MIA PaCa-2 Pancreatic Cancer Cells Research Articles

Abstract PO-108: Evaluation of antitumor activity of modified-gemcitabine solid-lipid nanoparticle in pancreatic pdx models

Abstract Purpose: Pancreatic cancer (PCa) is one of the most aggressive and devastating malignancies in the United States, with a high mortality rate. Despite the remarkable advances in cancer therapy, there are limited treatment options for PCa patients. Gemcitabine (Gem), a nucleoside analog, remains the preferred choice, either alone or in combination with other chemotherapeutic agents, to treat PCa. While Gem has proven to be effective against PCa in the early stages of treatment, PCa cells resistance to Gem and rapid metabolism of Gem has resulted in poor treatment outcomes. The objective of this study was to address the issues associated with Gem via chemical modification of Gem to 4NSG. This significantly reduced the metabolism and highly sensitized PCa cells to Gem. Methods: Gem was modified by linking the 4-amino group of Gem and stearoyl acyl derivative to form 4NSG and characterized using nuclear magnetic resonance (NMR) for) to determine new bond formation, micro-elemental analysis micro-elemental analysis used to ascertain the presence of elemental composition and purity, high liquid chromatography (HPLC) used to determine percent purity of 4NSG. The 4NSG was developed into solid lipid nanoparticles (4NSG-SLN), and the size was determined using a particle size analyzer. Patient-derived primary pancreatic cancer cells (CMZ and G68Ca) and MiaPaCa-2 cells were treated with free Gem and 4NSG-SLN. Percent cell viability was determined using resazurin assay and antitumor efficacy testing of Gem, and 4NSG-SLN was performed in patient-derived xenograft (PDX) mouse models bearing G68Ca PCa. Results: Analysis of the H-NMR spectra displayed an amide bond at 11ppm, confirming the conjugated bond between the 4-amino group of Gem and stearoyl derivative. The half-maximal inhibitory concentration (IC50 =11±1.3µM) of 4NSG-SLN-treated CMZ culture was significantly higher than that of Gem treated CMZ culture (IC50 = 56±2 µM, p<0.001). We found a similar trend of higher growth inhibition of 4NSG-SLN treated G68Ca and MiaPaCa-2 cultures (IC50 (4NSG-SLN-G68Ca) = 12±2.1 µM; IC50(Gem-MiaPaCa-2) = 27±4 µM) respectively. Where p< 0.001 (4NSG-SLN-G68Ca vs. Gem-G68Ca) and p<0.001 (4NSG-SLN-MiaPaCa-2 vs Gem-MiaPaCa-2) compared with Gem treated G68Ca and Mia-PaCa-2 cultures (IC50 (G68Ca) = 68±26 µM; IC50 (Mia-PaCa-2) =54±5 µM) respectively. Put together, the anticancer activity of 4NSG-SLN demonstrated enhanced efficacy in CMZ, G68Ca, and MiaPaCa-2 treated cultures compared with their corresponding Gem treated cultures. For antitumor efficacy testing, 4NSG-SLN demonstrated significant tumor growth inhibition in PDX models harboring G68Ca pancreatic tumors compared with Gem treated with PDX models. Immunohistostaining studies of 4NSG-SLN treated pancreatic tumors significantly reduced vascular endothelial growth factor receptor (VEGFR) expression compared with Gem treated pancreatic tumors. Conclusion: This study demonstrated that 4NSG might be a novel approach to significantly enhance the therapeutic efficacy of Gem in the treatment of PCa. Citation Format: Edward Agyare, Taylor Smith, Andriana Inkoom, Bo Han, Jose Trevino, Nkafu Bechem Ndemazie. Evaluation of antitumor activity of modified-gemcitabine solid-lipid nanoparticle in pancreatic pdx models [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-108.

