Impact of LMWH and Specific Factor Xa Inhibitors, Apixaban and Fondaparinux, on Cancer Cell Biology and Procoagulant Properties of Cancer Microenvironment

Abstract Background Cancer patients with venous thromboembolism (VTE) or at risk of VTE are treated with antithrombotic agents. Cancer cells express procoagulant properties and induce hypercoagulability in the microenvironment, that could impact the efficiency of the antithrombotic agents. Aims In the present study, we investigated the interaction between antithrombotic agents with pancreatic cancer cells, as well as with their microenvironment. The impact of apixaban, fondaparinux, enoxaparin and tinzaparin on the procoagulant properties of pancreatic cancer cells BXPC3 was examinated. Reciprocally, we also investigated the impact of BXPC3 on the potency of these antithrombotic agents. Methods BXPC3 cells (400 cells/μl) were exposed for 48 hours to apixaban (2 µg/ml), fondaparinux (2 µg/ml), enoxaparin, tinzaparin (2 anti-Xa IU/ml) or NaCL (control). Then, conditioned media (CM) and BXPC3 cells were harvested, separated and put in contact with normal platelet-poor plasma (PPP). Subsequently, thrombin generation (TG) was assessed using Thrombogram-Thrombinoscope® assay (Diagnostica Stago). Cells' viability was also assessed with the MTT assay. Gene expression for Tissue Factor (TF), Vascular Endothelial Growth Factor (VEGF), Thrombospondin 1 (THSB1) was assessed with RT-qPCR at the cells exposed or not to the antithrombotic agents. Expression of TF protein and activity of cancer cells was assessed using ELISA method. Residual anti-Xa activity in CM was measured using specific amidolytic assays for each antithrombotic agent. Results Apixaban, fondaparinux, enoxaparin, and tinzaparin significantly reduced cell viability by 25%, 12%, 14%, and 11% respectively. In the control experiment non treated BXPC3 cells enhanced TG. Pre-treatment of BXPC3 with the antithrombotic agents did not significantly modify their capacity to trigger and enhance TG. Among the studied agents only apixaban resulted in significant decrease of TF mRNA expression. However, protein expression of TF was not significantly modified by any of the antithrombotic agents. VEGF's mRNA expression was significantly decreased by fondaparinux and enoxaparin. THBS1's mRNA expression was significantly increased by apixaban. The concentrations of the anti-Xa activity of fondaparinux, enoxaparin and tinzaparin in the CM obtained at 48h after exposure of cells were reduced by 27%, 48% and 26% respectively as compared to those initially added in the culture medium. In contrast, the concentration of apixaban in the CM did not significantly change. The CM obtained by cells exposed to apixaban, fondaparinux, enoxaparin and tinzaparin inhibited TG by 70%, 30%, 40% and 90% respectively. Conclusion. Antithrombotic agents reduced the viability of BXPC3 cells. Among the studied agents, apixaban had the most pronounced effect on cells' viability. The antithrombotic agents had a potential downregulating effect on the proangiogenetic properties of BXPC3 via the decrease of VEGF gene expression (fondaparinux and enoxaparin) and enhancement of THBS1 gene expression (apixaban). Nevertheless, preincubation of BXPC3 with the antithrombotic agents did not alter the expression of TF protein and their effect on thrombin generation. Moreover, BXPCE exerted a "degradation" effect on LMWH and fondaparinux. Apixaban appeared to escape from this effect of the cancer cells. A significant inhibitory effect on thrombin generation was exerted by the residual concentrations of the antithrombotic agents in the microenvironment of cancer cells. The ensemble of these data highlight for the first time that the presence of antithrombotic agents in cancer cell microenvironment alters the biology of cancer cells and offer a constant antithrombotic effect in the microenvironment. Disclosures No relevant conflicts of interest to declare.

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Differential Gemcitabine Sensitivity in Primary Human Pancreatic Cancer Cells and Paired Stellate Cells Is Driven by Heterogenous Drug Uptake and Processing

Cancers ◽

10.3390/cancers12123628 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3628

Author(s):

Manoj Amrutkar ◽

Nils Tore Vethe ◽

Caroline S. Verbeke ◽

Monica Aasrum ◽

Anette Vefferstad Finstadsveen ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Cell ◽

