title: Sensitizing osteosarcoma stem cells to doxorubicin-induced apoptosis through retention of doxorubicin and modulation of apoptotic-related proteins.
abstract: OBJECTIVE
  Osteosarcoma is the most common pediatric bone malignancy with high propensity to metastasize and relapse. Emerging evidence suggest that osteosarcoma is sustained by a subset of self-renewing cancer stem like cells (CSCs) relying on mechanisms to evade apoptosis and survive in response to drugs-induced DNA damage. We proposed to decipher the mechanisms underlying the resistance of CSCs to doxorubicin-induced apoptosis.
  
  
  METHODS
  CSCs were isolated using a sphere-forming assay and tested for sensitivity to doxorubicin-induced apoptosis, using MTT cell viability and BrdU proliferation assays, TUNEL staining and caspases 3/7 activity. Bcl-2 family proteins were analyzed by Western blot. Doxorubicin uptake was determined by confocal microscopy and bioluminescence imaging.
  
  
  RESULTS
  We showed that osteosarcoma sphere stem-like cells expressed the multidrug-related efflux transporters P-glycoprotein and BCRP and are highly resistant to doxorubicin-induced apoptosis. Conversely after exposure to doxorubicin, these cells displayed an up-regulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL with concomitant down-regulation of Bak and decreased caspase 3/7 activity. Inhibition of drug efflux transporters enhanced the cellular uptake of doxorubicin, being encompassed by an up-regulation the pro-apoptotic protein Bak and suppression of Bcl-2, favoring the commitment of CSCs towards apoptosis.
  
  
  CONCLUSIONS
  These results seemingly suggest that the high apoptotic threshold of CSCs to doxorubicin-induced cell dead stimuli is mainly dependent on the drug concentration reaching tumor cells that are governed by efflux transporter activity. Therefore, modulation of these transporters may be effective in potentiating the proapoptotic effects of doxorubicin, and emerges as an attractive strategy to sensitize osteosarcoma CSCs to chemotherapy.
authors.affiliation: Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
authors.forename: Cláudia
authors.initials: C
authors.lastname: Gonçalves
authors.affiliation: Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
authors.forename: Sara R
authors.initials: SR
authors.lastname: Martins-Neves
authors.affiliation: Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
authors.forename: Daniela
authors.initials: D
authors.lastname: Paiva-Oliveira
authors.affiliation: Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
authors.forename: Vitor E B
authors.initials: VE
authors.lastname: Oliveira
authors.affiliation: Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
authors.forename: Carlos
authors.initials: C
authors.lastname: Fontes-Ribeiro
authors.affiliation: Laboratory of Pharmacology and Experimental Therapeutics, Institute for Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugal; Center of Investigation in Environment, Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra, Coimbra, Portugal. Electronic address: cgomes@fmed.uc.pt.
authors.forename: Célia M F
authors.initials: CM
authors.lastname: Gomes
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western; D001859:Bone Neoplasms / Q000188:drug therapy* / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D006801:Humans; D018613:Microscopy, Confocal; D014411:Neoplastic Stem Cells / Q000378:metabolism; D012516:Osteosarcoma / Q000188:drug therapy* / Q000473:pathology; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D051017:Apoptosis Regulatory Proteins; D004317:Doxorubicin
keywords: Apoptosis; BCRP; Cancer stem cells; Chemoresistance; Doxorubicin; Osteosarcoma; P-glycoprotein