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Cited by 148Cancer Cells and MetastasisCancer, Hypoxia, and MetabolismEndometrial and Cervical Cancer Treatments

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The resistance of intracellular mediators to doxorubicin and cisplatin are distinct in 3D and 2D endometrial cancer

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Chitcholtan, Kenny, Sykes, Peter H, Evans, John J·2012·Journal of Translational Medicine
DOI 10.1186/1479-5876-10-38PMC PMC3316127
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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.

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Figures

Figure 1 Morphology, growth pattern, and expression of β1-integrin. (A) Differential interface contrast (DIC) images of 3D multicellular morphologies from structures formed by epithelial endometrial cancer cell lines after 5 days cultured on poly-HEMA coated cultureware. Ishikawa cell line formed tightly compact spheroids, which were resistant to trypsin-EDTA digestion. RL95-2 and KLE cell lines formed cell aggregates and clusters respectively, and they were easily dissociated by trypsin-EDTA treatment. The scale bar is 100 μm. (B) The growth of Ishikawa, RL95-2 and KLE cell lines cultured in 3D structures. The total cell number was counted by a haemocytometer at intervals of 2 days. The open circle (○), dark circle (●) and triangle (▼) represent Ishikawa, RL95-2 and KLE cell lines, respectively. (C) The expression of β1-integrin was observed in Ishikawa, RL95-2 and KLE cell lines.
Figure 1 Morphology, growth pattern, and expression of β1-integrin. (A) Differential interface contrast (DIC) images of 3D multicellular morphologies from structures formed by epithelial endometrial cancer cell lines after 5 days cultured on poly-HEMA coated cultureware. Ishikawa cell line formed tightly compact spheroids, which were resistant to trypsin-EDTA digestion. RL95-2 and KLE cell lines formed cell aggregates and clusters respectively, and they were easily dissociated by trypsin-EDTA treatment. The scale bar is 100 μm. (B) The growth of Ishikawa, RL95-2 and KLE cell lines cultured in 3D structures. The total cell number was counted by a haemocytometer at intervals of 2 days. The open circle (○), dark circle (●) and triangle (▼) represent Ishikawa, RL95-2 and KLE cell lines, respectively. (C) The expression of β1-integrin was observed in Ishikawa, RL95-2 and KLE cell lines.CC BY · PubMed Central
Figure 2 Cell viability, apoptosis and distribution of doxorubicin (DOX). (A) Cell viability after 48 hours treatment with doxorubicin (DOX) and cisplatin (CIS). The treated cells were stained with PI and analysed with FACS. The PI positive cell staining was calculated relatively to the control. The (■) dark and (grey square symbol) grey bars represent multicellular structures and cell monolayers, respectively. (B) After 48 hours treated with anticancer agents, cells were stained with annexin-FITC and PI and analysed with FACS. Multicellular structures and cell monolayers of Ishikawa, RL95-2 and KLE cells were studied. The data are presented as mean ± SEM from at least four separate experiments. The (■) dark and (grey square symbol) grey bars represent 3D multicellular structures and cell monolayers respectively. (*) P < 0.05, compared to control multicellular structures. (#) P < 0.05, compared to control cell monolayers. (■) P < 0.05, comparison of between 3D cell cultures and cell monolayers. (C) Distribution of doxorubicin (DOX) within 3D multicellular structures of Ishikawa, RL95-2 and KLE after 48 hr incubations. DOX was located in the nucleus of cancer cells in multicellular structures. Images were obtained and representative of two independent experiments.
Figure 2 Cell viability, apoptosis and distribution of doxorubicin (DOX). (A) Cell viability after 48 hours treatment with doxorubicin (DOX) and cisplatin (CIS). The treated cells were stained with PI and analysed with FACS. The PI positive cell staining was calculated relatively to the control. The (■) dark and (grey square symbol) grey bars represent multicellular structures and cell monolayers, respectively. (B) After 48 hours treated with anticancer agents, cells were stained with annexin-FITC and PI and analysed with FACS. Multicellular structures and cell monolayers of Ishikawa, RL95-2 and KLE cells were studied. The data are presented as mean ± SEM from at least four separate experiments. The (■) dark and (grey square symbol) grey bars represent 3D multicellular structures and cell monolayers respectively. (*) P < 0.05, compared to control multicellular structures. (#) P < 0.05, compared to control cell monolayers. (■) P < 0.05, comparison of between 3D cell cultures and cell monolayers. (C) Distribution of doxorubicin (DOX) within 3D multicellular structures of Ishikawa, RL95-2 and KLE after 48 hr incubations. DOX was located in the nucleus of cancer cells in multicellular structures. Images were obtained and representative of two independent experiments.CC BY · PubMed Central