Stellate Cells ◽

Drug Uptake ◽

Pancreatic Stellate Cells ◽

Human Pancreatic Cancer ◽

Pancreatic Cancer Cells ◽

The Impact

Gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC) is attributed to cancer cell-intrinsic drug processing and the impact of the tumor microenvironment, especially pancreatic stellate cells (PSCs). This study uses human PDAC-derived paired primary cancer cells (PCCs) and PSCs from four different tumors, and the PDAC cell lines BxPC-3, Mia PaCa-2, and Panc-1, to assess the fate of gemcitabine by measuring its cellular uptake, cytotoxicity, and LC-MS/MS-based metabolite analysis. Expression analysis and siRNA-mediated knockdown of key regulators of gemcitabine (hENT1, CDA, DCK, NT5C1A) was performed. Compared to PSCs, both the paired primary PCCs and cancer cell lines showed gemcitabine-induced dose-dependent cytotoxicity, high uptake, as well as high and variable intracellular levels of gemcitabine metabolites. PSCs were gemcitabine-resistant and demonstrated significantly lower drug uptake, which was not influenced by co-culturing with their paired PCCs. Expression of key gemcitabine regulators was variable, but overall strong in the cancer cells and significantly lower or undetectable in PSCs. In cancer cells, hENT1 inhibition significantly downregulated gemcitabine uptake and cytotoxicity, whereas DCK knockdown reduced cytotoxicity. In conclusion, heterogeneity in gemcitabine processing among different pancreatic cancer cells and stellate cells results from the differential expression of molecular regulators which determines the effect of gemcitabine.

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TGF-βRII knock-down promotes tumor growth and chemoresistance to gemcitabine of pancreatic cancer cells via phosphorylation of STAT3

10.1101/352963 ◽

2018 ◽

Author(s):

Vincent Drubay ◽

Nicolas Skrypek ◽

Lucie Cordiez ◽

Romain Vasseur ◽

Céline Schulz ◽

...

Keyword(s):

Pancreatic Cancer ◽

Tumor Growth ◽

Cancer Cells ◽

Cancer Cell ◽

Pancreatic Cancer Cell ◽

Locally Advanced ◽

Pancreatic Cancer Cells ◽

The Impact

AbstractPancreatic adenocarcinoma (PDAC) is one of the most deadly cancers in the western countries because of a lack of early diagnostic markers and efficient therapeutics. At the time of diagnosis, more than 80% of patients have metastasis or locally advanced cancer and are therefore not eligible for surgical resection. Pancreatic cancer cell also harbour a high resistance to chemotherapeutic drugs such as gemcitabine that is one of the main palliative treatment for PDAC.TGF-β possesses both tumor-suppressive and oncogenic activities in pancreatic cancer. TGF-β signalling pathway plays complex role during carcinogenesis by initially inhibiting epithelial growth and later promoting the progression of advanced tumors and thus emerged as tumor suppressor pathway. TGF-β binds to its receptor TGF-βRII and activates different pathways: canonical pathway involving the Smad proteins and alternative pathways such as MAPKs. Smad4 is mutated in 50-80% of PDAC. Mutations of TGF-βRII also occurs (5-10%). In order to decipher the role of TGF-β in carcinogenesis and chemoresistance, we decided to characterize the knocking down of TGF-βRII that is the first actor of TGF-β signalling. We developed pancreatic cancer cell lines stably invalidated for TGF-βRII and studied the impact on biological properties of pancreatic cancer cells both in vitro and in vivo. We show that TGF-βRII silencing alters tumor growth and migration as well as resistance to. TGF-βRII silencing also leads to S727 STAT3 and S-63 c-Jun phosphorylation, decrease of MRP3 and increase of MRP4 ABC transporter expression and induction of a partial EMT phenotype.In the future, the better understanding TGF-β signaling pathways and underlying cellular mechanisms in chemoresistance to gemcitabine may bring new therapeutic tools to clinicians.

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DNAJA1 dysregulates metabolism promoting an anti-apoptotic phenotype in pancreatic ductal adenocarcinoma

10.21203/rs.3.rs-76631/v1 ◽

2020 ◽

Author(s):

Heidi Roth ◽

Fatema Bhinderwala ◽

Rodrigo Franco ◽

You Zhou ◽

Robert Powers

Keyword(s):

Pancreatic Cancer ◽

Pancreatic Ductal Adenocarcinoma ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Cell ◽

