HPDE6-C7 Cells Research Articles

Role of miR-92a-3p/PTEN axis in regulation of pancreatic cancer cell proliferation and metastasis.

To explore the effect of miR-92a-3p on the proliferation and metastasis of pancreatic cancer cells via targeting phosphatase and tension homolog deleted on chromosome ten (PTEN). MiR-92a-3p expression and PTEN protein levels were quantified in a normal pancreatic cells (HPDE6-C7) and 5 pancreatic cancer cell lines (Panc-1, BxPC-3, AsPC-1,MIA Paca-2, and Capan-2) by real-time PCR and Western blotting, respectively. BxPC-3 and Panc-1 cells were selected for further experiment. After transfection of normal control (NC) mimics (NC mimics group), miR-92a-3p mimics (miR-92a-3p mimics group), NC inhibitor or miR-92a-3p inhibitor (NC inhibitor group or miR-92a-3p inhibitor group), the proliferation of BxPC-3 and Panc-1 cells was measured by cell counting kit-8 (CCK-8), and the migration of them was measured by Transwell assay, and the levels of PTEN protein were measured by Western blotting. In addition, wild-type PTEN 3'-UTR (wt-PTEN 3'UTR) and mutant-type PTEN 3'-UTR (mut-PTEN 3'UTR) luciferase reporter vectors were constructed and co-transfected with NC mimics, miR-92a-3p mimic, NC inhibitor or miR-92a-3p inhibitor into 293T tool cells, and then the dual luciferase reporter assay was performed to examine the regulative correlation between miR-92a-3p and PTEN. The BxPC-3 cells were divided into 4 groups: a NC inhibitor+si-NC group, a miR-92a-3p inhibitor+si-NC group, a NC inhibitor+si-PTEN group, and a miR-92a-3p inhibitor+si-PTEN group. The Panc-1 cells were also assigned into 4 groups: a NC mimics+NC group, a miR-92a-3p mimics+si-NC group, a NC mimics+ PTEN group, and a miR-92a-3p mimics+PTEN group. The proliferation of Panc-1 cells was measured by CCK-8; the cell migration was measured by Transwell assay, and the levels of PTEN protein were measured by Western blotting. The miR-92a-3p was highly expressed in pancreatic cancer cell lines (allP<0.01), while the PTEN protein levels were lower in pancreatic cancer cell lines (allP<0.05)compared with that in the HPDE6-C7 cells. Compared with the NC mimics group, the cell viability of BxPC-3 and Panc-1 cells were both increased in the miR-92a-3p mimics group (bothP<0.01); compared with the inhibitor group, the cell viability of BxPC-3 and Panc-1 cells were both decreased in the miR-92a-3p inhibitor group (bothP<0.01). Compared with the NC mimics group, the cell number of BxPC-3 and Panc-1 cells through micropores were increased in the miR-92a-3p mimics group (bothP<0.01); compared with the inhibitor group, the cell number of BxPC-3 and Panc-1 cells through micropores were decreased in the miR-92a-3p inhibitor group (bothP<0.01). Compared with NC mimics group, the activity of dual luciferaseof wt-PTEN3'-UTR was inhibited in the miR-92a-3p mimics group (P<0.01); compared with the NC inhibitor group, the activity of dual luciferase of wt-PTEN3'-UTR was promoted in the miR-92a-3p inhibitor group (P<0.01). Compared with the miR-92a-3p inhibitor+si-NC group, the suppressive effects of miR-92a-3p on the proliferation and metastasis of BxPC-3 cells was restored in the miR-92a-3p inhibitor+si-PTEN group; while compared with the miR-92a-3p mimics+NC group, the positive effects of miR-92a-3p overexpression on the proliferation and metastasis of Panc-1 cells was restored in the miR-92a-3p mimics+PTEN group. The highly expressed miR-92a-3p in pancreatic cancer cells can decrease the protein levels of PTEN, thereby enhancing the proliferation and metastasis activity of pancreatic cancer cells.

