PaTu8988 Cells Research Articles

Dihydrotanshinone I Inhibits Pancreatic Cancer Progression via Hedgehog/ Gli Signal Pathway.

Pancreatic cancer is highly fatal and its incidence is rising worldwide. Its poor prognosis is attributed to a lack of effective diagnostic and therapeutic strategies. Dihydrotanshinone I (DHT), a phenanthrene quinone liposoluble compound from Salvia miltiorrhiza Bunge (Danshen), exerts anti-tumor effects by inhibiting cell proliferation, enhancing apoptosis, and inducing cell differentiation. However, its effects on pancreatic cancer are unclear. > Methods: The role of DHT in the growth of tumor cells was explored using real-time cell analysis (RTCA), colony formation assay, and CCK-8. The effects of DHT on tumor cells invasion as well as migration were assessed by Transwell and migration assays. Expressions of pro-apoptosis and metastasis factors in tumor cells were examined using western blot. Tumor apoptosis rates were studied using flow cytometry. The anticancer effect of DHT in vivo was assessed by tumor transplantation into nude mice. Our analyses show that DHT has a suppressive role in epithelial-mesenchymal transition (EMT), invasiveness, proliferation, as well as migratory ability of Patu8988 and PANC-1 cells via Hedgehog/Gli signaling. Moreover, it drives apoptosis via caspases/BCL2/BAX signaling. Experiments in nude mice transplanted with tumors have shown DHT to have anticancer effects in vivo. > Conclusion: Our data show that DHT effectively suppresses pancreatic cancer cell proliferation as well as metastasis, and induces apoptosis via Hedgehog/Gli signaling. These effects have been reported to be dose- and time-dependent. Therefore, DHT can be exploited as a potential treatment for pancreatic cancer.>.

Circadian Genes MBOAT2/CDA/LPCAT2/B4GALT5 in the Metabolic Pathway Serve as New Biomarkers of PACA Prognosis and Immune Infiltration.

Pancreatic cancer (PACA) is a highly malignant tumor with a poor prognosis. Recent studies have discovered substantial differences in the expression levels of several circadian genes in PACA samples compared to normal samples. The goal of this research was to find differentially expressed rhythm genes (DERGs) in PACA samples and determine their role in the development of PACA. A total of 299 DERGs were identified in PACA, including 134 downregulated genes and 165 upregulated genes. DERGs were significantly abundant in the metabolic pathway and immune response pathways, according to GO and KEGG analyses. Survival analyses showed that PACA patients who had higher expression levels of MBOAT2/CDA/LPCAT2/B4GALT5 had shorter overall survival times. Using cell assay verification, the mRNA levels of MBOAT2/CDA/LPCAT2/B4GALT5 in Patu-8988 and PNAC-1 cells were found to be significantly higher than those in HPDE6-C7 cells, which was in line with previous studies on PACA patient data. Through conducting univariate Cox analysis, it was determined that MBOAT2/CDA/LPCAT2/B4GALT5 expression, age and grade were all high-risk factors. The MBOAT2/CDA/LPCAT2/B4GALT5 genes were independently correlated with overall survival, according to the multivariate Cox analysis. The proportion of immune cells in PACA and normal samples significantly changed, according to the immune infiltration analysis. Furthermore, MBOAT2/CDA/LPCAT2/B4GALT5 expression levels were significantly related to the level of immune cell infiltration. The protein-protein interaction network of the MBOAT2/CDA/LPCAT2/B4GALT5 genes included 54 biological nodes and 368 interacting genes. In conclusion, the finding of these DERGs adds to the investigation of the molecular processes underlying the onset and progression of PACA. In the future, DERGs may serve as prognostic and diagnostic biomarkers as well as drug targets for chronotherapy in PACA patients.

Transcriptome and proteome analysis of the antitumor activity of maslinic acid against pancreatic cancer cells.