Nuclear Receptor 4A2 (NR4A2/NURR1) Regulates Autophagy and Chemoresistance in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor prognosis and chemotherapy with gemcitabine has limited effects and is associated with development of drug resistance. Treatment of Panc1 and MiaPaca2 pancreatic cancer cells with gemcitabine induced expression of the orphan nuclear receptor 4A2 (NURR1) and analysis of the cancer genome atlas indicated the NURR1 is overexpressed in pancreatic tumors and is a negative prognostic factor for patient survival. Results of NURR1 knockdown or treatment with the NURR1 antagonist 1,1-bis(3΄-indolyl)-1-(p-chlorophenyl)methane (C-DIM 12) demonstrated that NURR1 was pro-oncogenic in pancreatic cancer cells and regulated cancer cell and tumor growth and survival. NURR1 is induced by gemcitabine and serves as a key drug-resistance factor and is also required for gemcitabine-induced cytoprotective autophagy. NURR1 regulated genes were determined by RNA sequencing of mRNAs expressed in MiaPaCa2 cells expressing NURR1 and in CRISPR/Cas9 gene edited cells for NURR1 knockdown and KEGG enrichment analysis of the differentially expressed genes showed that autophagy was the major pathway regulated by NURR1. Moreover, NURR1 regulated expression of two major autophagic genes ATG7 and ATG12 which are also overexpressed in pancreatic tumors and like NURR1 are negative prognostic factors for patient survival. Thus, gemcitabine-induced cytoprotective autophagy is due to the NURR1 - ATG7/ATG12 axis and this can be targeted and disrupted by NURR1 antagonist C-DIM12 demonstrating the potential clinical applications for combination therapies with gemcitabine and NURR1 antagonists.

Retraction Note to: Linc‑ROR confers gemcitabine resistance to pancreatic cancer cells via inducing autophagy and modulating the miR‑124/PTBP1/PKM2 axis.

In this study, we investigated the regulation of linc-ROR on autophagy and gemcitabine resistance of pancreatic cancer cells and further studied the underlying involvement of the miR-124/PTBP1/PKM2 axis in this regulation. Pancreatic cancer cell lines PANC-1 and MIAPaCa-2 cells were used as in vitro model. Autophagy was assessed by western blot of LC3 I/II and observation GFP-LC3 puncta. Cell viability was examined using CCK-8 assay. Cell apoptosis was examined by flow cytometric analysis of Annexin V/PI staining. QRT-PCR, RNA fluorescence in situ hybridization and dual luciferase assay were used to study the expression and the binding between linc-ROR and miR-124. Linc-ROR siRNA significantly sensitized PANC-1 and MIAPaCa-2 cells to gemcitabine, while linc-ROR overexpression significantly reduced the sensitivity. Linc-ROR knockdown reduced basal autophagy, while linc-ROR overexpression markedly increased basal autophagy in the cells. Linc-ROR siRNA showed similar effect as 3-MA on enhancing gemcitabine-induced cell apoptosis and also reduced PKM2 expression. MiR-124 overexpression restored PKM1 and reduced PKM2 levels in the cells. In addition, miR-124 mimics also alleviated autophagy in pancreatic cancer cells. Both miR-124 mimics and PKM2 siRNA enhanced gemcitabine-induced cell apoptosis. In both pancreatic cell lines and PADC tissues, linc-ROR is negatively correlated with miR-124 expression. In addition, dual luciferase assay verified two 8mer binding sites between miR-124 and linc-ROR. Linc-ROR confers gemcitabine resistance to pancreatic cancer cells at least partly via inducing autophagy. There is a linc-ROR/miR-124/PTBP1/PKM2 axis involved in regulation of gemcitabine resistance in pancreatic cancer cells.

Integrin β1 Promotes Pancreatic Tumor Growth by Upregulating Kindlin-2 and TGF-β Receptor-2

The tumor microenvironment plays a critical role in defining the growth and malignancy of solid tumors. Extracellular matrix (ECM) proteins such as collagen, vitronectin, and fibronectin are major components of the tumor microenvironment. Tumor growth-promoting reciprocal interaction between ECM and cytoplasmic proteins is regulated by the cell surface receptors called integrins. This study investigated the mechanism by which integrin β1 promotes pancreatic tumor growth. In MIA PaCa-2 pancreatic cancer cell line, the loss of integrin β1 protein reduced the ability of cells to proliferate in a 3D matrix and compromised the ability to form a focal adhesion complex. Decreased expression of integrin α5 was observed in KO cells, which resulted in impaired cell spreading and adhesion on vitronectin and fibronectin. Reduced expression of the integrin-associated protein, kindlin-2 was also recorded. The downregulation of kindlin-2 decreased the phosphorylation of Smad2/3 by reducing the expression of TGF-β receptor 2. These results unravel a new mechanism of integrin β1 in tumor growth by modifying the expression of kindlin-2 and TGF-β receptor 2 signaling.