Basic research of pancreatic cancer cell-derived extracellular vesicles

Objective To isolate extracellular vesicles (VEs) from the culture supernatant of pancreatic cancer (PANC-1) and human pancreatic ductal epithelial cells (HPDE6-C7), and to compare the differential expression of microRNA (miRNA, miR)-483-5p in the two kinds of cells and their secreted EVs. Methods Ultracentrifugation method was used to prepare the complete culture medium of depletion of serum-derived vesicles. Cell count Kit (CCK-8) was used to detect the effect of complete medium on cell proliferation without ultracentrifugation (control group) and with ultracentrifugation (experimental group). Cells were cultured in vesicle-depleted complete medium, and EVs were extracted by ultracentrifugation. The concentration of EVs was detected by BCA protein concentration determination kit and nanoparticles tracking analysis (NTA). Transmission electron microscope, NTA, and Western blotting were used to identify whether they, obtained by ultracentrifugation, were in accordance with the characteristics of EVs. The expression of miR-483-5p was detected by quantitative real time polymerase chain reaction (qPCR). The results of CCK-8 proliferation test and qPCR test were expressed by mean±standard deviation (SD). The statistical method was t test. Results The results of CCK-8 proliferation test showed that there was no significant difference between the experimental group and the control group after 48 h and 72 h in HPDE6-C7 cells (HPDE6-C7, 0.674±0.036 vs. 0.671±0.016, t=0.315, P48 h>0.05; 0.890±0.027 vs. 0.925±0.099, t=0.581, P72 h>0.05), as well as the PANC-1 (0.759±0.004 vs. 0.761±0.016, t=0.249, P48 h>0.05; 1.114±0.025 vs. 1.145±0.014, t=1.898, P72 h>0.05). The transmission electron microscopy showed that the EVs were tea-cup tray . The results of NTA showed that the diameter of 98% PANC-1 derived EVs was 130.0 nm, and that of 98% HPDE6-C7 derived EVs was 129.7 nm. Western blotting showed that CD63 and TSG101 were positive, but GM130 was negative. The concentration of EVs secreted by PANC-1 and HPDE6-C7 was measured by protein concentration assay kit and NTA as follows: 1.5 g/L, 1.510 11 particles/mland 1.3 g/L, 1.610 11 particles/ml, respectively. Compared with HPDE6-C7, the expression of miR-483-5p in PANC-1 was significantly increased (2.820±0.180 vs. 1.000±0.006, t=-17.539, P<0.01). Compared with HPDE6-C7 derived EVs, the expression of miR-483-5p in PANC-1 derived EVs was significantly increased (3.503±0.265 vs. 1.002±0.084, t=-15.582, P<0.01). Conclusion The ultracentrifugation method does not affect the normal proliferation of cells while removing fetal bovine serum-derived vesicles in the complete medium; the identification results show that the isolated pellets correspond to the characteristics of EVs. According to the results of NTA, it can be judged that they should be classified as small EVs. The results of qPCR show that miR-483-5p is highly expressed in PANC-1 and the secreted EVs. Key words: Pancreatic cancer; Extracellular vesicles; Ultracentrifugation; MicroRNA-483-5p

5-Aminolevulinic acid hydrochloride loaded microbubbles-mediated sonodynamic therapy in pancreatic cancer cells

5-Aminolevulinic acid hydrochloride (ALA)-mediated sonodynamic therapy (SDT) had anti-tumour effect on pancreatic cancer cells. Hence, ALA loaded lipid/poly(lactic-co-glycolic acid) (PLGA) microbubbles (MBs)-mediated SDT for pancreatic cancer has great potential. The average size of ALA-lipid MBs and ALA-PLGA MBs was about 3.0 µm. The two kinds of MBs had good biocompatibility to normal HPDE6-C7 cells and were not toxic to pancreatic cancer cells. Compared with ALA-induced SDT, a statistically significant decrease in cell viability was observed in ALA lipid/PLGA MBs combined with ultrasound groups in AsPC-1 and BxPC-3 cells (p < .05). Obvious effect on the apoptotic rate, apoptosis and pyroptosis morphology, enhanced reactive oxygen species was found in ALA-lipid/PLGA MBs mediated SDT in vitro. Through in vivo study, we found ALA-lipid/PLGA MBs-mediated SDT was a promise treatment for pancreatic cancer.

MiR-203 regulates JAK-STAT pathway in affecting pancreatic cancer cells proliferation and apoptosis by targeting SOCS3.