Maslinic acid (MA) is a triterpenoid compound of natural abundance in olive plants possessing numerous biological activities. The effect and molecular mechanism of MA on pancreatic cancer cells remain elusive. Here, we explored the anti-tumor activity of MA on human pancreatic cancer cells and the potential underlying molecular mechanism. The anti-cancer effects of MA on whole-cell processes, including proliferation, migration, and invasion in pancreatic cancer cells, were systematically assessed by colony formation, transwell, and migration assays. The search for potential therapeutic targets was achieved via transcriptomics and proteomics analyses. MA was demonstrated to inhibit the proliferation, migration, and invasion of PANC-1 and Patu-8988 cells, but induced apoptosis of these cells. Several key candidate genes and proteins of functional relevance for the anti-tumor activity of MA were identified through the association analysis of transcriptomics and proteomics. To our knowledge, this is the first transcription and proteomics-based comprehensive analysis of the mechanism of MA against pancreatic cancer. The findings demonstrate that MA holds promise as a therapeutic drug for managing pancreatic cancer.

Inhibition of JAK2/STAT3 signaling pathway by panaxadiol limits the progression of pancreatic cancer.

Pancreatic cancer is the fourth leading cause of cancer-related death with the characteristics of chemoresistance and early metastasis. Panaxadiol, a triterpenoid saponin extracted from the roots of American ginseng, has been proved to display anti-tumor activity in colon cancer. In this study, we found panaxadiol significantly inhibited proliferation, and induced apoptosis in human pancreatic cancer cell lines PANC-1 and Patu8988 in a dose-dependent manner. Furthermore, the expression of apoptosis-related proteins (Bax, Bcl2, Cleaved-caspase3) was detected via western blot and immunofluorescence staining. In addition, panaxadiol was also found to inhibit the migration of pancreatic cancer cells by wound healing and transwell assays. In vivo, the growth of xenograft pancreatic cancer models was also notably suppressed by panaxadiol compared to the control group. Moreover, the down-regulation of JAK2-STAT3 signaling pathway was responsible for the underlying pro-apoptosis mechanism of panaxadiol, and this result was in good agreement with molecular docking analysis between panaxadiol and STAT3. In conclusion, our work comprehensively explored the anti-tumor ability in PANC-1 and Patu8988 cells of panaxadiol and provided a potential choice for the clinical treatment of pancreatic cancer patients.

NUDCD1 knockdown inhibits the proliferation, migration, and invasion of pancreatic cancer via the EMT process.

NudC domain containing 1 (NUDCD1) is an oncoprotein frequently activated or upregulated in various human cancers, but its role in pancreatic cancer (PC) remains unknown. Thus, we aimed to determine the function and mechanism of NUDCD1 in PC. We employed Western blot and quantitative real-time polymerase chain reaction to assess NUDCD1 expression in cells and PC tissues. NUDCD1 was knocked down in Patu8988 and PANC-1 cells. We conducted real-time cell analysis, wound healing assay, transwell assay and colony formation assay to evaluate the metastatic and proliferative abilities of PC cells. Western blot was conducted to assess the expression of markers associated with apoptosis and epithelial–mesenchymal transition (EMT). Also, we established a tumor xenograft model to determine the role of NUDCD1 in vivo. NUDCD1 was overexpressed in PC tissues and cells. NUDCD1 knockdown suppressed the invasion, migration, and proliferative abilities of the cells and induced PC cell apoptosis. The specific mechanism of NUDCD1 was related to the modulation of the EMT process. Data obtained from in vivo experiments revealed that NUDCD1 knockdown inhibited the tumor growth, proliferation, and metastasis by modulating the EMT and inducing the apoptosis of PC cells.

FUBP1 mediates the growth and metastasis through TGFβ/Smad signaling in pancreatic adenocarcinoma.