Caveolin‐1 increases glycolysis in pancreatic cancer cells and triggers cachectic states

In pancreatic cancer, autocrine insulin-like growth factor-1 (IGF-1) and paracrine insulin stimulate both IGF-1 receptor (IGF1R) and insulin receptor (IR) to increase tumor growth and glycolysis. In pancreatic cancer patients, cancer-induced glycolysis increases hepatic gluconeogenesis, skeletal muscle proteolysis, and fat lipolysis and, thereby, causes cancer cachexia. As a protein coexisting with IGF1R and IR, caveolin-1 (cav-1) may be involved in pancreatic cancer-induced cachexia. We undertook the present study to test this hypothesis. Out of wild-type MiaPaCa2 and AsPC1 human pancreatic cancer cell lines, we created their stable sub-lines whose cav-1 expression was diminished with RNA interference or increased with transgene expression. When these cells were studied in vitro, we found that cav-1 regulated IGF1R/IR expression and activation and also regulated cellular glycolysis. We transplanted the different types of MiaPaCa2 cells in growing athymic mice for 8 weeks, using intact athymic mice as tumor-free controls. We found that cav-1 levels in tumor grafts were correlated with expression levels of the enzymes that regulated hepatic gluconeogenesis, skeletal muscle proteolysis, and fat lipolysis in the respective tissues. When the tumors had original or increased cav-1, their carriers’ body weight gain was less than the tumor-free reference. When cav-1 was diminished in tumors, the tumor carriers’ body weight gain was not changed significantly, compared to the tumor-free reference. In conclusion, cav-1 in pancreatic cancer cells stimulated IGF1R/IR and glycolysis in the cancer cells and triggered cachectic states in the tumor carrier.

Panax notoginseng Saponins Promote Cell Death and Chemosensitivity in Pancreatic Cancer through the Apoptosis and Autophagy Pathways.

Panax notoginseng Saponins (PNS) is used as a traditional Chinese medicine for ischemic stroke and cardiovascular disease; it has been proven to possess anticancer activity recently. In this study, we aimed to explore the curative anticancer effect and potential mechanisms of PNS in pancreatic cancer cells. Pancreatic cancer Miapaca2 and PANC-1 cells were treated with PNS and Gemcitabine (Gem), respectively. Then the cell viability was assessed by CCK-8 assay, cell proliferation was tested by colony formation assay and EdU cell proliferation assay, cell migration and invasiveness were tested by wound healing assay and transwell assay, respectively, and cell apoptosis was detected by flow cytometry. Finally, we detected the expression levels of proteins related to migration, apoptosis and autophagy through Western blotting. PNS not only inhibited the proliferation, migration, invasion and autophagy of Miapaca2 and PANC-1 cells, but also induced apoptosis and promoted chemosensitivity of pancreatic cancer cells to Gem. PNS may exhibit cytotoxicity and increase chemosensitivity of pancreatic cancer cells to Gem by inhibiting autophagy and inducing apoptosis, providing a new strategy and potential treatment option for pancreatic cancer.

Abstract 3121: Characterization of nab-paclitaxel resistant and gemcitabine resistant BxPC3 and MIA PaCa-2 pancreatic cancer cells

Abstract The gemcitabine (Gem) and nab-paclitaxel (Nab-p) combination is currently the FDA approved standard of care for patients with Stage IV metastatic pancreatic cancer. The response rate of the Gem and Nab-p regimen ranges from 23% to 46%, with many patients eventually becoming refractory. The specific aim of this research project is to determine which genes and pathways differ in Gem resistant (GemR) or Nab-p resistant (Nab-pR) MIA PaCa2 (MP2) and BxPC3 cells when compared with their parental cell lines. Cell proliferation assays were carried out to confirm resistance of the cell lines. The IC50 values of gemcitabine in the Gem resistant BxPC3 (BxPC3-GemR) and MP2 (MP2-GemR) are 26 and 64 fold higher than those in the corresponding parental cell lines, respectively. The IC50 values of nab-paclitaxel in the Nab-p resistant BxPC3 (BxPC3-Nab-pR) and MP2 (MP2-Nab-pR) are 13 and 140 fold higher than those in the corresponding parental cell lines, respectively. RNA sequencing (RNA-seq) was then performed to compare the gene expression profiles of the parental and resistant cell lines. Gene set enrichment analysis (GSEA) revealed that differentially expressed genes between Gem resistant and parental BxPC3 cell lines are often enriched in pathways associated with endoplasmic reticulum (ER) stress response (e.g. ATF-6 and XBP1) whereas in MP2 cells, The AMPK signaling pathway (e.g. PRKAB1, and PRKAG1) seemed to be activated in Gem resistant cells. NFkB signaling pathway (e.g. RIPK2 and NFKB2) was elevated in both Nab-p resistant BxPC3 and MP2 cell lines compared to their corresponding parental cell lines. Genes from these pathways were selected for further validation of their differential expression using RT-PCR. ATF6 which is a stress sensor and transducer in the ER is upregulated in BxPC3-GemR. PRKAB1 and PRKAG1 which regulate AMPK activity are upregulated in MP2-GemR. RIPK2 and NFKB1 are both up in the Nab-p resistant cell lines. Results from this study could provide insights on how to devise new therapeutic regimens to overcome resistance to gemcitabine and nab-paclitaxel. Citation Format: Danielle M. Arias, John Collins, Pawan Noel, Daniel D. Von Hoff, Haiyong Han. Characterization of nab-paclitaxel resistant and gemcitabine resistant BxPC3 and MIA PaCa-2 pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3121.

