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Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network

Significance Statement

In the manuscript “Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network” a combination therapy comprising chemotherapeutics and death receptor agonists was analyzed in an in vitro model of pancreatic cancer.  This co-treatment led to a strong synergistic effect, killing a high amount of cancer cells. More detailed analysis revealed a sensitization mechanism via down-regulation of the anti-apoptotic proteins c-FLIP and Mcl-1, which are typically overexpressed in cancer cells thereby causing defects in the cell death machinery. This gemcitabine-mediated decrease of c-FLIP protein levels changed the composition of the death-inducing signaling complex (DISC) after CD95L-treatment towards a higher amount of the apoptosis-inducing proteins Caspase-8 and Caspase-10. This modified protein complex composition is linked to the higher rate of cell death induction. Cell death assays in presence of inhibitors of apoptosis (zVAD-fmk) and necroptosis (Necrostatin-1) indicated that the combination of gemcitabine and CD95L induced both cell death modes, apoptosis and necroptosis, while the stimulation with CD95L alone led solely to apoptosis. Additionally, the usage of the cutting edge technology “imaging flow cytometry” confirmed that the combinatorial treatment not only sensitized cells to death, but also switched them to an alternative cell death pathway – necroptosis via RIPK1. These results demonstrate that this cell death network is regulated via caspase-dependent and caspase-independent pathways. The combined therapy of chemotherapeutics and death receptor agonists might offer a possibility to sensitize cells with defects in the apoptotic machinery towards necroptosis-mediated cell death.  These findings might improve treatment strategies and are important for the development of the systems biology-based approaches for the personalized medicine.

 

About The Author

Dr. Sabine Pietkiewicz studied Biology at the Heinrich-Heine-University in Düsseldorf, Germany and already started focusing on apoptosis and necroptosis after death receptor stimulation during her diploma thesis in the Institute of Molecular Medicine, under the supervision of Prof. Schulze-Osthoff. In Düsseldorf she obtained her Dr. rer. nat. at the Laboratory of Molecular Radiooncology, Clinic and Policlinic for Radiation Therapy and Radiooncology, and has characterized the role of JNK isoforms in cell death pathways after treatment with the proteasomal inhibitor and chemotherapeutic Bortezomib. She currently conducts research in the department Translational Inflammation Research of Prof. Inna N. Lavrik at the Medical Faculty, Otto von Guericke University, Magdeburg, Germany, analyzing the CD95/APO-1/Fas signaling networks.  


About The Author

Prof. Inna N. Lavrik is a head of department Translational Inflammation Research at the Medical Faculty, Otto von Guericke University, Magdeburg, Germany. She did her Postdoctoral training in Heidelberg, at German Cancer Research Center, at the Division of Immunogenetics, Head Prof. Dr. Peter H. Krammer.  Afterwards she had a group leader position at DKFZ/Bioquant, Heidelberg, Germany, which she held before moving to Magdeburg.

Combinatorial treatment of CD95L and gemcitabine in pancreatic cancer cells induces apoptotic and RIP1-mediated necroptotic cell death network

Journal Reference

Exp Cell Res. 2015;339(1):1-9.

Pietkiewicz S1, Eils R2, Krammer PH3, Giese N4, Lavrik IN5. 

Show Affiliations
  1. Department of Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany.
  2. Bioquant, Heidelberg University, 69120 Heidelberg, Germany; Department for Bioinformatics and Functional Genomics, Institute for Pharmacy and Molecular Biotechnology, University of Heidelberg, 69120 Heidelberg, Germany; Division of Theoretical Bioinformatics, GermanCancer Research Center (DKFZ), 69120 Heidelberg, Germany.
  3. Division of Immunogenetics, GermanCancer Research Center (DKFZ), 69120 Heidelberg, Germany.
  4. Department of General Surgery, University of Heidelberg, Germany (g)Federal Research Center Institute of Cytology and Genetics, Novosibirsk, Russia.
  5. Department of Translational Inflammation Research, Institute of Experimental Internal Medicine, Otto von Guericke University, Magdeburg, Germany; Federal Research Center Institute of Cytology and Genetics, Novosibirsk, Russia. Electronic address: [email protected]

Abstract

Combination therapy of cancer is based on the cumulative effects mediated by several drugs. Although molecular mechanisms of action of each particular drug are partially elucidated, understanding of the dynamic cross-talk between different cell death pathways at the quantitative level induced by combination therapy is still missing. Here, we exemplified this question for the death receptor (DR) networks in pancreatic cancer cells. We demonstrate that the combined action of CD95L and gemcitabine in pancreatic cancer cells leads to the simultaneous induction of caspase-dependent and caspase-independent cell death. The pro-apoptotic effects are mediated through down-regulation of the anti-apoptotic proteins c-FLIP and Mcl-1, while caspase-independent cell death was blocked by inhibition of the kinase activity of RIP1. Furthermore, gemcitabine co-treatment strongly increased the amount of cells undergoing CD95-induced RIP1-regulated necrosis. Imaging flow cytometry has enabled us to get the quantitative insights into the apoptosis-necroptosis network and reveal that the majority of the cells upon the CD95L/gemcitabine co-treatment undergoes necroptosis. Our data underlie the importance of the quantitative understanding of the interplay between different cell death modalities, which is essential for the development of anti-cancer therapies. Taken together, our results are important for combination therapy of pancreatic cancer comprising chemotherapeutics and DR-agonists and offer a possibility to sensitize cells with defects in the apoptotic machinery towards necroptosis-type-mediated death.

Copyright © 2015 Elsevier Inc. All rights reserved.

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