Recent studies have reported that the expression levels of far upstream element-binding protein 1 (FUBP1) were upregulated and served a crucial role in several types of cancer. However, the underlying molecular mechanisms and clinical significance of FUBP1 in pancreatic adenocarcinoma (PAAD) remain unclear. The present study aimed to determine the expression levels of FUBP1 in patients with PAAD and subsequently investigated the biological functions and mechanisms of FUBP1 using in vitro assays. FUBP1 expression levels and survival outcomes in patients with PAAD were analyzed using The Cancer Genome Atlas and starBase databases. Reverse transcription-quantitative PCR was used to analyze the mRNA expression levels of FUBP1 in PAAD and adjacent normal tissues. In addition, the expression of FUBP1 was knocked down with small interfering RNA and overexpressed using FUBP1-overexpressed plasmids, and the effects on biological functions, including cell proliferation, migration and invasion, were investigated. Western blotting and immunofluorescence assays were used to determine the role of FUBP1 in epithelial-mesenchymal transition (EMT). The results of the present study revealed that the expression levels of FUBP1 were upregulated in PAAD tissues compared with adjacent normal tissues and the upregulated expression was significantly associated with poor survival. The knockdown of FUBP1 expression significantly inhibited the proliferative, migratory and invasive abilities of the PAAD PaTu8988 cell line, while the overexpression of FUBP1 promoted cell proliferation, migration and invasion in the PAAD SW1990 cell line. Furthermore, the knockdown of FUBP1 downregulated the expression levels of EMT-related markers, including N-cadherin, β-catenin and vimentin, while the expression levels of E-cadherin were upregulated. The knockdown of FUBP1 was also revealed to regulate the TGFβ/Smad signaling cascade by downregulating phosphorylated-Smad2/3 and TGFβ1 expression levels. Conversely, the overexpression of FUBP1 reversed these effects. In conclusion, the findings of the present study indicated that FUBP1 may be a potential oncogene that mediates the EMT of PAAD via TGFβ/Smad signaling. These data suggested that FUBP1 may represent a potential biomarker for the diagnosis of PAAD or a target for the treatment of patients with PAAD.

The PPARγ Agonist Rosiglitazone Enhances the Radiosensitivity of Human Pancreatic Cancer Cells.

PurposeAs radiation therapy is widely used for the management of pancreatic cancer, identifying novel targets to improve the radiosensitivity of cancer cells is beneficial. Rosiglitazone, a specific peroxisome proliferator-activated receptor γ (PPARγ) agonist, has an inhibitory effect on various types of cancer cells. The purpose of this paper is to investigate the effect of rosiglitazone on the radiosensitivity of pancreatic cancer cells and the potential mechanism.Materials and MethodsPPARγ expression in pancreatic cancer and adjacent tissues was evaluated using immunohistochemistry analysis. The viability, migration and invasion ability of PANC1 and PaTu8988 cells were detected using MTT assay, scratch-wound assay and transwell invasion assay. The effect of rosiglitazone on radiosensitivity of the cells was determined using the clonogenic assay. PANC1 cells were inoculated into BALB/c mice to establish tumors. Microarray was used to investigate changes of genes involved.ResultsHigher PPARγ expression was demonstrated in pancreatic cancer tissues compared with para-carcinoma tissues. Rosiglitazone inhibited the cell viability and enhanced the radiation-induced anti-migration and anti-invasion effect. Rosiglitazone potentiated the radiosensitivity of pancreatic cancer cells and PANC1 xenografts. Microarray analysis revealed that rosiglitazone plus radiation altered the expression of multiple genes and affected multiple pathways.ConclusionRosiglitazone enhances the radiosensitivity of human pancreatic cancer cells in vitro and in vivo via complex mechanisms.

PRMT5 promotes epithelial-mesenchymal transition via EGFR-β-catenin axis in pancreatic cancer cells.

Protein arginine methyltransferase 5 (PRMT5) has been implicated in the development and progression of human cancers. However, few studies reveal its role in epithelial‐mesenchymal transition (EMT) of pancreatic cancer cells. In this study, we find that PRMT5 is up‐regulated in pancreatic cancer, and promotes proliferation, migration and invasion in pancreatic cancer cells, and promotes tumorigenesis. Silencing PRMT5 induces epithelial marker E‐cadherin expression and down‐regulates expression of mesenchymal markers including Vimentin, collagen I and β‐catenin in PaTu8988 and SW1990 cells, whereas ectopic PRMT5 re‐expression partially reverses these changes, indicating that PRMT5 promotes EMT in pancreatic cancer. More importantly, we find that PRMT5 knockdown decreases the phosphorylation level of EGFR at Y1068 and Y1172 and its downstream p‐AKT and p‐GSK3β, and then results in down‐regulation of β‐catenin. Expectedly, ectopic PRMT5 re‐expression also reverses the above changes. It is suggested that PRMT5 promotes EMT probably via EGFR/AKT/β‐catenin pathway. Taken together, our study demonstrates that PRMT5 plays oncogenic roles in the growth of pancreatic cancer cell and provides a potential candidate for pancreatic cancer treatment.