MCT4 as a potential therapeutic target to augment gemcitabine chemosensitivity in resected pancreatic cancer

Pancreatic cancer is a devastating disease with a high relapse rate, even in resectable pancreatic cancer. This study aimed to identify the prognostic significance and therapeutic chance of the metabolic subtypes for resectable pancreatic cancer. We obtained transcriptomic data from the TCGA-PAAD cohort via the The Cancer Genome Atlas (TCGA) data portal (n = 182). After integrative analysis of transcriptomic data in the discovery cohort, immunohistochemical (IHC) staining was performed in the independent cohort to validate the molecule of interest. Experimental testing for the molecule of interest was performed using pancreatic cancer cell lines, including AsPC1, BxPC3, MIA PaCa-2, and PANC-1 in vitro. Two subtypes showing distinct gene expression patterns in the TCGA-PAAD dataset were identified. The active glucose metabolism subtype showed significantly lower survival regarding cancer relapse after surgical resection. The genes SLC2A1 (GLUT1) and SLC16A3 (MCT4) were highly enriched in the active metabolism subtype. The validation cohort showed high IHC staining intensity for MCT4 and a significantly high relapse rate (p = 0.01). Several molecular pathways associated with aggressive tumor biology regarding cell cycle, Myc, and mTOR downstream signaling were highly enriched in the high glucose uptake subtype as well as distinct response for immunotherapy. MCT4 inhibition suppressed pancreatic cancer cell lines in vitro and showed a synergetic effect with gemcitabine treatment. MCT4 was identified from integrative analysis as a potential therapeutic target in resectable pancreatic cancer. The precision strategy for resectable pancreatic cancer should be validated in a clinical setting with a prospective study design.

TGF-β1 Facilitates TAp63α Protein Lysosomal Degradation to Promote Pancreatic Cancer Cell Migration.

Simple SummaryNumerous studies demonstrate that the activation of transforming growth factor-β (TGF-β) signaling is a critical driving force for promoting cancer cell migration and tumor metastasis. Our recent study indicates that TGF-β1 promotes FBXO3-mediated ΔNp63α protein degradation to facilitate cancer metastasis. In this study, we show that TGF-β1 can inhibit TAp63α protein stability in a lysosome-dependent, but canonical Smad pathway-independent manner, which leads to upregulation of p53-R248W expression, and consequently results in increased pancreatic cancer cell migration.TGF-β signaling plays a pivotal role in promoting tumor cell migration and cancer metastasis. ΔNp63α and TAp63α are two major isoforms of p53-related p63 protein. Our recent study has shown that TGF-β1 promotes ΔNp63α protein degradation to facilitate cancer metastasis. However, whether TAp63α is involved in TGF-β1-induced cancer metastasis remains unclear. In this study, we show that, in human pancreatic cancer MIA PaCa-2 cells harboring p53-R248W allele, TGF-β1 can significantly inhibit TAp63α protein stability in a Smad pathway-independent manner. Lysosome inhibitor, chloroquine, but not proteasome inhibitor MG132, can rescue TGF-β1-induced downregulation of TAp63α protein. In addition, we show that either TGF-β1 treatment or silencing of TAp63α can dramatically increase migration of MIA PaCa-2 cells. Importantly, the restored expression of TAp63α can effectively block TGF-β1-induced migration of MIA PaCa-2 cells. Mechanistically, we show that TGF-β1 promotes TAp63α protein degradation, leading to upregulation of p53-R248W protein expression, and consequently resulting in elevated MIA PaCa-2 cell migration. Together, this study indicates that lysosomal degradation is an important way for regulating TAp63α protein fate and highlights that TGF-β1-TAp63α-mutant p53 axis is critically important in pancreatic cancer metastasis.