Janus kinase (JAK)- signal transducer and transcriptional activator (STAT) pathway overactivation is closely related to tumorigenesis. Cytokine signal transduction inhibitor 3 (SOCS3) is a negative regulator of JAK-STAT. It is shown that miR-203 is significantly elevated in the pancreatic cancer tissues. The bioinformatics analysis revealed a targeted binding site between miR-203 and the 3'-UTR of SOCS3 mRNA. This study investigated the role of miR-203 in regulating SOCS3 expression and the proliferation and apoptosis of the pancreatic cancer cells. Quantitative Real Time-PCR (qRT-PCR) was used to detect the expressions of miR-203 and SCOS3 mRNA in tumor tissues and paracancerous tissues. The Dual-Luciferase reporter gene assay was adopted to validate the target interaction between miR-203 and SOCS3. The PANC-1 cells were cultured in vitro and divided into miR-NC group and miR-203 inhibitor group followed by an analysis of the expressions of SOCS3, p-JAK2, and p-STAT3, cell apoptosis by flow cytometry, and cell proliferation by EdU staining. Compared with the adjacent tissues, miR-203 expression was significantly increased, while SOCS3 mRNA level was significantly declined in the tumor tissues of pancreatic cancer patients. There was a targeted regulatory relationship between miR-203 and SOCS3 mRNA. Compared with those in HPDE6-C7 cells, miR-203 level was upregulated, whereas SOCS3 mRNA and the protein expressions were reduced in pancreatic cancer PANC-1 and BXPC3 cells. The transfection of miR-203 inhibitor significantly increased SOCS3 mRNA and the protein levels, decreased p-JAK2 and p-STAT3 protein expressions, enhanced cell apoptosis, and inhibited cell proliferation in the pancreatic cancer PANC-1 cells. Increased miR-203 expression and reduced SOCS3 level are associated with the pathogenesis of pancreatic cancer. MiR-203 can regulate the proliferation and apoptosis of the pancreatic cancer cells by targeting the inhibited SOCS3 expression and regulating the JAK-STAT pathway activity.

Targeting Pancreatic Cancer Cells with Peptide-Functionalized Polymeric Magnetic Nanoparticles.

Pancreatic cancer is a concealed and highly malignant tumor, and its early diagnosis plays an increasingly weighty role during the course of cancer treatment. In this study, we developed a polymeric magnetic resonance imaging (MRI) nanoplatform for MRI contrast agents. To improve tumor-targeting delivery of MRI contrast agents, we employed a pancreatic cancer targeting CKAAKN peptide to prepare a peptide-functionalized amphiphilic hyaluronic acid–vitamin E succinate polymer (CKAAKN–HA–VES) for delivering ultra-small superparamagnetic iron oxide (USPIO), namely, CKAAKN–HA–VES@USPIO. With the modification of the CKAAKN peptide, CKAAKN–HA–VES@USPIO could specifically internalize into CKAAKN-positive BxPC-3 cells. The CKAAKN–HA–VES@USPIO nanoparticles presented a more specific accumulation into pancreatic cancer cells than normal pancreatic cells, and an obvious decrease in signal intensity was observed in CKAAKN-positive BxPC-3 cells, compared with CKAAKN-negative HPDE6-C7 cells and non-targeting HA–VES@USPIO nanoparticles. The results demonstrated that our polymeric MRI nanoplatform could selectively internalize into CKAAKN-positive pancreatic cancer cells by the specific binding of CKAAKN peptide with pancreatic cancer cell membrane receptors, which provided a novel polymeric MRI contrast agent with high specificity for pancreatic cancer diagnosis, and makes it a very promising candidate for magnetic resonance imaging contrast enhancement.

MiR-155 regulates the proliferation and apoptosis of pancreatic cancer cells through targeting SOCS3.

