Zhaohui Lu

Retinoic acid inhibits pancreatic cancer cell migration and EMT through the downregulation of IL-6 in cancer associated fibroblast cells

Retinoic acid (RA) is a small molecular derivative of vitamin A that is stored in quiescent stellate cells in pancreas stroma. Cancer associated fibroblasts (CAFs) are activated fibroblast cells in pancreatic ductal adenocarcinoma tumor microenvironment. We treated CAFs with RA and found that these cells became static due to the low expression of α-SMA, FAP, and IL-6 and decreased production of extracellular matrix (ECM). Furthermore, we verified that the low secretion of IL-6 from CAFs was related to RA-induced inhibition of migration and epithelial-mesenchymal transition (EMT) of tumor cells. However, RA could not inhibit the migration and EMT of tumor cells directly. Therefore, our study showed that one of the therapeutic effects of RA on tumor cells is through its modulation of CAFs in tumor microenvironment. The tumor microenvironment plays an important role in promoting tumor migration and might be a promising target of biological treatment.

Plasma proteomic analysis of pancreatic cancer by 2-dimensional gel electrophoresis.

Objective: Late diagnosis is the major reason for poor prognosis of pancreatic cancer (PC). Developing new biomarkers of early stage detection is critical. Methods: Proteomic analysis with 2-dimensional gel electrophoresis was used to identify differentially expressed proteins in plasma of PC patients. The 2-dimensional gel electrophoresis plasma protein profiles of 11 PC patients (preoperative and postoperative) were compared with those of 10 patients with chronic pancreatitis (CP) and 11 healthy controls. Results: Five proteins were found to be constantly changed. Haptoglobin (Hp) &bgr; chain and leucine-rich &agr;2 glycoprotein up-regulated slightly in PC plasma. Pancreatic cancer had a higher frequency of Hp2-2 phenotype but lacked Hp1-1 phenotype. Hemoglobin was increased significantly in plasma samples of PC and CP. &agr;1 Antitrypsin gradually increased its expression level in healthy control, PC, and CP. Immunoglobin J chain was elevated in CP plasma samples. Haptoglobin, &agr;1 antitrypsin, and leucine-rich &agr;2-glycoprotein were all greatly elevated after tumor resection in PC patients. Conclusions: Proteomic analysis can simultaneously detect changes of multiproteins in plasma of PC, but detected proteins are abundant and common plasma proteins and their diagnostic value may be limited.Abbreviations: 2-DE - 2-dimensional gel electrophoresis, &agr;1-AT - &agr;1 antitrypsin, CP - chronic pancreatitis, Hb - hemoglobin, Hp - haptoglobin, Ig - immunoglobulin, LRG - leucine-rich &agr;2-glycoprotein, MS - mass spectrometry, PC - pancreatic cancer, SELDI-TOF - surface-enhanced laser desorption ionization time- of-flight, sIgA - secretory immumoglobin A

Deregulation of the MiR-193b-KRAS Axis Contributes to Impaired Cell Growth in Pancreatic Cancer

Modulation of KRAS activity by upstream signals has revealed a promising new approach for pancreatic cancer therapy; however, it is not clear whether microRNA-associated KRAS axis is involved in the carcinogenesis of pancreatic cancer. Here, we identified miR-193b as a tumor-suppressive miRNA in pancreatic ductal adenocarcinoma (PDAC). Expression analyses revealed that miR-193b was downregulated in (10/11) PDAC specimens and cell lines. Moreover, we found that miR-193b functioned as a cell-cycle brake in PDAC cells by inducing G1-phase arrest and reducing the fraction of cells in S phase, thereby leading to dampened cell proliferation. miR-193b also modulated the malignant transformation phenotype of PDAC cells by suppressing anchorage-independent growth. Mechanistically, KRAS was verified as a direct effector of miR-193b, through which the AKT and ERK pathways were modulated and cell growth of PDAC cells was suppressed. Taken together, our findings indicate that miR-193b-mediated deregulation of the KRAS axis is involved in pancreatic carcinogenesis, and suggest that miR-193b could be a potentially effective target for PDAC therapy.

Antisense bcl-2 transfection up-regulates anti-apoptotic and anti-oxidant thioredoxin in neuroblastoma cells.

AbstractAntisense bcl-2 therapy combined with chemotherapy has been proved to be effective in various tumors. However, the role played by antisense bcl-2 therapy alone is not clear. In this study, we compared the apoptosis and the protein profiles of antisense bcl-2 transfected human neuroblastoma SK-N-MC cells to the control cells. Flow cytometric data indicated that antisense bcl-2 transfection did not lead to more extensive apoptosis in SK-N-MC cells (14.9 ± 3.8%) than the control cells (10.3 ± 2.3%). The above observation was confirmed by fluorescence microscopy using Hoechst 33258 staining. However, antisense bcl-2 induced changes in the expression of various proteins as shown by proteomic comparison, which included the up-regulation of the anti-apoptotic and anti-oxidant protein thioredoxin. By western blot validation, thioredoxin was found to be up-regulated by 2.9-folds with the corresponding down-regulation of Bcl-2 by 2.1-folds. The up-regulation of thioredoxin may be a compensating mechanism for cell survival in neuroblastoma when Bcl-2 expression is suppressed, and it may to some extent attenuate the effectiveness of antisense bcl-2 therapy.

Adenovirus-mediated Ink4a/ARF gene transfer significantly suppressed the growth of pancreatic carcinoma cells.