Pancreatic Cancer Cell ◽

Cancer Cell Lines ◽

Ductal Adenocarcinoma ◽

Pancreatic Cancer Cells ◽

The Impact

Abstract BackgroundAt less than 7%, pancreatic ductal adenocarcinoma (PDAC) has one of the poorest 5-year cancer survival rates and is set to be the leading cause of cancer related deaths by 2030. The co-chaperone protein DNAJA1 (HSP40) is downregulated four-fold in pancreatic cancer cells, but its impact on pancreatic ductal adenocarcinoma (PDAC) progression remains unclear.MethodsDNAJA1 was overexpressed in pancreatic cancer cell lines, BxPC-3 and MIA PaCa-2, through retroviral transfection. The impact of overexpressing DNAJA1 was investigated using a combination of untargeted metabolomics, stable isotope resolved metabolomics (SIRM), confocal microscopy, flow-cytometry, and cell-based assays.ResultsPancreatic cancer cells overexpressing DNAJA1 exhibited a global metabolomic change. Specifically, differential output from Warburg glycolysis, an increase in redox currency, and an alteration in amino acid levels were observed in both overexpression cell lines. DNAJA1 overexpression also led to mitochondrial fusion, an increase in the expression of Bcl-2, a modest protection from redox induced cell death, a loss of structural integrity due to the loss of actin fibers, and an increase in cell invasiveness in BxPC-3. These differences were more pronounced in BxPC-3, which contains a loss-of-function mutation in the tumor suppressing gene SMAD4.ConclusionsThe overexpression of DNAJA1 promoted cellular proliferation, redox tolerance, invasiveness, and anti-apoptosis, which suggests DNAJA1 has numerous regulatory roles. Overall, our findings suggest a proto-oncogenic role of DNAJA1 in PDAC progression and suggests DNAJA1 may function synergistically with other proteins with altered activity in pancreatic cancer cell lines.

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The impact of pancreatic cancer cell exosomes on the phenotype of pancreatic cancer cells

Pancreatology ◽

10.1016/j.pan.2017.05.071 ◽

2017 ◽

Vol 17 (3) ◽

pp. S22-S23

Author(s):

Stuart Robinson ◽

Derek Mann ◽

Richard Charnley ◽

Jelena Mann ◽

Steve White

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

Cancer Cell ◽

Pancreatic Cancer Cell ◽

Pancreatic Cancer Cells ◽

The Impact

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Effect of Low Molecular Weight Heparins and Fondaparinux Upon Thrombin Generation Triggered by Human Pancreatic Cancer Cells BXPC3

Current Vascular Pharmacology ◽

10.2174/157016111206141210121441 ◽

2014 ◽

Vol 12 (6) ◽

pp. 893-902 ◽

Cited By ~ 5

Author(s):

Grigoris Gerotziafas ◽

Vassiliki Galea ◽

Elisabeth Mbemba ◽

Mouna Sassi ◽

Marie-Paule Roman ◽

...

Keyword(s):

Pancreatic Cancer ◽

Molecular Weight ◽

Cancer Cells ◽

Thrombin Generation ◽

Human Pancreatic Cancer ◽

Low Molecular Weight ◽

Low Molecular Weight Heparins ◽

Pancreatic Cancer Cells

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Galectin-3 is modulated in pancreatic cancer cells under hypoxia and nutrient deprivation

Biological Chemistry ◽

10.1515/hsz-2019-0413 ◽

2020 ◽

Vol 401 (10) ◽

pp. 1153-1165 ◽

Cited By ~ 2

Author(s):

Antônio F. da Silva Filho ◽

Lucas B. Tavares ◽

Maira G. R. Pitta ◽

Eduardo I. C. Beltrão ◽

Moacyr J. B. M. Rêgo

Keyword(s):

Pancreatic Cancer ◽

Cell Death ◽

Mrna Expression ◽

Cancer Cells ◽

Ductal Adenocarcinoma ◽

Reverse Transcription Pcr ◽

Pancreatic Cancer Cells ◽

Through Flow ◽

Galectin 3

AbstractPancreatic ductal adenocarcinoma is one of the most aggressive tumors with a microenvironment marked by hypoxia and starvation. Galectin-3 has been evaluated in solid tumors and seems to present both pro/anti-tumor effects. So, this study aims to characterize the expression of Galectin-3 from pancreatic tumor cells and analyze its influence for cell survive and motility in mimetic microenvironment. For this, cell cycle and cell death were accessed through flow cytometry. Characterization of inside and outside Galectin-3 was performed through Real-Time Quantitative Reverse Transcription PCR (qRT-PCR), immunofluorescence, Western blot, and ELISA. Consequences of Galectin-3 extracellular inhibition were investigated using cell death and scratch assays. PANC-1 showed increased Galectin-3 mRNA expression when cultivated in hypoxia for 24 and 48 h. After 24 h in simultaneously hypoxic/deprived incubation, PANC-1 shows increased Galectin-3 protein and secreted levels. For Mia PaCa-2, cultivation in deprivation was determinant for the increasing in Galectin-3 mRNA expression. When cultivated in simultaneously hypoxic/deprived condition, Mia PaCa-2 also presented increasing for the Galectin-3 secreted levels. Treatment of PANC-1 cells with lactose increased the death rate when cells were incubated simultaneously hypoxic/deprived condition. Therefore, it is possible to conclude that the microenvironmental conditions modulate the Galectin-3 expression on the transcriptional and translational levels for pancreatic cancer cells.

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Mesothelin blockage by Amatuximab suppresses cell invasiveness, enhances gemcitabine sensitivity and regulates cancer cell stemness in mesothelin-positive pancreatic cancer cells

BMC Cancer ◽

10.1186/s12885-020-07722-3 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Fumihiko Matsuzawa ◽

Hirofumi Kamachi ◽

Tatsuzo Mizukami ◽

Takahiro Einama ◽

Futoshi Kawamata ◽

...