Reduced expression of suppressors of cytokine signaling 3 (SOCS3) is associated with a variety of tumors. The elevated miR-155 expression is associated with the onset of pancreatic cancer. Bioinformatics analysis revealed a targeted relation between miR-155 and the 3'-UTR of SOCS3. This study investigated whether miR-155 regulates SOCS3 expression and affects the biological effects of pancreatic cancer cells. QRT-PCR was used to detect the expression of miR-155 and SCOS3 mRNA in tumor tissues and paracancerous tissues of patients with pancreatic cancer. The dual luciferase reporter gene assay validated the target interaction between miR-155 and SOCS3. Pancreatic cancer cell line SW1990 cells were divided into miR-NC group and miR-155 inhibitor group followed by an analysis of the expressions of SOCS3, p-JAK2 and p-STAT3, cell apoptosis by flow cytometry, and cell proliferation by EdU staining. Compared with adjacent tissues, miR-155 expression was increased in tumor tissues of patients with pancreatic cancer, and SOCS3 expression was decreased. There was a targeted regulatory relationship between miR-155 and SOCS3 mRNA. Compared with HPDE6-C7 cells, miR-155 expression in pancreatic cancer SW1990 and Capan-1 cells was increased, and SOCS3 expression was decreased. Transfection of miR-155 inhibitor significantly increased SOCS3 expression in pancreatic cancer SW1990 cells, decreased the expression of p-JAK2 and p-STAT3, increased cell apoptosis, and decreased cell proliferation. Increased miR-155 expression and decreased SOCS3 expression are related to the pathogenesis of pancreatic cancer. miR-155 inhibits the proliferation and apoptosis of pancreatic cancer cells by inhibition of SOCS3 expression.

Targeting liquid-liquid phase separation in pancreatic cancer

Background: Accumulated evidence suggests a critical role of protein-mediated liquid-liquid phase separation (LLPS) in many key biological processes through organization of membrane-less compartments. However, the pathological role of protein-mediated LLPS remains elusive and it is unknown whether protein-mediated LLPS contributes to carcinogenesis. Methods: HPDE6-C7, BxPC-3, PANC-1, AsPC-1 and CFPAC-1 cells were treated with the LLPS inhibitor, 1,6-hexanediol. Cell proliferation was determined by CCK8 assay. An atopic BxPC-3 xenograft mouse model was established and treated with 1,6-hexanediol. Gene expression profiling was achieved by RNA-sequencing. Results: 1,6-hexanediol significantly inhibited cell proliferation and induced cell death in all tested pancreatic cancer cells (BxPC-3, PANC-1, AsPC-1 and CFPAC-1) but had a minimal effect on control cells (HPDE6-C7) at low doses. In an atopic BxPC-3 xenograft model, 1,6-hexanediol significantly limited pancreatic cancer growth. In whole genomic transcriptional analysis, 1,6-hexanediol significantly downregulated the expression of a set of genes that were enriched in cytokine-cytokine receptor interactions, WNT signaling pathway, ECM-receptor interaction, MAPK signaling pathway and focal adhesion. Furthermore, 1,6-hexanediol treatment specifically reduced the expression of MYC but not IL-6 and KLF5. Conclusions: Together, we provided evidence to demonstrate an important role of protein-mediated LLPS in pancreatic cancer. Cancer cells might be more addictive to LLPS and thus disruption of such a process might be a novel way to treat cancer.

Long non-coding RNA CASC2 upregulates PTEN to suppress pancreatic carcinoma cell metastasis by downregulating miR-21

BackgroundThe mechanism of pancreatic cancer metastasis remains poorly understood. Recently, lncRNA CASC2 has been demonstrated to be a tumor suppressor in various types of cancer. This study aimed to explore the mechanism of CASC2 in the regulation of pancreatic cancer metastasis.MethodsThe expression levels of CASC2 and miR-21 in pancreatic cells were detected by qRT-PCR. Using specific expression vectors, including mimics or shRNA, the expression levels of CASC2, miR-21 and PTEN in pancreatic cells were altered. The association between CASC2, miR-21 and PTEN was detected. Then, cell migration and invasion were assessed using the transwell assay.ResultsCASC2 expression was downregulated in the pancreatic cancer cell lines CAPAN-1, BxPC-3, JF305, PANC-1 and SW1990 compared with levels in normal human pancreatic HPDE6-C7 cells. CACS2 overexpression inhibited the migration and invasion of PANC-1 cells and significantly inhibited the expression of miR-21 and PTEN. MiR-21 was a direct target of CACS2. The overexpression of miR-21 significantly abolished the antimetastatic effects of CASC2 on PANC-1 cells. Moreover, the downregulation of PTEN significantly abolished the antimetastatic effects of CASC2.ConclusionCASC2 functions as a tumor suppressor in pancreatic cancer cells to inhibit tumor cell migration and invasion. Our work revealed a novel regulatory mechanism of the CASC2/miR-21/PTEN axis that may be important in pancreatic cancer.