Role of GRP78 inhibiting artesunate-induced ferroptosis in KRAS mutant pancreatic cancer cells.

Objective: To investigate the exact role of GRP78 in artesunate-induced ferroptosis in KRAS mutant pancreatic cancer cells.Methods: Artesunate-induced KRAS mutant human pancreatic cancer cells (AsPC-1 and PaTU8988) ferroptosis was confirmed by fluorescent staining experiments and CCK8. Western blot and short-hairpin RNA-based knockdown assays were conducted to detect GRP78 activity and its role in artesunate-induced ferroptosis.Results: Artesunate induced AsPC-1 and PaTU8988 cell death in ferroptosis manner, rather than necrosis or apoptosis. In addition, artesunate increased the mRNA and protein levels of GRP78 in a concentration-dependent manner in AsPC-1 and PaTU8988 cells. Knockdown GRP78 enhanced artesunate-induced ferroptosis of pancreatic cancer cells in vitro and in vivo.Conclusion: Combining artesunate with GRP78 inhibition may be a novel maneuver for effective killing of KRAS mutant pancreatic ductal adenocarcinoma cells.

Diosgenin exhibits tumor suppressive function via down-regulation of EZH2 in pancreatic cancer cells

ABSTRACT Pancreatic cancer (PC) is one of the most aggressive and lethal malignancies worldwide. Although significant progress has been made in oncology treatment, this refractory disease is still become intractable. Natural herb product diosgenin is described to exhibit vast range of pharmacological activities in preclinical studies, including anti-cancer activities. Accumulating data demonstrated that Enhancer of zeste homolog 2 (EZH2) as an oncogenic protein is over-expressed in various human cancers, including PC. However, the underlying mechanism has not been fully understood. In this study, we aim to investigate the anti-cancer properties and molecular basis of diosgenin in PC cells. Significant inhibition of cell proliferation was observed in diosgenin treated Patu8988 and Panc-1 cells in a dose- and time-dependent manner. Apoptotic cell death and G2/M phase arrest were also induced by diosgenin treatment in PC cells. Moreover, obvious inhibition of cell migration and invasive capacities was detected in diosgenin treated PC cells. Mechanistically, the expression levels of EZH2 and its target Vimentin were reduced, and PTEN was promoted after diosgenin exposure. Our results further supported that EZH2 signaling was closely associated with the anti-tumor characteristics of diosgenin in PC cells. Therefore, inhibition of EZH2 by diosgenin could be a promising therapeutic method for PC treatment.

Lentivirus-mediated RNA interference targeting programmed death receptor ligand 1 increases the immunologic anti-tumor effect of dendritic cell vaccination against pancreatic cancer in SCID-hu mice.

Programmed death receptor ligand 1 (PD-L1), which belongs to the B7 family, is overexpressed in a variety of human cancer types and serves a crucial role in immune escape by malignant cells. Programmed death receptor 1 (PD-1) is a specific PD-L1 receptor. PD-1/PD-L1 signaling inhibits the antitumor effects of dendritic cell (DC) immunization for tumor treatment. The aim of the present study was to determine whether inhibiting PD-L1 may increase the immunologic anti-tumor effect of dendritic cells against pancreatic cancer. In the present study, PD-L1 levels in non-cancerous and malignant tissue samples were compared, and the impact of PD-L1 downregulation on human pancreatic cancer PaTu8988 cells was determined by lentivirus-based RNA interference and DC immunotherapy. PD-L1 expression in pancreatic specimens was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. PaTu8988 cells expressing reduced levels of PD-L1 were generated by lentivirus-based knockdown to assess the mechanism by which the inhibition of PD-L1 signaling in DC immunization affects therapeutic outcomes in pancreatic cancer-bearing SCID-hu mice. PD-L1 levels were markedly elevated in pancreatic adenocarcinoma samples compared with in non-cancerous tissue. PD-L1 silencing in pancreatic adenocarcinoma cells resulted in improved treatment outcomes of DC immunization in vitro and in vivo compared with traditional DC immunization. PD-L1 silencing enhances the antitumor response of cytotoxic T cells by increasing interferon γ production in vitro. In vivo, this method prevented tumor growth and lung metastasis, and prolonged survival in the SCID-hu model. In conclusion, the results of the present study suggested that suppressing PD-L1 in malignant cells during DC immunization may be a useful tool for immunotherapy in pancreatic adenocarcinoma.