Gemcitabine-Based Chemoradiotherapy Enhanced by a PARP Inhibitor in Pancreatic Cancer Cell Lines.

Pancreatic ductal adenocarcinoma is a devastating disease with a 5-year overall survival of 9% for all stages. Gemcitabine-based chemoradiotherapy for locally advanced pancreatic cancer is highly toxic. We conducted an in vitro study to determine whether poly(ADP-ribose) polymerase-1 inhibition radiosensitized gemcitabine-based chemotherapy. Human pancreatic cancer cell lines, MIA PaCa-2, AsPC-1, BxPC-3 and PANC-1 were treated with gemcitabine (10 nM) and/or olaparib (1 µM). Low-LET gamma single dose of 2, 5 and 10 Gy radiations were carried out. Clonogenic assay, PAR immunoblotting, cell cycle distribution, γH2Ax, necrotic and autophagic cell death quantifications were performed. Treatment with olaparib alone was not cytotoxic, but highly radiosensitized cell lines, particularly at high dose per fraction A non-cytotoxic concentration of gemcitabine radiosensitized cells, but less than olaparib. Interestingly, olaparib significantly enhanced gemcitabine-based radiosensitization in PDAC cell lines with synergistic effect in BxPC-3 cell line. All cell lines were radiosensitized by the combination of gemcitabine and olaparib, through an increase of unrepaired double-strand, a G2 phase block and cell death. Radiosensitization was increased with high dose of radiation. The combination of olaparib with gemcitabine-based chemoradiotherapy could lead to an enhancement of local control in vivo and an improvement in disease-free survival.

Impact of DNA repair and reactive oxygen species levels on radioresistance in pancreatic cancer

PurposeRadioresistance in pancreatic cancer patients remains a critical obstacle to overcome. Understanding the molecular mechanisms underlying radioresistance may achieve better response to radiotherapy and thereby improving the poor treatment outcome. The aim of the present study was to elucidate the mechanisms leading to radioresistance by detailed characterization of isogenic radioresistant and radiosensitive cell lines. MethodsThe human pancreatic cancer cell lines, Panc-1 and MIA PaCa-2 were repeatedly exposed to radiation to generate radioresistant (RR) isogenic cell lines. The surviving cells were expanded, and their radiosensitivity was measured using colony formation assay. Tumor growth delay after irradiation was determined in a mouse pancreatic cancer xenograft model. Gene and protein expression were analyzed using RNA sequencing and Western blot, respectively. Cell cycle distribution and apoptosis (Caspase 3/7) were measured by FACS analysis. Reactive oxygen species generation and DNA damage were analyzed by detection of CM-H2DCFDA and γH2AX staining, respectively. Transwell chamber assays were used to investigate cell migration and invasion. ResultsThe acquired radioresistance of RR cell lines was demonstrated in vitro and validated in vivo. Ingenuity pathway analysis of RNA sequencing data predicted activation of cell viability in both RR cell lines. RR cancer cell lines demonstrated greater DNA repair efficiency and lower basal and radiation-induced reactive oxygen species levels. Migration and invasion were differentially affected in RR cell lines. ConclusionsOur data indicate that repeated exposure to irradiation increases the expression of genes involved in cell viability and thereby leads to radioresistance. Mechanistically, increased DNA repair capacity and reduced oxidative stress might contribute to the radioresistant phenotype.