Peroxiredoxin 1 silencing inhibited the growth and promoted apoptosis of pancreatic cancer cells via targeting FOXO3 gene.

ObjectiveOur study aimed to investigate the interaction between peroxiredoxin 1 (Prx1) and forkhead box O3 (FOXO3) and to explore the role of PI3K/AKT pathway in the development of pancreatic cancer.Material and methodsHuman pancreatic normal cells HPDE6-C7 and pancreatic cancer cells PANC-1 were randomly divided into control group, Prx1-silencing (si-Prx1) group, Prx1/FOXO3 dual-silencing (si-Prx1/FOXO3) group, and negative control group. Cell proliferation assay, clone formation assay, and cell apoptosis assay were performed to investigate the effects of Prx1 silencing and FOXO3 silencing on the proliferation and apoptosis ability of pancreatic cancer cells. qRT-PCR and Western blot were performed to study the Prx1 and FOXO3 mRNA in the two cells and FOXO3 protein expression in PANC-1 cells.ResultWe found Prx1 silencing could inhibit growth and promote apoptosis of PANC-1 cells. And Prx1 silencing could decrease the Prx1 mRNA level and increase FOXO3 mRNA level. To further explore the role of Prx1 in PI3K/AKT, we study the cell proliferation and apoptosis ability after adding the PI3K inhibitor and PI3K activator. We observed that PI3K inhibitor could inhibit tumor cell growth and promote cell apoptosis. And PI3K inhibitor also downregulated Prx1 protein expression.ConclusionWe concluded that the Prx1 silencing inhibited the growth and promoted apoptosis of pancreatic cancer cells via modulation of PI3K/AKT pathway by targeting FOXO3 gene.

PKCι and YAP1 are crucial in promoting pancreatic tumorigenesis.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignant disease with 5-year survival rate of less than 6%. Activating mutations of Kras (mu-Kras) are often detected in most of PDAC patients. Although it has been known that oncogenic Kras is the driver of pancreatic cancer initiation and development, the underlying mechanisms by which mu-Kras promotes PDAC remain poorly understood. Here, we identify that PKCι is one of the crucial factors for supporting the survival of pancreatic cancer cells expressing mu-Kras. Our study demonstrates that after the knockdown of PKCι, the expression of the transcriptional co-activator YAP1 is decreased, which hinders the expression of the downstream target gene Mcl-1, and subsequently sensitizes pancreatic cancer MiaPaCa and PANC-1 cells experssing mu-Kras to apoptosis. In comparison, the suppression of PKCι has little impact on the viability of non-neoplastic pancreatic HPDE6-C7 cells. Moreover, the transient overexpression of oncogenic Kras in HPDE6-C7 elevates the expression of PKCι and YAP1 concomitantly. The upregulated YAP1 in HPDE6-C7/ mu-Kras cells is abolished once PKCι is suppressed, suggesting the linear relationship among mu-Kras, PKCι and YAP1. This phenomenon is further proven by the co-upregulation of PKCι and YAP1 in HPDE6-C7 cells stably transfected with mu-Kras. Taken together, our findings suggest that PKCι acts through promoting YAP1 function to promote the survival of pancreatic cancer cells expressing mu-Kras. It appears that targeting PKCι-YAP1 signaling is a feasible strategy for developing new therapeutics for treating pancreatic cancer patients.

MiR-221 inhibits proliferation of pancreatic cancer cells via down regulation of SOCS3.