Figure 3 The effect of doxorubicin (DOX) and cisplatin (CIS) on cell proliferation. (A) Effects of doxorubicin and cisplatin on cell proliferation in multicellular structures and cell monolayers. After 48 hours treatment of cells with anticancer agents, BrdU was added to the cultures for 5 hours. Cells were trypsinised, fixed, permeabilised, exposed to anti-BrdU antibody, stained with anti-mouse conjugated to Alexa™ 488 nm and analysed with FACS. The (■) dark and (grey square symbol) grey bars represent multicellular structures and cell monolayers respectively. (*) P < 0.05, compared to control multicellular structures. (#) P < 0.05, compared to control cell monolayers. The data are presented as mean ± SEM from at least four separate experiments. (B) Effects on staining patterns of proliferating cell nuclear antigen (PCNA) by doxorubicin and cisplatin. Frozen sections of multicellular structures were cut, fixed and stained with anti-PCNA antibody. Ishikawa, RL95-2 and KLE 3D multicellular structures were studied. PCNA is stained green and the nucleus is blue. The scale bar is 50 μm. The images were representative of at least two separate experiments. (C) Fluorescent intensity profile of PCNA of control and DOX treated Ishikawa spheroids.
Figure 3 The effect of doxorubicin (DOX) and cisplatin (CIS) on cell proliferation. (A) Effects of doxorubicin and cisplatin on cell proliferation in multicellular structures and cell monolayers. After 48 hours treatment of cells with anticancer agents, BrdU was added to the cultures for 5 hours. Cells were trypsinised, fixed, permeabilised, exposed to anti-BrdU antibody, stained with anti-mouse conjugated to Alexa™ 488 nm and analysed with FACS. The (■) dark and (grey square symbol) grey bars represent multicellular structures and cell monolayers respectively. (*) P < 0.05, compared to control multicellular structures. (#) P < 0.05, compared to control cell monolayers. The data are presented as mean ± SEM from at least four separate experiments. (B) Effects on staining patterns of proliferating cell nuclear antigen (PCNA) by doxorubicin and cisplatin. Frozen sections of multicellular structures were cut, fixed and stained with anti-PCNA antibody. Ishikawa, RL95-2 and KLE 3D multicellular structures were studied. PCNA is stained green and the nucleus is blue. The scale bar is 50 μm. The images were representative of at least two separate experiments. (C) Fluorescent intensity profile of PCNA of control and DOX treated Ishikawa spheroids.CC BY · PubMed Central
Figure 4 Expression of Erk1/2 and p-Erk1/2. (A) Effects on immunohistochemistry staining of active form of Erk (p-Erk) by doxorubicin and cisplatin. Multicellular structures of Ishikawa, RL95-2 and KLE cells were cut, fixed and stained with anti-p-Erk antibody. p-Erk was stained green and the nucleus is blue. The scale bar is 50 μm. (B) Western blotting of active form of Erk (p-Erk) and total expression of Erk. After 48 hours treatment with anticancer drugs, cells were lysed and proteins were separated and analysed by SDS-PAGE and Western blotting. The active form (p-Erk) and total expression of Erk from multicellular structures of Ishikawa (B:a-b,), RL95-2 (B:e-f), KLE (B:i-j) and cell monolayers of Ishikawa (B:c-d), RL95-2 (B:g-h), KLE (B:k-l).
Figure 4 Expression of Erk1/2 and p-Erk1/2. (A) Effects on immunohistochemistry staining of active form of Erk (p-Erk) by doxorubicin and cisplatin. Multicellular structures of Ishikawa, RL95-2 and KLE cells were cut, fixed and stained with anti-p-Erk antibody. p-Erk was stained green and the nucleus is blue. The scale bar is 50 μm. (B) Western blotting of active form of Erk (p-Erk) and total expression of Erk. After 48 hours treatment with anticancer drugs, cells were lysed and proteins were separated and analysed by SDS-PAGE and Western blotting. The active form (p-Erk) and total expression of Erk from multicellular structures of Ishikawa (B:a-b,), RL95-2 (B:e-f), KLE (B:i-j) and cell monolayers of Ishikawa (B:c-d), RL95-2 (B:g-h), KLE (B:k-l).CC BY · PubMed Central
Figure 5 Densitometry of Erk1/2 and p-Erk1/2. Western blotting analysis of cell protein lysate was from 3D cell cultures and cell monolayers. GAPDH was used as control for loading. Densitometric analyses were performed using Alliance 4.7, Unitec (Cambridge, UK) and are presented as a ratio of Erk/GAPDH and pErk/GAPDH. The (■) dark and (grey square symbol) grey bars represent multicellular structures and cell monolayers respectively. (*) P < 0.05, compared to control multicellular structures. (#) P < 0.05, compared to control cell monolayers. (■) P < 0.05, comparison of between multicellular structures and cell monolayers. The data are presented as mean ± SEM from at least four separate experiments.