High-Fat Diet Drives an Aggressive Pancreatic Cancer Phenotype

Emerging evidence indicates associations between high-fat diet (HFD), metabolic syndrome (MetS), and increased risk of pancreatic cancer. However, individual components of an HFD that increase cancer risk have not been isolated. In addition, a specific pattern of cytokine elevation by which MetS drives pancreatic tumor progression is not well described. We hypothesized that oleic acid (OA), a major component of HFD, would augment pancreatic neoplastic processes. An orthotopic pancreatic cancer model with Panc02cells was used to compare the effect of low-fat diet to OA-based HFD on cancer progression. Tumors were quantitated, analyzed by immunohistochemistry. In addition, serum cytokine levels were quantitated. Proliferation, migration assays, and expression of epithelial-to-mesenchymal transition factors were evaluated on Panc02 and MiaPaCa-2 pancreatic cancer cells cultured in high concentrations of OA. HFD tumor-bearing mice (n=8) had an 18% weight increase (P<0.001) and increased tumor burden (P<0.05) compared with the low-fat diet tumor-bearing group (n=6). HFD tumors had significantly increased angiogenesis (P<0.001) and decreased apoptosis (P<0.05). Serum of HFD mice demonstrated increased levels of glucagon and glucagon-like peptide-1. Two pancreatic cancer cell lines cultured in OA demonstrated significant increases in proliferation (P<0.001) and a >2.5-fold increase in cell migration (P<0.001) when treated with OA. Panc02 treated with OA had increased expression of epithelial-to-mesenchymal transition factors SNAI-1 (Snail) and Zeb-1(P<0.01). High-fat conditions invitro and invivo resulted in an aggressive pancreatic cancer phenotype. Our data support further investigations elucidating molecular pathways augmented by MetS conditions to identify novel therapeutic strategies for pancreatic cancer.

A Combined Activity of Thrombin and P-Selectin Is Essential for Platelet Activation by Pancreatic Cancer Cells.

Pancreatic cancer patients have an elevated risk of suffering from venous thrombosis. Among several risk factors that contribute to hypercoagulability of this malignancy, platelets possess a key role in the initiation of clot formation. Although single mechanisms of platelet activation are well-known in principle, combinations thereof and their potential synergy to mediate platelet activation is, in the case of pancreatic cancer, far from being clear. Applying an inhibitor screening approach using light transmission aggregometry, dense granule release, and thrombin formation assays, we provide evidence that a combination of tissue factor-induced thrombin formation by cancer cells and their platelet P-selectin binding is responsible for AsPC-1 and Capan-2 pancreatic cancer cell-mediated platelet activation. While the blockade of one of these pathways leads to a pronounced inhibition of platelet aggregation and dense granule release, the simultaneous blockade of both pathways is inevitable to prevent platelet aggregation completely and minimize ATP release. In contrast, MIA PaCa-2 pancreatic cancer cells express reduced levels of tissue factor and P-selectin ligands and thus turn out to be poor platelet activators. Consequently, a simultaneous blockade of thrombin and P-selectin binding seems to be a powerful approach, as mediated by heparin to crucially reduce the hypercoagulable state of pancreatic cancer patients.

AMPK Is the Crucial Target for the CDK4/6 Inhibitors Mediated Therapeutic Responses in PANC-1 and MIA PaCa-2 Pancreatic Cancer Cell Lines

The survival rate of pancreatic ductal adenocarcinoma (PDAC) patients is short, and PDAC is a cancer type that ranks fourth in the statistics regarding death due to cancer. Mutation in the KRAS gene, which plays a role in pancreatic cancer development, activates the PI3K/AKT/mTOR signaling pathway. The activity of the AMPK as a cellular energy sensor is one of the fundamental mechanisms that can induce effective therapeutic responses against CDK4/6 inhibitors via adjusting the cellular and tumor microenvironment stress management. The phosphorylation of AMPKα at the different phosphorylation residues such as Thr172 and Ser 377 causes metabolic differentiation in the cells following CDK4/6 inhibitor treatment in accordance with an increased cell cycle arrest and senescence under the control of different cellular players. In this study, we examined the competencies of the CDK4/6 inhibitors LY2835219 and PD-0332991 on the mechanism of cell survival and death based on AMPK signaling. Both CDK4/6 inhibitors LY2835219 and PD-0332991 modulated different molecular players on the PI3K/AKT/mTOR and AMPK signaling axis in different ways to reduce cell survival in a cell type dependent manner. These drugs are potential inducers of apoptosis and senescence that can alter the therapeutic efficacy cells.

