title: Enhanced cytotoxic activity of doxorubicin through the inhibition of autophagy in triple negative breast cancer cell line.
abstract: The outcome of triple negative breast cancer is still poor and requires improvement with better therapy options. Autophagy has recently been shown to play a role in anticancer drug resistance. Therefore, we investigated if the effectiveness of doxorubicin was augmented by the inhibition of autophagy.
  
  
  
  MDA-MB-231 was used as a model cell line for triple negative breast cancer and 3-methyladenine was used as an inhibitor of autophagy. Cells were treated with 0.46-1.84μM doxorubicin and 2.5-10μM 3-methyladenine for 48h. Cell death mode was examined with M30 and M65 ELISA assays. ROS level and LDH activity was examined and the cellular acidic compartment of cells was monitored by acridine orange staining. The expression of various autophagy and apoptosis related proteins/genes were evaluated with Western blotting and RT-qPCR respectively.
  
  
  
  Synergism was observed between the compounds (CI value<1.0). RT-qPCR analysis revealed that the combination resulted in a down-regulation of autophagy-related genes. Moreover, the combination resulted in a different cell death modality, upregulating necroptosis-related genes. This suggests that the mode of cell death may switch from apoptosis to necroptosis, which is a more severe form of cell death, when autophagy is inhibited. These results were further confirmed at protein level by Western blotting.
  
  
  
  Inhibition of autophagy seems to sensitize triple negative breast cancer cells to doxorubicin, warranting further in vivo studies for the proof of this concept.
  
  
  
  Autophagy has a key role in drug resistance in MDA-MB-231 cells. Therefore combinatorial approaches may effectively overcome resistance.
authors.affiliation: Uludag University, Faculty of Arts and Science, Department of Biology, Bursa, Turkey.
authors.forename: Seyma
authors.initials: S
authors.lastname: Aydinlik
authors.affiliation: Uludag University, Faculty of Arts and Science, Department of Biology, Bursa, Turkey.
authors.forename: Merve
authors.initials: M
authors.lastname: Erkisa
authors.affiliation: Uludag University, Faculty of Arts and Science, Department of Biology, Bursa, Turkey.
authors.forename: Buse
authors.initials: B
authors.lastname: Cevatemre
authors.affiliation: Uludag University, Faculty of Arts and Science, Department of Biology, Bursa, Turkey.
authors.forename: Mehmet
authors.initials: M
authors.lastname: Sarimahmut
authors.affiliation: Uludag University, Faculty of Arts and Science, Department of Biology, Bursa, Turkey.
authors.forename: Egemen
authors.initials: E
authors.lastname: Dere
authors.affiliation: Uludag University, Faculty of Arts and Science, Department of Biology, Bursa, Turkey.
authors.forename: Ferda
authors.initials: F
authors.lastname: Ari
authors.affiliation: Istinye University, Faculty of Medicine, Department of Clinical Biochemistry, Istanbul, Turkey. Electronic address: eulukaya@istinye.edu.tr.
authors.forename: Engin
authors.initials: E
authors.lastname: Ulukaya
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects*; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology*; D005260:Female; D006801:Humans; D017382:Reactive Oxygen Species / Q000378:metabolism; D064726:Triple Negative Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D051017:Apoptosis Regulatory Proteins; D017382:Reactive Oxygen Species; D004317:Doxorubicin
keywords: Autophagy; Breast cancer; Drug resistance; Necroptosis; Synergism