Construction and effects of functionalized graphene oxide nonparticles-mediated RNA interference of hypoxia-inducible factor-1α gene

Objective To explore the effects of graphene oxide (GO)-polyethylene glycol (PEG)-folic acid (FA)-pyrenemethylamine hydrochloride (PyNH2)-mediated RNA interference (RNAi) of hypoxia-inducible factor-1α (HIF-1α) on the biological behaviors of human pancreatic cancer Patu8988 cells. Methods GO-PEG-FA-PyNH2 and RNAi targeting HIF-1α gene (GO-PEG-FA-PyNH2-HIF-1α-RNAi) was constructed. The expressions of HIF-1α and glucose transporter 1 (Glut-1) in Patu8988 cells were determined after knockdown of HIF-1α by RNAi. The invasive ability, the proliferation and the cell cycle of Patu8988 cells were investigated. The effect of HIF-1α knockdown on the uptake of 18F-fluorodeoxyglucose (FDG) in Patu8988 cells was also detected. Comparison of data was conducted by one-way analysis of variance and least significant difference t test. Results The GO-PEG-FA-PyNH2 was successfully constructed, and no cytotoxicity was found. Under the hypoxia or normoxia state, the mRNA and protein levels of HIF-1α and mRNA level of Glut-1 in cells transfected with GO-PEG-FA-PyNH2-HIF-1α-RNAi (study group) were lower than those in cells transfected with GO-PEG-FA-PyNH2(negative group) and cells transfected with Opti-minimal essential medium (Opti-MEM, control group; F=30.25-32.58, t=3.66-5.81, all P<0.05); the numbers of migrated cells in the study group were much lower than those in the negative group and the control group (F=38.63 and 41.35, t=20.51-35.25, all P<0.01); the cell proliferation in the study group was significantly lower than that in the negative group and the control group (F=35.19 and 38.11, t=15.11-22.19, all P<0.05). The proportions of G0/G1 cells in the study group were higher than those in the negative group and the control group (F=34.60 and 34.83, t=11.55-34.56, all P<0.05); the 18F-FDG uptake in the study group was lower than that in the negative group and control group (F=29.85 and 31.69, t=3.35-4.35, all P<0.05). Conclusion GO-PEG-FA-PyNH2-mediated HIF-1α RNAi inhibits the expression of HIF-1α in pancreatic cancer cells, leading to changes in related biological behaviors. Key words: Pancreatic neoplasms; Tumor cells, cultured; RNA, small interfering; Hypoxia-inducible factor 1, alpha subunit; Graphene oxide; Polyethylene glycols; Folic acid; 1-Pyrenemethylamine

The Distribution and Imaging of 99mTc-nGO-PEG-FA in Human Patu8988 Tumor-Bearing Nude Mice.