SWATH-MS proteomics of PANC-1 and MIA PaCa-2 pancreatic cancer cells allows identification of drug targets alternative to MEK and PI3K inhibition

Pancreatic cancer remains one of the most lethal diseases with dismal five-year survival rates. Although mutant KRas protein-driven activation of downstream MAPK Raf/MEK/ERK and PI3K/Akt signaling pathways represent major oncogenic alterations, signaling blockade with MEK and PI3K inhibitors has shown that intrinsic resistance may hamper the effectiveness of this targeted approach. However, there have been no mass spectrometry-based proteomic studies for in-depth comparison of protein expression differences between pancreatic cancer cells with sensitivity and resistance to MEK and PI3K kinase inhibitors. In this work, we compared PANC-1 and MIA PaCa-2 pancreatic cancer cells which are, respectively, resistant and sensitive to MEK- and PI3K-targeted therapy. We conducted a label-free data-independent acquisition mass spectrometry (SWATH-MS) study with extensive peptide fractionation to quantitate 4808 proteins and analyze differential expression of 743 proteins between resistant and sensitive cells. This allowed identification of the tumor suppressor protein phosphatase 2A (PP2A) and proteins from mitochondrial respiratory complex I implicated in oxidative phosphorylation as alternative candidate drug targets for cells resistant to MEK and PI3K inhibition. PP2A activator DT-061 decreased viability of PANC-1 cells and this was accompanied by reduced expression of c-Myc. PANC-1 cells also showed response to metformin and the novel complex I inhibitor IACS-010759. These findings provide insights into the distinct cellular proteomes and point out alternative pharmacological targets for MEK and PI3K inhibition-resistant pancreatic cancer cells.

Predicting the bioactivity of 2-alkoxycarbonylallyl esters as potential antiproliferative agents against pancreatic cancer (MiaPaCa-2) cell lines: GFA-based QSAR and ELM-based models with molecular docking

BackgroundThe number of cancer-related deaths is on the increase, combating this deadly disease has proved difficult owing to resistance and some serious side effects associated with drugs used to combat it. Therefore, scientists continue to probe into the mechanism of action of cancer cells and designing novel drugs that could combat this disease more safely and effectively. Here, we developed a genetic function approximation model to predict the bioactivity of some 2-alkoxyecarbonyl esters and probed into the mode of interaction of these molecules with an epidermal growth factor receptor (3POZ) using the three-dimensional quantitative structure activity relationship (QSAR), extreme learning machine (ELM), and molecular docking techniques. ResultsThe developed QSAR model with predicted (R2pred) of 0.756 showed that the model was fit to be validated parameter for a built model and also proved that the developed model could be used in practical situation, R2 for training set (0.9929) and test set (0.8397) confirmed that the model could successfully predict the activity of new compounds due to its correlation with the experimental activity, the models generated with ELM models showed improved prediction of the activity of the molecules. The lead compounds (22 and 23) had binding energies of −6.327 and −7.232 kcalmol−1 for 22 and 23 respectively and displayed better inhibition at the binding sites of 3POZ when compared with that of the standard drug, chlorambucil (−6.0 kcalmol−1). This could be attributed to the presence of double bonds and the α-ester groups. ConclusionThe QSAR and ELM models had good prognostic ability and could be used to predict the bioactivity of novel anti-proliferative drugs.

Protopine/Gemcitabine Combination Induces Cytotoxic or Cytoprotective Effects in Cell Type-Specific and Dose-Dependent Manner on Human Cancer and Normal Cells.

The natural alkaloid protopine (PRO) exhibits pharmacological properties including anticancer activity. We investigated the effects of PRO, alone and in combination with the chemotherapeutic gemcitabine (GEM), on human tumor cell lines and non-tumor human dermal fibroblasts (HDFs). We found that treatments with different PRO/GEM combinations were cytotoxic or cytoprotective, depending on concentration and cell type. PRO/GEM decreased viability in pancreatic cancer MIA PaCa-2 and PANC-1 cells, while it rescued the GEM-induced viability decline in HDFs and in tumor MCF-7 cells. Moreover, PRO/GEM decreased G1, S and G2/M phases, concomitantly with an increase of subG1 phase in MIA PaCa-2 and PANC-1 cells. Differently, PRO/GEM restored the normal progression of the cell cycle, altered by GEM, and decreased cell death in HDFs. PRO alone increased mitochondrial reactive oxygen species (ROS) in MIA PaCa-2, PANC-1 cells and HDFs, while PRO/GEM increased both intracellular and mitochondrial ROS in the three cell lines. These results indicate that specific combinations of PRO/GEM may be used to induce cytotoxic effects in pancreatic tumor MIA PaCa-2 and PANC-1 cells, but have cytoprotective or no effects in HDFs.

Determining the effect of ellagic acid on the proliferation and migration of pancreatic cancer cell lines.