Keyword(s):

Pancreatic Cancer ◽

Mouse Model ◽

Cancer Cells ◽

Cancer Cell ◽

Morphological Changes ◽

Pancreatic Cancer Cells ◽

Cell Invasiveness ◽

And Migration ◽

Migration Capacity

Abstract Background Mesothelin is a 40-kDa glycoprotein that is highly overexpressed in various types of cancers, however molecular mechanism of mesothelin has not been well-known. Amatuximab is a chimeric monoclonal IgG1/k antibody targeting mesothelin. We recently demonstrated that the combine therapy of Amatuximab and gemcitabine was effective for peritonitis of pancreatic cancer in mouse model. Methods We discover the role and potential mechanism of mesothelin blockage by Amatuximab in human pancreatic cells both expressing high or low level of mesothelin in vitro experiment and peritonitis mouse model of pancreatic cancer. Results Mesothelin blockage by Amatuximab lead to suppression of invasiveness and migration capacity in AsPC-1 and Capan-2 (high mesothelin expression) and reduce levels of pMET expression. The combination of Amatuximab and gemcitabine suppressed proliferation of AsPC-1 and Capan-2 more strongly than gemcitabine alone. These phenomena were not observed in Panc-1 and MIA Paca-2 (Mesothelin low expression). We previously demonstrated that Amatuximab reduced the peritoneal mass in mouse AsPC-1 peritonitis model and induced sherbet-like cancer cell aggregates, which were vanished by gemcitabine. In this study, we showed that the cancer stem cell related molecule such as ALDH1, CD44, c-MET, as well as proliferation related molecules, were suppressed in sherbet-like aggregates, but once sherbet-like aggregates attached to peritoneum, they expressed these molecules strongly without the morphological changes. Conclusions Our work suggested that Amatuximab inhibits the adhesion of cancer cells to peritoneum and suppresses the stemness and viability of those, that lead to enhance the sensitivity for gemcitabine.

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610 POSTER Synergistic cytotoxicity, inhibition of Akt and c-Kit phosphorylation and modulation of gene expression by sorafenib and gemcitabine in human pancreatic cancer cells

European Journal of Cancer Supplements ◽

10.1016/s1359-6349(06)70615-2 ◽

2006 ◽

Vol 4 (12) ◽

pp. 184

Author(s):

R. Danesi ◽

E. Giovannetti ◽

S. Nannizzi ◽

V. Mey ◽

S. Ricciardi ◽

...

Keyword(s):

Gene Expression ◽

Pancreatic Cancer ◽

Cancer Cells ◽

Human Pancreatic Cancer ◽

Pancreatic Cancer Cells ◽

Synergistic Cytotoxicity ◽

Modulation Of Gene Expression

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Differential mucin gene expression in human pancreatic and colon cancer cells

Biochemical Journal ◽

10.1042/bj2760599 ◽

1991 ◽

Vol 276 (3) ◽

pp. 599-605 ◽

Cited By ~ 42

Author(s):

S Yonezawa ◽

J C Byrd ◽

R Dahiya ◽

J J L Ho ◽

J R Gum ◽

...

Keyword(s):

Pancreatic Cancer ◽

Colon Cancer ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Cell ◽

Cancer Cell Lines ◽

Human Pancreatic Cancer ◽

Northern Blots ◽

Western Blots ◽

Pancreatic Cancer Cells

The purpose of this study was to determine the quantity and nature of the mucins synthesized and secreted by four different pancreatic cancer cell lines. Well- to moderately-differentiated SW1990 and CAPAN-2 human pancreatic cancer cells were found to produce more high-Mr glycoprotein (HMG) than less-differentiated MIA PaCa-2 and PANC-1 cells. Most of the labelled HMG was secreted within 24 h. The results of chemical and enzymic degradation, ion-exchange chromatography and density-gradient centrifugation indicated that the HMG in SW1990 and CAPAN-2 cells has the properties expected for mucins, whereas much of the HMG in MIA PaCa-2 and PANC-1 cells may not be mucin, but proteoglycan. These results are consistent with immunoblots and Northern blots showing the presence of apomucin and apomucin mRNA in SW1990 and CAPAN-2 cells, but not in MIA PaCa-2 and PANC-1 cells. The Western blots and Northern blots also show that SW1990 and CAPAN-2 cells, like breast cancer cells, have the mammary-type apomucin and mRNA coded by the MUC1 gene, but lack the intestinal type apomucin and mRNA coded by the MUC2 gene. In contrast, the colon cancer cell lines tested in culture express apomucin and mRNA coded by MUC2 but not by MUC1.

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