Background: To study the distribution and imaging of 99mTc-nGO-PEG-FA in human pancreatic cancer Patu8988 tumor-bearing nude mice, and to explore its usefulness as an imaging reagent for pancreatic cancer. Materials and Methods: Natural graphite powder was used as raw material to prepare the nanosized graphene oxide (nGO) by using the modified Hummers method, and then was covalently modified by polyethylene glycol (PEG) on the surface of nGO. The nGO was further optimized by in vitro cell experiment, and then conjugated with the targeting molecule folic acid (FA) to form nGO-PEG-FA system. The nGO-PEG-FA was finally labeled by radioactive nuclide 99mTc by direct labeling method to form the 99mTc-nGO-PEG-FA molecular imaging probe. Nude mice bearing patu8988 pancreatic cancer xenografts were intravenous injection (I.V.) injected with 99mTc-nGO-PEG-FA, and the distribution of 99mTc-nGO-PEG-FA in nude mice at different time course was investigated by determination of tissue uptake of radioactivity (%ID/g), as well as the single photon emission computed tomography (SPECT) imaging at different time course. Results: The labeling rate of nGO-PEG-FA with 99mTc was (90.08 ± 2.34)%, and the highest binding rate of 99mTc-nGO-PEG-FA with Patu8988 cells was (3.15 ± 0.31)%. The radioactive uptake in tumor reached (5.11 ± 1.23)%ID/g at 6 h after I.V. injection of 99mTc-nGO-PEG-FA in nude mice. Meanwhile, the radioactive uptake in liver, spleen, and lung was also high and reached (10.33 ± 1.22)%ID/g, (5.86 ± 0.59)%ID/g, and (3.55 ± 0.93)%ID/g, respectively, whereas less radioactivity uptake was observed in the heart (1.12 ± 0.33)%ID/g and blood (2.76 ± 0.39)%ID/g, respectively. The tumors can be clearly imaged at 4.0-6.0 h after 99mTc-nGO-PEG-FA injection. Conclusions: 99mTc-nGO-PEG-FA can efficiently target pancreatic cancer, which may be developed as an imaging agent for pancreatic cancer.

The interaction of long non-coding RNA MIAT and miR-133 play a role in the proliferation and metastasis of pancreatic carcinoma

Pancreatic cancer (PC) is one of the most aggressive malignancies in humans. Despite advances in early detection and treatment of PC, the prognosis is still limited. LncRNA myocardial infarction–associated transcript (MIAT) is found abnormally expressed in a variety of cancers. However, the role of MIAT in PC is still unknown. This study aimed to explore whether MIAT was related to the progression of PC and the underlying mechanism. Bioinformatics analysis and luciferase reporter assay validated that miR-133 may target MIAT 3′-UTR. MIAT expression was found remarkably increased and miR-133 expression was significantly decreased in PC tissues and PC cell lines (PATU-8988, BxPC-3, PANC-1, SW1990 and AsPC-1 cells). PC patients with high MIAT level had poor prognosis than that with low MITA level. Besides that, PATU-8988 cells transfected with siMIAT and/or miR-133 inhibitor. The results exhibited that the inhibition of miR-133 expression reversed the inhibition effect of MIAT down-regulation in the growth, migration and invasion of PC cells. Moreover, tumor growth was tremendously suppressed in nude rats received injection of PATU-8988 cells transfected with siMIAT. Taken together, our results suggest that the interaction of MIAT and miR-133 play a role in the proliferation and metastasis of pancreatic carcinoma.

Furin inhibitor D6R suppresses epithelial-mesenchymal transition in SW1990 and PaTu8988 cells via the Hippo-YAP signaling pathway.

Hexa-D-arginine (D6R), an inhibitor of furin, has potential therapeutic applications in different types of human tumor. However, the function of D6R in targeting pancreatic cancer cells remains to be elucidated. In the present study, the proliferation, invasion and migration abilities of SW1990 and PaTu8988 cells were examined using a Cell Counting Kit-8, and Transwell and wound healing assays. Subsequently, the expression of proteins associated with epithelial-mesenchymal transition (EMT) and the Hippo-yes-associated protein (YAP) pathway were detected using western blot analysis. It was revealed that D6R significantly inhibited the proliferation, migration and invasion abilities of SW1990 and PaTu8988 cells. Additionally, D6R led to the upregulation of E-cadherin (an epithelial marker), and the downregulation of N-cadherin and vimentin (mesenchymal markers) in SW1990 and PaTu8988 cells. Furthermore, the results of the present study revealed that D6R significantly affected the YAP phosphorylation level and the total YAP protein level, indicating that D6R was functionally involved in the Hippo-YAP signaling pathway. It has been suggested that D6R-suppressed EMT in SW1990 and PaTu8988 cells may occur via the Hippo-YAP pathway and that it may be a feasible drug to ameliorate the malignant phenotype of SW1990 and PaTu8988 cells.