The over-activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway induced by cytokines are closely correlated with tumorigenesis. Suppressor of cytokine signaling 3 (SOCS3) serves as a negative regulator for JAK-STAT, and its down-regulation is involved in the oncogenesis of pancreatic cancer. We aimed at investigating the effect of miR-221 on the expression and proliferation, cycle and apoptosis of pancreatic cancer cells and determine the related mechanism. Dual luciferase reporter gene assay was used to analyze the regulation between miR-221 and SOCS3. The expressions of miR-221, SOCS3, p-JAK and p-STAT3 in normal human pancreatic epithelial cell HPDE6-C7 and pancreatic cancer cell PANC-1 were quantified by qPCR and Western blot. Flow cytometry was used to identify cell cycle and proliferation. In vitro cultured PANC-1 cells were transfected with miR-221 inhibitor or pIRES2-SOCS3. The expressions of miR-221, SOCS3, p-JAK and p-STAT3, along with the cell proliferation or apoptosis, were compared. Bioinformatics analysis showed the existence of binding site between miR-221 and 3'-UTR of SOCS3 mRNA. Dual luciferase gene reporter assay confirmed the targeted regulation between miR-221 and SOCS3. Compared to HPDE6-C7 cells, higher levels of miR-221, p-JAK and p-STAT3 expression, and lower expression of SOCS3, were found in PANC-1 cells, along with the increase of cell proliferation. Transfection of miR-221 inhibitor or pIRES2-SOCS3 remarkably enhanced SOCS3 expression, inhibited the levels of p-JAK and p-STAT3 expression, and impeded the proliferation of PANC-1 cells. MiR-221 decreases proliferation potency of PANC-1 cells and affects JAK-STAT3 signaling pathway via inhibiting SOCS3.

Phosphamide-containing diphenylpyrimidine analogues (PA-DPPYs) as potent focal adhesion kinase (FAK) inhibitors with enhanced activity against pancreatic cancer cell lines

A family of phosphamide-containing diphenylpyrimidine analogues (PA-DPPYs) were synthesized as potent focal adhesion kinase (FAK) inhibitors. The PA-DPPY derivatives could significantly inhibit the FAK enzymatic activity at concentrations lower than 10.69 nM. Among them, compounds 7a and 7e were two of the most active FAK inhibitors, possessing IC50 values of 4.25 nM and 4.65 nM, respectively. In particular, compound 7e also displayed strong activity against AsPC cell line, with an IC50 of 1.66 μM, but show low activity against the normal HPDE6-C7 cells (IC50 > 20 μM), indicating its low cell cytotoxicity. Additionally, flow cytometry analysis showed that after treatment with 7e (8 μM, 72 h), both AsPC and Panc cells growth were almost totally inhibited, with a cell viability rate of 16.8% and 18.1%, respectively. Overall, compound 7e may be served as a valuable FAK inhibitor for the treatment of pancreatic cancer.

High Expression of Cell Division Cycle 42 Promotes Pancreatic Cancer Growth and Predicts Poor Outcome of Pancreatic Cancer Patients.

Cell division cycle 42 (CDC42), an important member of the Rho family, is overexpressed in various human cancers. However, its expression and role in pancreatic cancer (PC) are not well understood. The present study was designed to investigate the expression patterns and underlying cellular mechanisms of CDC42 in PC. First, immunohistochemical analysis, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were performed to detect CDC42 expression in clinical pancreatic carcinoma and adjacent tissues. Second, differential expression of CDC42 between PC cells and normal cells was evaluated by qRT-PCR and Western blotting. Third, the correlation between CDC42 expression as well as clinicopathological characteristics and patient survival was analyzed. Finally, CDC42 was knocked down to examine its role both in vivo and in vitro. The results showed significantly increased CDC42 expression in pancreatic tumor tissues compared with adjacent normal tissues, as revealed by qRT-PCR, Western blotting and immunostaining. Compared to PanC-1 cells, CDC42 expression was downregulated in HPDE6-C7 cells as shown by qRT-PCR and Western blotting. High CDC42 expression was observed in 69.2% (83/120) of pancreatic adenocarcinoma patients and was significantly associated with tumor differentiation (p=0.013), median tumor size (p=0.005), tumor infiltration (pT stage, p=0.04), lymph nodal status (pN stage, p=0.044) and TNM staging (p=0.003). Multivariate Cox regression analysis revealed CDC42 expression to be an independent predictor of survival of PC patients (HR 3.0, 95% CI 1.60-5.61, p=0.001). Finally, we found that CDC42 promoted the proliferation of PanC-1 cells both in vivo and in vitro. Our findings reveal that CDC42 might play an important role in promoting PC development, and the findings suggest that CDC42 might serve as a potential prognostic indicator of PC.

