI1 expression at approximately 29.1 kDa. Cytoplasm staining was positive and uniformly spread in transfected cancer cells, using immunohistochemistry and immunofluorescence. The most obvious difference was present after 30 h （MiaPaca Ⅱ 43.6 ± 9.42, pCMV-MiaPaca Ⅱ 44.8 ± 8.56, pCMV-KAI1-MiaPaca Ⅱ 22.0 ± 4.69, P 〈 0.05）. Gelatinolysis revealed a wider and clearer band of gelatinolytic activity in non-transfected than in transfected cells （MiaPaCa Ⅱ cells 30.8 ± 0.57, transfected cells 28.1 ± 0.65, P 〈 0.05）. In vivo tumor growth rates of KAI1 transfectants with KAI1-Lipofectamine 1.22 ± 0.31 in A group were lower than control 4.61 ± 1.98 and pCMV-KAI 11.67 ± 0.81. Analyses of metastases with and without KAI1 transfection in mice were different in liver and lung between controls 1.62 ± 0.39, 0.45 ± 0.09, pCMV-KAI 1.01 ± 0.27, 0.33 ± 0.09 and KAI1-Lipofectamine 0.99 ± 0.21, 0.30 ± 0.09 respectively （P 〈 0.05）.CONCLUSION： High expression of KAI1 gene was found in transfected MiaPaCa Ⅱ human pancreatic cancer cells with lower metastatic ability. KAI1 gene plays an important role in inhibiting     

PDZK1 induces resistance to apoptosis in esophageal adenocarcinoma cells

Background

Esophageal cancer is a lethal malignancy with a poor prognosis. The incidence of esophageal adenocarcinoma, which develops from Barrett’s esophagus (BE), has recently been increasing. In a previous study, we found that PDZK1 expression is higher in long segment BE compared to that in short-segment BE. However, the function of PDZK1 in the mucosa of BE is unclear.

Aims

Clarify the role of PDZK1 in BE mucosa using PDZK1 overexpressed cells.

Methods

Human adenocarcinoma-derived OE33 cells were used as a parental cell line and transfected to generate PDZK1 overexpressed OE33 cells (PC cells) or transfected with empty vector as control cells (NC cells). Cell growth of NC and PC cells in 10% fetal bovine serum was evaluated by cell counting. The effect of PDZK1 on proteasome inhibitor (PSI)-induced apoptosis was qualified by fluorescence microscopy and quantified by flow cytometry. Expression of apoptosis-related proteins was evaluated by western blotting.

Results

There were no significant differences in cell growth between NC and PC cells. PSI significantly increased apoptosis in NC cells, but not in PC cells. In response to PSI, increased levels of cleaved-caspase3 and decreased pro-caspase3 levels were found in NC cells, but not in PC cells. In NC cells, PSI significantly decreased Bcl-2 expression without affecting Bax levels. In contrast, high expression of both Bcl-2 and Bax was observed in PC cells.

Conclusion

Overexpression of PDZK1 protein induces an apoptosis-resistant phenotype in BE cells, which may be a potential therapeutic target.

     

Par-4 activation restrains EMT-induced chemoresistance in PDAC by attenuating MDM-2

BackgroundWe recently reported prostate apoptosis response 4 (Par-4), a potential tumor suppressor protein restrains epithelial-mesenchymal transition (EMT) properties and promotes mesenchymal-epithelial transition (MET) in invasive cancer cells by repressing Twist-1 promoter activity. Here, we demonstrate that genetic as well as pharmacological modulation of Par-4 by NGD16 (a small molecule antimetastatic agent), limits EMT-induced chemoresistance in aggressive cancer cells by suppressing MDM-2, a downstream effector of Twist-1.MethodsMatrigel invasion assay, gelatin degradation assay, cell scattering assay, MTT assay and colony formation assay were used to study the proliferation and migration abilities of invasive cancer cells. Immunoblotting, immunocytochemistry, and immunoprecipitation analysis were utilized for determining protein expression and protein-protein interaction. 4T1 aggressive mouse carcinoma model was employed to evaluate tumor growth and lung metastasis.ResultsTreatment of gemcitabine (nucleoside analogue anticancer agent) to pancreatic cancer (Panc-1, MiaPaca-2) and breast cancer (MDA-MB-231) cells amplified MDM-2 expression along with increase in EMT properties. Conversely, NGD16 boosted expression of tumor suppressor Par-4 and inhibited invasion and migration abilities of these cells. Moreover, induction of Par-4 effectively diminished MDM-2 along with pro-EMT markers, whereas, augmented the expression of epithelial markers. Furthermore, siRNA-mediated silencing of Par-4 divulged that NGD16 exerts its EMT inhibitory effects in a Par-4-dependent manner. Mechanistically, Par-4 activation provokes p53 by disrupting MDM-2-p53 interaction, which restored epithelial characteristics in cancer cells. Additionally, partial knockdown of MDM-2 through siRNA pronounced the anti-proliferative and anti-invasive effects of NGD16. Finally, NGD16 efficiently inhibited tumor growth and lung metastasis in mouse mammary carcinoma model without showing any undesirable effects.ConclusionOur findings unveil Par-4 as a key therapeutic target and NGD16 (the pharmacological modulator of Par-4) are potential tools to suppress EMT and associated chemoresistance, which could be exploited clinically for the treatment of aggressive cancers.