Effects of Smoothened gene targeting small interfering RNA lentiviral vector on biological characteristics of human pancreatic cancer cells

Objective To observe the effect of lentiviral vector carrying Smoothened (SMO) gene targeting small interfering RNA (siRNA) on biological characteristics of pancreatic cancer cells in vitro. Methods Based on previous research methods, 3 lentiviral vectors with SMO siRNA (titer: 5×108, 6×108, 5×108 TU/ml) were synthesized and transfected into Patu-8988 cells. The expression of SMO gene in cells was detected by real-time quantitative polymerase chain reaction (Real-time PCR), and the inhibition rate of gene expression was calculated. Interference vectors with the highest inhibition rate were screened to transfect into Patu-8988 for 72 h. Cell growth was detected by methyl thiazol tetrazolium (MTT), cell apoptosis by flow cytometry, cell invasion and metastasis ability by Transwell invasion assay, and the expression of pathway target gene B cell lymphoma/leukemia-2 (bcl-2) by Western blotting, respectively. Results The inhibition rate of 3 lentiviral vectors of SMO siRNA on SMO gene expression in Patu-8988 cells was 82.4%, 93.1% and 89.1%, respectively. With the selection of interference vector with the highest inhibition rate, it was transfected into Patu-8988 cells for 72 h, and cells grew well. As compared with the negative control group, the growth inhibition rate of Patu-8988 cells in transfected group was (7.46±0.32)%, (25.41±0.28)%, (25.94±0.51)%, (26.01±0.94)% and (43.23±0.88)% from the first to the fifth day, respectively; the apoptotic rate was (1.35±0.11)%, which was lower than that of the control group in which no significant apoptosis was observed (t=-4.523, P=0.011). The number of cells observed in the Invasion chamber and Transwell chamber was (38.00±4.73)/HP and (78.00±3.71)/HP in the transfected group, respectively, significantly lower than those in the control group (128.00±10.91)/HP and (306.00±9.12)/HP, respectively (t=-22.808, P=0.000; t=-69.256, P=0.000); the relative expression of bcl-2 protein was down-regulated in the cells (t=-27.386, P=0.000). Conclusion SMO small interfering RNA lentiviral vector can effectively block the Hedgehog signaling pathway, and significantly inhibit the growth, invasion and metastasis of Patu-8988 cells. Key words: Pancreatic carcinoma; Hedgehog signal transduction pathway; Smoothened gene; Small interfering RNA

Quercetin inhibits epithelial-mesenchymal transition, decreases invasiveness and metastasis, and reverses IL-6 induced epithelial-mesenchymal transition, expression of MMP by inhibiting STAT3 signaling in pancreatic cancer cells.

Quercetin, a flavone, is multifaceted, having anti-oxidative, anti-inflammatory, and anticancer properties. In the present study, we explored the effects of quercetin on the epithelial–mesenchymal transition (EMT) and invasion of pancreatic cancer cells and the underlying mechanisms. We noted that quercetin exerted pronounced inhibitory effects in PANC-1 and PATU-8988 cells. Moreover, quercetin inhibited EMT and decreased the secretion of matrix metalloproteinase (MMP). Meanwhile, we determined the activity of STAT3 after quercetin treatment. STAT3 phosphorylation decreased following treatment with quercetin. We also used activating agent of STAT3, IL-6, to induce an increase in cell malignancy and to observe the effects of treatment with quercetin. As expected, the EMT and MMP secretion increased with activation of the STAT3 signaling pathway, and quercetin reversed IL-6-induced EMT, invasion, and migration. Therefore, our results demonstrate that quercetin triggers inhibition of EMT, invasion, and metastasis by blocking the STAT3 signaling pathway, and thus, quercetin merits further investigation.