The small-molecule IAP antagonist AT406 inhibits pancreatic cancer cells in vitro and in vivo

In the present study, we tested the anti-pancreatic cancer activity by AT406, a small-molecule antagonist of IAP (inhibitor of apoptosis proteins). In established (Panc-1 and Mia-PaCa-2 lines) and primary human pancreatic cancer cells, treatment of AT406 significantly inhibited cell survival and proliferation. Yet, same AT406 treatment was non-cytotoxic to pancreatic epithelial HPDE6c7 cells. AT406 increased caspase-3/-9 activity and provoked apoptosis in the pancreatic cancer cells. Reversely, AT406′ cytotoxicity in these cells was largely attenuated with pre-treatment of caspase inhibitors. AT406 treatment caused degradation of IAP family proteins (cIAP1 and XIAP) and release of cytochrome C, leaving Bcl-2 unaffected in pancreatic cancer cells. Bcl-2 inhibition (by ABT-737) or shRNA knockdown dramatically sensitized Panc-1 cells to AT406. In vivo, oral administration of AT406 at well-tolerated doses downregulated IAPs (cIAP1/XIAP) and inhibited Panc-1 xenograft tumor growth in severe combined immunodeficient (SCID) nude mice. Together, our preclinical results suggest that AT406 could be further evaluated as a promising anti-pancreatic cancer agent.

MicroRNA-183-5p promotes the proliferation, invasion and metastasis of human pancreatic adenocarcinoma cells.

The aim of the current study was to investigate the potential role of microRNA-183-5p (miR-183-5p) in the proliferation, invasion and metastasis of pancreatic cancer, and to identify promising target genes of oncogenic miR-183-5p. Western blotting and quantitative polymerase chain reaction (qPCR) were used to investigate whether these oncogenic microRNAs may be useful as biomarkers in pancreatic carcinoma (PaCa). Potential target genes were verified using miRDB, PicTar and TargetSCAN, and qPCR was used to detect the expression of miR-183 and suppressor of cytokine signaling 6 (SOCS-6; a potential target of miR-183) in PANC-1 PaCa cells and in the HPDE6-C7 pancreatic ductal cell line for comparison. The function of miR-183 in cell proliferation, wound healing, invasion and migration was also investigated using a miR-183 inhibitor. Western blot analysis was used to confirm SOCS-6 as a tumor suppressor and qPCR was used to detect and confirm that this potential target gene is directly regulated by miR-183. The results indicated that the expression of miR-183 in PANC-1 cells was upregulated compared with that in HPDE6-C7 cells, whilst the expression of SOCS-6 was downregulated. SOCS-6 expression was also significantly lower in PaCa tissues compared with that in matched normal pancreatic tissues from PaCa patients. Furthermore, expression of miR-183 was inversely correlated with that of SOCS-6. miR-183 knockdown decreased cell growth and motility in pancreatic cancer cells and significantly increased the expression of SOCS-6. These data suggest that oncogenic miR-183 may be useful as a pancreatic cancer biomarker. In addition, inhibition of miR-183 expression may be beneficial as PaCa treatment. SOCS-6 is a potential target gene of miR-183.

Targeting pancreatic cancer cells by a novel hydroxamate-based histone deacetylase (HDAC) inhibitor ST-3595.

In the current study, we tested the potential anti-pancreatic cancer activity of a novel hydroxamate-based histone deacetylase (HDAC) inhibitor ST-3595. We showed that ST-3595 exerted potent anti-proliferative and cytotoxic activities against both established pancreatic cancer cell lines (PANC-1, AsPC-1, and Mia-PaCa-2), and patient-derived primary cancer cells. It was, however, generally safe to non-cancerous pancreatic epithelial HPDE6c7 cells. ST-3595-induced cytotoxicity to pancreatic cancer cells was associated with significant apoptosis activation. Reversely, the pan caspase inhibitor z-VAD-fmk and the caspase-8 inhibitor z-ITED-fmk alleviated ST-3595-mediated anti-pancreatic cancer activity in vitro. For the mechanism study, ST-3595 inhibited HDAC activity, and induced mitochondrial permeability transition pore (MPTP) opening in pancreatic cancer cells. Inhibition of MPTP, by cyclosporin A, sanglifehrin A, or by cyclophilin-D (Cyp-D) siRNA knockdown, dramatically inhibited ST-3595-induced pancreatic cancer cell apoptosis. Meanwhile, we found that a low concentration of ST-3595 dramatically sensitized gemcitabine-induced anti-pancreatic cancer cell activity in vitro. In vivo, ST-3595 oral administration inhibited PANC-1 xenograft growth in nude mice, and this activity was further enhanced when in combination with gemcitabine. In summary, the results of this study suggest that targeting HDACs by ST-3595 might represent as a novel and promising anti-pancreatic cancer strategy.

