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. 2018 Mar 12;9(3):392.
doi: 10.1038/s41419-018-0395-2.

Paraoxonase 2 overexpression inhibits tumor development in a mouse model of ovarian cancer

Asokan Devarajan  1 , Feng Su  2 , Victor Grijalva  3 , Meghna Yalamanchi  3 , Ashna Yalamanchi  3 , Feng Gao  2   4 , Hannah Trost  3 , Josephine Nwokedi  3 , Gina Farias-Eisner  5 , Robin Farias-Eisner  2 , Alan M Fogelman  3 , Srinivasa T Reddy  6   7   8
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V体育ios版 - Paraoxonase 2 overexpression inhibits tumor development in a mouse model of ovarian cancer

Asokan Devarajan (V体育官网入口) et al. Cell Death Dis. .

V体育安卓版 - Abstract

Ovarian cancer (OC) is most lethal malignancy among all gynecological cancer. Large bodies of evidences suggest that mitochondrial-derived ROS play a critical role in the development and progression of OC. Paraoxonase 2 (PON2) is a membrane-associated lactonase with anti-oxidant properties. PON2 deficiency aggravates mitochondrial ROS formation, systemic inflammation, and atherosclerosis. The role of PON2 in cancer development remains unknown. In this report, in human, we identified that PON2 expression is higher in early stages (but not in late stages) of OC when compared to normal tissue. Using a mouse xenograft model of OC, we demonstrate that overexpression of PON2 prevents tumor formation VSports手机版. Mechanistically, PON2 decreases OC cell proliferation by inhibiting insulin like growth factor-1 (IGF-1) expression and signaling. Intriguingly, PON2 reduces c-Jun-mediated transcriptional activation of IGF-1 gene by decreasing mitochondrial superoxide generation. In addition, PON2 impairs insulin like growth factor-1 receptor (IGF-1R) signaling in OC cells by altering cholesterol homeostasis, which resulted in reduced caveolin-1/IGF-1R interaction and IGF-1R phosphorylation. Taken together, we report for the first time that PON2 acts as a tumor suppressor in the early stage of OC by reducing IGF-1 production and its signaling, indicating PON2 activation might be a fruitful strategy to inhibit early stage ovarian tumor. .

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Conflict of interest statement

Conflict of interest statement: A. M. F. , and S. T. R. are principals in Bruin Pharma. A. M. F V体育安卓版. is an officer in Bruin Pharma.

Figures

Fig. 1
Fig. 1. PON2 protein is induced in early stage ovarian cancer.
Immunohistochemistry was performed on ovarian carcinoma tissue array slides (US Biomax Rockville, MD, USA) using PON2 antibody as described under the Materials and methods section. PON2 protein expression was quantified using Definiens Tissue Studio Software. Representative photographs of the staining are shown in a, and the quantification of the data is presented in b. Values are expressed as percentage of PON2-positive staining cells. