title: The resistance of intracellular mediators to doxorubicin and cisplatin are distinct in 3D and 2D endometrial cancer.
abstract: BACKGROUND
  Advanced endometrial cancer often shows resistance to clinical chemotherapy although potencies of anticancer drugs in vitro are promising. The disparity suggests that in vivo microenvironments are not recapitulated by in vitro models used for preclinical testing. However, spheroids replicate some important properties of tumours in vivo. Therefore, for the first time, we compared effects of doxorubicin and cisplatin on 3D multicellular structures and 2D cell monolayers of endometrial cancer cells.
  
  
  METHODS
  3D multicellular structures were generated by culturing cancer cells on non-adherent surfaces; and for comparison cell monolayers were cultured on adherent culture plates. Ishikawa, RL95-2, and KLE cell lines were studied. Morphologies of 3D multicellular structures were examined. After 48 hours treatment with anticancer drugs, apoptosis, proliferation, glucose metabolism and vascular endothelial growth factor (VEGF) were analysed. Immunostaining of PCNA, Glut-1, p-Erk1/2, SOD-1 and p-Akt1/2/3 was also performed.
  
  
  RESULTS
  Distinct 3D multicellular morphologies were formed by three different endometrial cancer cell lines. Doxorubicin induced less apoptosis in 3D multicellular structures of high grade cancer cells (RL95-2 and KLE cell lines) than in cell monolayers. Parallel alterations in Erk1/2 phosphorylation and cell proliferation might suggest they were linked and again doxorubicin had less effect on 3D multicellular structures than cell monolayers. On the other hand, there was no correlation between altered glucose metabolism and proliferation. The responses depended on cancer cell lines and were apparently not mediated by altered Glut-1 levels. The level of SOD-1 was high in 3D cell cultures. The effects on VEGF secretion were various and cancer cell line dependent. Importantly, both doxorubicin and cisplatin had selective paradoxical stimulatory effects on VEGF secretion. The microenvironment within 3D multicellular structures sustained Akt phosphorylation, consistent with it having a role in anchorage-independent pathways.
  
  
  CONCLUSIONS
  The cancer cells responded to microenvironments in a distinctive manner. 3D multicellular structures exhibited greater resistance to the agents than 2D monolayers, and the differences between the culture formats were dependent on cancer cell lines. The effects of anticancer drugs on the intracellular mediators were not similar in 3D and 2D cultures. Therefore, using 3D cell models may have a significant impact on conclusions derived from screening drugs for endometrial carcinomas.
authors.affiliation: Department of Obstetrics and Gynaecology, University of Otago, 2 Riccarton Avenue, Christchurch 8011, New Zealand. kenny.chitcholtan@otago.ac.nz
authors.forename: Kenny
authors.initials: K
authors.lastname: Chitcholtan
authors.forename: Peter H
authors.initials: PH
authors.lastname: Sykes
authors.forename: John J
authors.initials: JJ
authors.lastname: Evans
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D003720:Densitometry; D003847:Deoxyglucose / Q000031:analogs & derivatives / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D016889:Endometrial Neoplasms / Q000145:classification / Q000201:enzymology / Q000473:pathology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D005947:Glucose / Q000378:metabolism; D051272:Glucose Transporter Type 1 / Q000378:metabolism; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D018874:Spheroids, Cellular / Q000187:drug effects / Q000473:pathology; D013482:Superoxide Dismutase / Q000378:metabolism; D046509:Tissue Culture Techniques / Q000379:methods*; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D000975:Antioxidants; D051272:Glucose Transporter Type 1; D019012:Integrin beta1; C495677:SLC2A1 protein, human; D042461:Vascular Endothelial Growth Factor A; D004317:Doxorubicin; D003847:Deoxyglucose; D013482:Superoxide Dismutase; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D009327:4-Chloro-7-nitrobenzofurazan; D005947:Glucose; C098340:2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; D002945:Cisplatin