Crocus sativus L. Causes a Non Apoptotic Calpain Dependent Death in C6 Rat Glioma Cells, Exhibiting a Synergistic Effect with Temozolomide

Abstract

Crocus sativus L., a dietary herb, has been used for various diseases including cancer. This is an in vitro study investigating the antineoplastic effect of the extract of the plant against C6 glioma rat cell line. The mechanism of cellular death and the synergistic effect of the extract with the alkylating agent temozolomide (TMZ) were investigated. Cellular viability was examined in various concentrations of the extract alone or in combination with TMZ. Apoptosis was determined with flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and autophagy by western blotting of the light chain 3 (LC3)-II. Cellular viability was reduced after exposure to the extract with half maximal inhibition concentration at 3 mg/ml. Flow cytometry and TUNEL assay suggested that the extract does not induce apoptosis. Moreover, their combination increased the ratio dead/apoptotic cells 10-fold (P < 0.001). LC3-II protein levels reduced after Crocus extract while this effect was reversed when the calpain inhibitor MDL28170 was added, suggesting a calpain-dependent death possibly through autophagy. We concluded that the extract of Crocus increases dead cell number after 48 h of exposure. Our results suggest that the cell undergoes calpain-dependent programmed cell death while co-exposure to Crocus extract and TMZ enhances the antineoplastic effect of the latter.

Acknowledgments

The authors thank the Association of Crocus Producers (Crocos, Kozani, Greece), who provided (as a gift) the stigmas of Crocus. We also thank Dr. Eugene Kokkalou, Dr. Catherine Chatzimeletiou, and Dr. Zafiroula Iakovidou-Kritsi Head Professor of 1st Department of Biology & Genetics for their excellent technical support.

Disclosure Statement

The authors declare that they have no competing interests.

Authors’ Contribution

Dimitrios Giakoumettis: Conception and design of the study, acquisition of data, analysis and interpretation of the data, drafted the manuscript, and has provided final approval of the version to be published.

Chryssa Pourzitaki: Conception and design of the study, acquisition of data, analysis and interpretation of the data, drafted the manuscript, has provided final approval of the version to be published, and has agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Theofanis Vavilis: Conception and design of the study, analysis and interpretation of the data, drafted the manuscript, and has provided final approval of the version to be published.

Anastasia Tsingotjidou: Conception and design of the study, analysis and interpretation of the data, drafted the manuscript, and has provided final approval of the version to be published.

Anastasia Kyriakoudi: acquisition of data, drafted the manuscript, and has provided final approval of the version to be published.

Maria Tsimidou: acquisition of data, drafted the manuscript, and has provided final approval of the version to be published.

Marina Boziki: acquisition of data, drafted the manuscript, and has provided final approval of the version to be published.

Antonia Sioga; acquisition of data, drafted the manuscript, and has provided final approval of the version to be published.

Nikolaos Foroglou: Conception and design of the study, drafted the manuscript, and has provided final approval of the version to be published.

Aristeidis Kritis: Conception and design of the study, analysis and interpretation of the data, drafted the manuscript, and has provided final approval of the version to be published.