Figure 5 Densitometry of Erk1/2 and p-Erk1/2. Western blotting analysis of cell protein lysate was from 3D cell cultures and cell monolayers. GAPDH was used as control for loading. Densitometric analyses were performed using Alliance 4.7, Unitec (Cambridge, UK) and are presented as a ratio of Erk/GAPDH and pErk/GAPDH. The (■) dark and (grey square symbol) grey bars represent multicellular structures and cell monolayers respectively. (*) P < 0.05, compared to control multicellular structures. (#) P < 0.05, compared to control cell monolayers. (■) P < 0.05, comparison of between multicellular structures and cell monolayers. The data are presented as mean ± SEM from at least four separate experiments.CC BY · PubMed Central
Figure 6 The uptake of 2-NBDG and staining of Glut-1 in multicellular structures and cell monolayers. (A) Effects on uptake of a fluorescent glucose analog 2-NBDG after cells were treated for 48 hours with doxorubicin and cisplatin. The (■) dark and (grey square symbol) grey bars represent multicellular structures and cell monolayers respectively. (*) P < 0.05, compared to control multicellular structures. (#) P < 0.05, compared to control cell monolayers. Data are expressed as the mean ± SEM from at least four independent experiments. (B) Effects on immunohistochemistry staining of Glut-1 by doxorubicin and cisplatin. Multicellular structures of Ishikawa, RL95-2, and KLE cells were cut, fixed and stained with anti-Glut-1 antibody. Glut-1 was stained green and the nucleus is blue. The scale bar is 50 μm.
Figure 6 The uptake of 2-NBDG and staining of Glut-1 in multicellular structures and cell monolayers. (A) Effects on uptake of a fluorescent glucose analog 2-NBDG after cells were treated for 48 hours with doxorubicin and cisplatin. The (■) dark and (grey square symbol) grey bars represent multicellular structures and cell monolayers respectively. (*) P < 0.05, compared to control multicellular structures. (#) P < 0.05, compared to control cell monolayers. Data are expressed as the mean ± SEM from at least four independent experiments. (B) Effects on immunohistochemistry staining of Glut-1 by doxorubicin and cisplatin. Multicellular structures of Ishikawa, RL95-2, and KLE cells were cut, fixed and stained with anti-Glut-1 antibody. Glut-1 was stained green and the nucleus is blue. The scale bar is 50 μm.CC BY · PubMed Central
Figure 7 Expression of superoxide dismutase-1 (SOD-1) and effects on secretion of vascular endothelial growth factor (VEGF) from multicellular structures and cells monolayers. (A) Expression of SOD-1 of cells cultured in 3D and 2D cell models. SOD-1 was maintained at high levels in cancer cells cultured as 3D multicellular structures compared with 2D cell monolayers. (B) Secreted VEGF in the media was collected and examined by ELISA. The (■) dark and (grey square symbol) grey bars represent 3D cell cultures and cell monolayers respectively. (*) P < 0.05, compared to control multicellular structures. (#) P < 0.05, compared to control cell monolayers. Data are expressed as the mean ± SEM.
Figure 7 Expression of superoxide dismutase-1 (SOD-1) and effects on secretion of vascular endothelial growth factor (VEGF) from multicellular structures and cells monolayers. (A) Expression of SOD-1 of cells cultured in 3D and 2D cell models. SOD-1 was maintained at high levels in cancer cells cultured as 3D multicellular structures compared with 2D cell monolayers. (B) Secreted VEGF in the media was collected and examined by ELISA. The (■) dark and (grey square symbol) grey bars represent 3D cell cultures and cell monolayers respectively. (*) P < 0.05, compared to control multicellular structures. (#) P < 0.05, compared to control cell monolayers. Data are expressed as the mean ± SEM.CC BY · PubMed Central
Figure 8 Expression of Akt and p-Akt. (A) Effects on immunohistochemistry staining of p-Akt by doxorubicin and cisplatin. Multicellular structures of Ishikawa, RL95-2 and KLE cells were cut, fixed and stained with anti-p-Akt antibody. p-Akt was stained green and the nucleus is blue. The scale bar is 50 μm. (B) Western blotting of active form Akt (p-Akt) and total expression of Akt. After 48 hours treatment with anticancer drugs, cells were lysed and proteins were separated and analysed by SDS-PAGE and Western blotting. The active form (p-Akt) and total expression of Akt of multicellular structures of Ishikawa (B:a-b,), RL95-2 (B:e-f), KLE (B:i-j) and cell monolayers of Ishikawa (B:c-d), RL95-2 (B:g-h), KLE (B:k-l).
Figure 8 Expression of Akt and p-Akt. (A) Effects on immunohistochemistry staining of p-Akt by doxorubicin and cisplatin. Multicellular structures of Ishikawa, RL95-2 and KLE cells were cut, fixed and stained with anti-p-Akt antibody. p-Akt was stained green and the nucleus is blue. The scale bar is 50 μm. (B) Western blotting of active form Akt (p-Akt) and total expression of Akt. After 48 hours treatment with anticancer drugs, cells were lysed and proteins were separated and analysed by SDS-PAGE and Western blotting. The active form (p-Akt) and total expression of Akt of multicellular structures of Ishikawa (B:a-b,), RL95-2 (B:e-f), KLE (B:i-j) and cell monolayers of Ishikawa (B:c-d), RL95-2 (B:g-h), KLE (B:k-l).CC BY · PubMed Central

