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European Journal of Gynaecological Oncology  2020, Vol. 41 Issue (3): 352-360    DOI: 10.31083/j.ejgo.2020.03.5094
Original Research Previous articles | Next articles
Morphological analysis of peritoneal dissemination of ovarian cancer based on levels of carbonyl reductase 1 expression
F. Oyama1, Y. Asano2, H. Shimoda2, 3, K. Horie4, J. Watanabe4, Y. Yokoayama1()
1Department of Obstetrics and Gynecology, Graduate School of Medicine, Hirosaki University, Hirosaki, Japan
2Department of Neuroanatomy, Cell Biology and Histology, Graduate School of Medicine, Hirosaki University, Hirosaki, Japan
3Department of Anatomical Science, Graduate School of Medicine, Hirosaki University, Hirosaki, Japan
4Department of Cellular and Histo-Pathology, Division of Medical Life Science, Hirosaki University Graduate School of Health Science, Hirosaki, Aomori, Japan
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Abstract  

Purpose of Investigation: When carbonyl reductase 1 (CR1) is highly expressed in human ovarian cancer cells in vivo, tumor growth is reported to be inhibited. Conversely, when expression of CR1 decreases, tumor growth, invasion, and metastasis are reported to increase. Thus, the aim of the current study was to examine dynamic changes in ovarian cancer cells under different CR1 expression levels in artificial human peritoneal tissue (AHPT). Materials and Methods: Serous ovarian cancer cells with different levels of CR1 expression were produced by transfection of HRA human ovarian carcinoma cells with CR1 DNA or CR1 siRNA. The transfected cells were seeded in AHPT and observed over time until peritoneal development of carcinomatosis. Apoptotic cells in the AHPT were compared using TUNEL staining and fluorescence-based flow cytometry. Results: Cells transfected with CR1 DNA or CR1 siRNA did not differ from control cells in terms of their adherence to the mesothelium. After 24 hours, when cells had invaded the tissue below the mesothelium, proliferation of CR1-overexpressing cells was inhibited while proliferation of CR1-suppressing cells increased. At 72 hours, CR1-suppressing cells had invaded the stroma. CR1-overexpressing cells had a markedly higher rate of apoptosis than control or CR1-suppressing cells. Moreover, electron microscopy revealed apoptotic bodies in cells overexpressing CR1. Differences in tumor growth depending on the extent of CR1 expression have been noted in vivo, and similar results were obtained in the present in vitro model of AHPT. High and low levels of CR1 expression did not affect cell adherence to the mesothelium, but low levels did result in cells invading and proliferating below the mesothelium. Conclusion: The present results have also demonstrated that tumor inhibition by CR1 involves an increase in apoptosis.

Key words:  Carbonyl reductase 1      Ovarian cancer cells      Artificial human peritoneal tissue      Transfection      Apoptosis     
Submitted:  03 December 2018      Accepted:  10 January 2019      Published:  15 June 2020     
*Corresponding Author(s):  Y. Yokoayama     E-mail:  yokoyama@hirosaki-u.ac.jp

Cite this article: 

F. Oyama, Y. Asano, H. Shimoda, K. Horie, J. Watanabe, Y. Yokoayama. Morphological analysis of peritoneal dissemination of ovarian cancer based on levels of carbonyl reductase 1 expression. European Journal of Gynaecological Oncology, 2020, 41(3): 352-360.

URL: 

https://ejgo.imrpress.com/EN/10.31083/j.ejgo.2020.03.5094     OR     https://ejgo.imrpress.com/EN/Y2020/V41/I3/352

Figure 1.  — Effect of CR1 expression levels on ovarian cancer cell proliferation. (A) Fluorescent images (overlay with the phase difference) of HRA cells transfected with the CR1 overexpression construct pCMV6-AC-GFP taken 48 hours post-transfection. (B) CR1 expression levels determined by western blot analysis. β-actin was used as an internal control. (C) Comparison of cell density (as assessed by microscopy) of HRA cells (control), CR1-DNA-transfected HRA cells and CR1-siRNA-transfected HRA cells. * p < 0.05 versus the control.

Figure 2.  In vitro peritoneal metastasis model comparing overexpression and suppression of CR1. (A) Cross-sections of AHPT seeded with HRA cells (red), CR1-DNA-transfected HRA cells (yellow), and CR1-siRNA-transfected HRA cells (green) stained with toluidine blue. Cell invasion in the stroma-like structure was observed in CR1-siRNA-transfected HRA cells at 72 hours (red arrow). Scale bars; 20 μm. (B) Quantification of HRA cell number. * p < 0.05, control HRA versus CR1-DNAtransfected HRA, § p < 0.05, control HRA versus CR1-siRNA-transfected HRA, # p < 0.05, CR1-DNA-transfected HRA versus CR1-siRNA-transfected HRA.

Figure 3.  — Effect of CR1 expression levels on apoptosis of ovarian cancer cells in an AHPT model. (A) TUNEL staining of AHPT and fluorescent detection of apoptotic cells 48 hours after seeding either control HRA cells. (B) CR1-DNA-transfected HRA cells. (C) CR1-siRNA-transfected HRA cells. In the microscopy images, yellow arrows show the TUNEL-positive cells (scale bar; 50 μm). On the fluorescence graphs, red boxes and arrows indicate apoptotic cells. (D) Caspase-3 expression in HRA cells, CR1-DNA-transfected HRA cells, and CR1-siRNA-transfected HRA cells. β-actin was used as an internal control.

Figure 4.  — Transmission electron microscopy (TEM) images showing the effect of CR1 expression in an AHPT model. TEM images are shown of AHPT 48 hours after seeding with either control HRA cells (A), CR1-siRNA-transfected HRA cells (B), or CR1-DNA-transfected HRA cells (C, D). (C) Note the cancer cells have changed to a ‘stone wall’ shape and display a decrease in cytoplasm (pink color shows cancer cells, scale bar; 5 μm). (D) A higher magnification of AHPT seeded with CR1-DNA-transfected HRA cells. Note the karyorrhexis in cancer cells and presence of apoptotic bodies (red arrows). Scale bar; 2.5 μm. M: mesothelium, LV: lymphatic vessel.

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