B7-H3 combats apoptosis induced by chemotherapy by delivering signals to pancreatic cancer cells

ObjectiveThis study aimed to investigate the role of B7-H3 in chemotherapy resistance of pancreatic cancer cells and discover the potential signal transduction pathway and molecular targets involved.MethodsImmunohistochemical staining and real-time polymerase chain reaction (PCR) were used to determine the expression of B7-H3 in clinical specimens. Clinical data were applied to survival analysis. Phosphoprotein was purified from cultured Patu8988 cells using the Phosphoprotein Purification Kit. Cell apoptosis was detected using propidium iodide–Annexin V staining to investigate the relation between the expression of B7-H3 and Patu8988 cells treated with gemcitabine. Western blot was used to determine the effect of B7-H3 on the expression of proteins including extracellular signal–regulated kinase (ERK)1/2, epidermal growth factor receptor (EGFR), and Inhibitor of NF-κB(IκB) in Patu8988 cells; B7-H3 was activated by 4H7, which as an agonist monoclonal antibody to B7-H3.ResultsThe expression of B7-H3 was found to be higher in tumor tissues than in normal tissues of pancreatic carcinoma. Survival analysis revealed that patients in the low-B7-H3 expression group were likely to have a longer overall survival compared with those in the high-expression group (P < 0.05). B7-H3 activated by 4H7 could reduce gemcitabine-induced apoptosis in Patu8988 cells. Activation of B7-H3 by 4H7 induced variations in p-ERK1/2, EGFR, and IκB protein levels. When B7-H3 was upregulated, the expression levels of EGFR and p-ERK1/2 proteins significantly increased (P < 0.05), but the expression level of IκB significantly decreased (P < 0.05), especially in the gemcitabine-treated group.ConclusionThis study demonstrated that B7-H3 could deliver signals to pancreatic cancer cells to combat apoptosis induced by gemcitabine.

Brusatol inhibits growth and induces apoptosis in pancreatic cancer cells via JNK/p38 MAPK/NF-κb/Stat3/Bcl-2 signaling pathway

Brusatol, isolated from brucea, has been proved to exhibit anticancer influence on various kind of human malignancies. However, the role that brusatol plays in pancreatic cancer is seldom known by the public. Through researches brusatol was proved to inhibit growth and induce apoptosis in both PATU-8988 and PANC-1 cells by decreasing the expression level of Bcl-2 and increasing the expression levels of Bax, Cleaved Caspase-3. Then we found the activation of the JNK, p38 MAPK and inactivation of the NF-κb, Stat3 are related with the potential pro-apoptotic signaling pathways. However, SP600125 could not only abrogated the JNK activation caused by brusatol, but also reverse the p38 activation and the decrease of Bcl-2 as SB203580 did. Besides, SP600125 and SB203580 also reversed the inactivation of NF-κb and Stat3. Furthermore, BAY 11–7082 and S3I-201 indeed had the similar effect as brusatol had on the expression of Phospho-Stat3 and Bcl-2. To sum up, we came to a conclusion that in pancreatic cancer, brusatol do inhibit growth and induce apoptosis. And we inferred that brusatol illustrates anticancer attribution via JNK/p38 MAPK/NF-κb/Stat3/Bcl-2 signaling pathway.

Furin promotes epithelial-mesenchymal transition in pancreatic cancer cells via Hippo-YAP pathway.

Furin, a well-characterized proprotein convertase, plays an important role in many diseases and links to tumor metastasis. However, the role of furin in pancreatic cancer progression remains to be elucidated. In the present study, we found that furin promotes the growth and the epithelial-mesenchymal transition (EMT) of pancreatic cancer cells. First, we found that furin knockdown significantly inhibited proliferation, invasion and migration in BxPC3 and SW1990 cells, while furin overexpression promoted the above behavior in PANC1 and PaTu8988 cells. Further evidence revealed that furin knockdown resulted in the upregulation of E-cadherin (epithelial marker), and the downregulation of N-cadherin and Vimentin (mesenchymal markers) in BxPC3 and SW1990 cells, whereas furin overexpression remarkably led to the opposite effects in PANC1 and PaTu8988 cells. Furthermore, our data showed that Furin knockdown, furin inhibitor D6R or overexpression significantly affected YAP phosphoration level and total YAP protein level, indicating that furin was involved in Hippo-YAP pathway. It is suggested that furin promotes epithelial-mesenchymal transition in pancreatic cancer cells probably via Hippo-YAP pathway and may be a potential target for anti-pancreatic cancer.