BackgroundEllagic acid is a natural dietary compound found in several berries and fruits. It inhibits fibrosis, inflammation and carcinogenesis. We tried to find out what role ellagic acid plays in pancreatic cancer to see if it can be used as an adjuvant for chemotherapy.MethodsThree pancreatic cancer cell lines, PANC-1, AsPC-1, and MIA PaCA-2, were used. Cell growth was measured by MTT assay. Activities of caspase-3 and caspase-8, and caspase-9 were evaluated to determine the apoptosis pathway. Epithelial to mesenchymal transition was identified through mRNA expression of associated genes, which were transforming growth factor-beta, matrix metalloproteinase-2 and matrix metalloproteinase-9. Quantitative real time PCR was used to verify mRNA expression. To determine the effect on migration, transwell system was used.ResultsEllagic acid inhibits pancreatic cancer cell growth by stimulating apoptosis. Apoptosis induced by ellagic acid in pancreatic cancer cells, is mediated by the activation of caspase-3 and caspase-9, and not caspase-8. Ellagic acid also suppresses migration via the inhibition of epithelial mesenchymal transition (EMT) in pancreatic cancer cells. Ellagic acid decreased the expression levels of transforming growth factor-beta, matrix metalloproteinase-2 and matrix metalloproteinase-9, while it increased that of E-cadherin.ConclusionsThese findings suggest that ellagic acid is useful in pancreatic cancer treatment.

Evaluating the in vitro therapeutic effects of human amniotic mesenchymal stromal cells on MiaPaca2 pancreatic cancer cells using 2D and 3D cell culture model

Human amniotic mesenchymal stromal cells (hAMSCs) are considered as a population of multipotent cells. The molecular events associated with mesenchymal stromal cell (MSC)/tumor cell interactions should be studied to identify the role of MSCs on suppressing or inducing the key signaling pathways of tumor cells. Thus, designing therapeutic approaches is considered as important. In the present study, hAMSCs and MiaPaca2 cells were first cultured separately. In addition, both cell lines were co-cultured by using 0.4 μm pore sized transwell membranes in different times. Further, the RNA of the cells was extracted, and Bcl2, Bax, epidermal growth factor receptor (EGFR), c-Src, C-terminal Src Kinase (CSK), and SGK223 expression were analyzed through quantitative real time PCR. Furthermore, the total cell lysates of the cells were prepared and analyzed by using western blot. Based on the results, the expression of EGFR, c-Src, SGK223, and CSK in MiaPaca2 cells reduced after treating with hAMSCs. Notably, the cellular apoptosis of MiaPaca2 cells was induced in 2D cell culture system. Further, the anti-cancer activity of conditioned medium from hAMSCs was confirmed in a 3D cell culture model by using hanging drop technique. Finally, hAMSCs have inhibitory effects on pancreatic cancer cells and can be considered as a therapeutic way to suppress EGFR, c-Src, and SGK223, as the potent targets in cancer cell signaling.

Annexin A1 Released in Extracellular Vesicles by Pancreatic Cancer Cells Activates Components of the Tumor Microenvironment, through Interaction with the Formyl-Peptide Receptors.

Pancreatic cancer (PC) is one of the most aggressive cancers in the world. Several extracellular factors are involved in its development and metastasis to distant organs. In PC, the protein Annexin A1 (ANXA1) appears to be overexpressed and may be identified as an oncogenic factor, also because it is a component in tumor-deriving extracellular vesicles (EVs). Indeed, these microvesicles are known to nourish the tumor microenvironment. Once we evaluated the autocrine role of ANXA1-containing EVs on PC MIA PaCa-2 cells and their pro-angiogenic action, we investigated the ANXA1 paracrine effect on stromal cells like fibroblasts and endothelial ones. Concerning the analysis of fibroblasts, cell migration/invasion, cytoskeleton remodeling, and the different expression of specific protein markers, all features of the cell switching into myofibroblasts, were assessed after administration of wild type more than ANXA1 Knock-Out EVs. Interestingly, we demonstrated a mechanism by which the ANXA1-EVs complex can stimulate the activation of formyl peptide receptors (FPRs), triggering mesenchymal switches and cell motility on both fibroblasts and endothelial cells. Therefore, we highlighted the importance of ANXA1/EVs-FPR axes in PC progression as a vehicle of intercommunication tumor cells-stroma, suggesting a specific potential prognostic/diagnostic role of ANXA1, whether in soluble form or even if EVs are captured in PC.