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Metadata

Authors
Chitcholtan, K., Sykes, P. H., & Evans, J. J.
Affiliations
  • University of Otago (NZ)
  • Christchurch Hospital (NZ)
Corresponding author
Chitcholtan, Kenny
ORCID
Venue
Journal of Translational Medicine
Publisher
Springer Science and Business Media LLC
Published
2012-03-06
Volume
10
Issue
1
Pages
38-38
ISSN
1479-5876 (ISSN-L)
DOI
10.1186/1479-5876-10-38
PMC
PMC3316127
OpenAlex
W2169461456
Cited by
148
References
54
Citations by year
2026: 4 · 2025: 8 · 2024: 6 · 2023: 7 · 2022: 16 · 2021: 7 · 2020: 16 · 2019: 11 · 2018: 14 · 2017: 16 · 2016: 13 · 2015: 7 · 2014: 8 · 2013: 13 · 2012: 2
FWCI
4.74
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Translational Medicine (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Topics
  • Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Health Sciences › Medicine › Obstetrics and Gynecology › Endometrial and Cervical Cancer Treatments
Keywords
endometrial cancer, doxorubicin, cisplatin, chemoresistance, multicellular spheroids, tumor microenvironment, apoptosis, glucose metabolism, VEGF secretion, Akt phosphorylation, ERK1/2 phosphorylation, SOD1, anchorage-independent growth, cancer cell lines, Ishikawa
MeSH terms
Antioxidants, Cell Survival, Cisplatin, Densitometry, Deoxyglucose, Doxorubicin, Female, Glucose, Humans, 4-Chloro-7-nitrobenzofurazan, Phosphorylation, Superoxide Dismutase, Tumor Cells, Cultured, Endometrial Neoplasms, Apoptosis, Spheroids, Cellular, Drug Resistance, Neoplasm, Integrin beta1, Vascular Endothelial Growth Factor A, Intracellular Space, Tissue Culture Techniques, Extracellular Signal-Regulated MAP Kinases, Cell Proliferation, Proto-Oncogene Proteins c-akt, Glucose Transporter Type 1

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