Ethanol and the Tobacco-specific Carcinogen, NNK, Contribute to Signaling in Immortalized Human Pancreatic Duct Epithelial Cells

Smoking is a well-documented risk factor for pancreatic cancer. The tobacco-specific nitrosamine, NNK (4-[methylnitrosamino]-1-[3-pyridyl]-1-butanone), significantly induces pancreatic ductal adenocarcinomas in laboratory rodents. Recent observations suggest that ethanol enhances the tumorigenic effects of smoking. Ethanol consumption is associated with the development of chronic pancreatitis, also considered a predisposing factor for pancreatic ductal adenocarcinoma. Because the precise role of ethanol in pancreatic carcinogenesis is not known, this study sought to elucidate the cumulative effects of ethanol and NNK on particular signal transduction pathways that might play a role in cell proliferation in immortalized human pancreatic duct epithelial cells. The HPDE6-c7 cells are developed from pancreatic duct epithelial cells, which are the putative cells of origin of pancreatic ductal adenocarcinoma. Cell proliferation assays, Western blot, and cyclic adenosine monophosphate assays were used to demonstrate the effects of ethanol and NNK treatments on these cells. Ethanol cotreatments enhanced the NNK-induced proliferation of these cells. This response was inhibited by the adenylyl cyclase, protein kinase A, mitogen-activated protein kinase (p42/p44), and epidermal growth factor receptor-specific tyrosine kinase inhibitors. Cotreatments of NNK and ethanol also increased cyclic adenosine monophosphate accumulation, cAMP response element-binding family of proteins and mitogen-activated protein kinase phosphorylation, and protein kinase A activation. These findings suggest a potential role for these pathways contributing to the development of smoking- and alcohol-related pancreatic carcinogenesis.

The tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone stimulates proliferation of immortalized human pancreatic duct epithelia through beta-adrenergic transactivation of EGF receptors.

Pancreatic ductal adenocarcinoma is an aggressive smoking-associated human cancer in both men and women. The nicotine-derived 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is thought to contribute to the development of these neoplasms in smokers through genotoxic effects. However, NNK has been recently identified as an agonist for both beta(1)- and beta(2)-adrenergic receptors. Binding of NNK to these receptors stimulates proliferation of pulmonary and pancreatic adenocarcinomas cells in vitro and in hamster models. The goal of this study was to elucidate the NNK effects on the signal transduction pathways downstream of both beta(1)- and beta(2)-adrenergic receptors in immortalized human pancreatic HPDE6-c7 cells. The HPDE6-c7 cells are developed from normal pancreatic duct epithelial cells which are the putative cells of origin of pancreatic ductal adenocarcinoma. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) cell proliferation assays, Western blot and cyclic AMP assays were employed to demonstrate the effects of NNK and other beta(1)- and beta(2)-adrenergic agonists and antagonist treatments on these cells. MTT cell proliferation assays demonstrated that NNK and the classic beta-adrenergic agonist, isoproterenol, increased cell proliferation in HPDE6-c7 cells. Western blot and cyclic AMP assays demonstrated that NNK treatments also resulted in: (1) transactivation of the epidermal growth factor receptor, EGFR, (2) an increase in intracellular cyclic AMP accumulation, and (3) phosphorylation of mitogen-activated protein kinase, Erk1/2. The proliferative response to NNK and isoproterenol were inhibited by the use of beta-blockers (propranolol), and the inhibitors of adenylyl cyclase (SQ 22536), EGFR-specific tyrosine kinase (AG 1478) and Erk (PD 98059). These findings suggest that the NNK -mediated beta-adrenergic receptor transactivation of the EGFR and phosphorylation of Erk1/2 in immortalized human pancreatic duct epithelial cells as a novel mechanism might contribute to the development of tobacco-associated pancreatic carcinogenesis.