Objectives: To determine the effects of adenovirus-mediated transfer of p14ARF and p16INK4a on growth and apoptosis of human pancreatic carcinoma cell lines. Results: Pancreatic carcinoma cell lines, PC-7, PANC-1 and MIA PaCa-2 (p14ARF and p16INK4a -/-), were used. PC-7 (p53 wt) and MIA PaCa-2 (p53 mt) cells infected with recombinant adenovirus vector expressing p14ARF gene (Adp14) showed significant inhibition of cell growth compared with control vector in vitro and in vivo, whereas PANC-1 cells (p53 mt) did not show such effects. Those three cell lines exhibited growth retardation and senescence phenotype after infected with the adenovirus vector containing p16INK4a gene (Adp16) in vivo and in vitro. Conclusions: Adenovirus-mediated transfer of p14ARF and p16INK4a produces significant growth suppression of pancreatic carcinoma cells in vitro and in vivo. The effects of Adp14 partly depend on the status of p53 gene in those cells.

The lncRNA MALAT1 acts as a competing endogenous RNA to regulate KRAS expression by sponging miR-217 in pancreatic ductal adenocarcinoma.

The long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript-1 (MALAT1) has been shown to play an important role in tumourigenesis. The aim of this study was to investigate the role of MALAT1 in pancreatic ductal adenocarcinoma. MALAT1 is expressed at higher levels in pancreatic ductal adenocarcinoma (PDAC) tissues than in nontumour tissues and in metastatic PDAC than in localized tumours. Patients with PDAC and high MALAT1 expression levels have shorter overall survival than patients with PDAC and low MALAT1 expression levels. Knocking down MALAT1 reduces pancreatic tumour cell growth and proliferation both in vitro and in vivo. Moreover, MALAT1 knockdown inhibits cell cycle progression and impairs tumour cell migration and invasion. We found that miR-217 can bind MALAT1 and regulate its expression in PDAC cell lines. We also found MALAT1 knockdown attenuates the protein expression of KRAS, a known target of miR-217. After MALAT1 knockdown, KRAS protein expression levels can be rescued through inhibition of miR-217 expression. More importantly, MALAT1 knockdown does not directly affect cellular miR-217 expression but decreases the miR-217 nucleus/cytoplasm ratio, suggesting that MALAT1 inhibits the translocation of miR-217 from the nucleus to the cytoplasm.

Loss of ARID1A Expression Correlates With Tumor Differentiation and Tumor Progression Stage in Pancreatic Ductal Adenocarcinoma

Mutations in the AT-rich interactive domain 1A gene, which encodes a subunit of the Switch/Sucrose nonfermentable chromatin remodeling complex, can result in loss of protein expression and are associated with different cancers. Here, we used immunohistochemistry to investigate the significance of AT-rich interactive domain 1A loss in 73 pancreatic ductal adenocarcinoma cases with paired paracancerous normal pancreatic tissues. The relationship between levels of the AT-rich interactive domain 1A protein product, BAF250a, and clinicopathological parameters in the 73 pancreatic cancer specimens was also analyzed. We found that the expression of AT-rich interactive domain 1A in normal pancreatic tissue was higher than that in tumor tissue. Loss of AT-rich interactive domain 1A expression in pancreatic tumors was associated with tumor differentiation (P = .002) and tumor stage (P = .048). Meanwhile, BAF250a protein levels were not related to lymph node metastasis, distant metastasis, sex, or age and were not associated with survival. Transfection of the pancreatic cancer cell lines AsPC-1 and PANC-1 with small-interfering RNA specific for AT-rich interactive domain 1A resulted in elevated messenger RNA and protein expression levels of B-cell lymphoma-2 (Bcl-2), CyclinD1, and Kirsten rat sarcoma viral oncogene (KRAS). The AT-rich interactive domain 1A expression level in the cells was increased following microRNA-31 (miR-31) inhibitor transfection. Our data provide additional evidence that AT-rich interactive domain 1A might function as a tumor suppressor gene in pancreatic carcinogenesis.

RUNX1 promote invasiveness in pancreatic ductal adenocarcinoma through regulating miR-93

Runt-related transcription factor 1(RUNX1), a key factor in hematopoiesis that mediates specification and homeostasis of hematopoietic stem and progenitor cells (HSPCs), is also overexpressed in several solid human cancers, and correlated with tumor progression. However, the expression and function of RUNX1 in pancreatic ductal adenocarcinoma were still unclear. Here, we show that RUNX1 is highly expressed in pancreatic adenocarcinoma tissues and knocking down of RUNX1 attenuated aggressiveness in pancreatic cell lines. Moreover, we found that RUNX1 could negatively regulate the expression of miR-93. Bioinformatics method showed that there are two binding sites in the the promotor region of miR-93 precursor and through ChIP-qPCR and firefly luciferase reporter assay, we vertified that these two binding sites each have transcriptive activity in one pancreatic cell lines. This result supported our presumption that RUNX1 regulate miR-93 through binding to the promotor region of miR-93. Besides, the expression and function of miR-93 is quite the opposite, miR-93 overexpression suppresses migration and invasiveness in pancreatic cell lines supporting that RUNX1 negatively regulated miR-93. Our findings provided evidence regarding the role of RUNX1 as an oncogene through the inhibition of miR-93. Targeting RUNX1 can be a potential therapeutic strategy in pancreatic cancer.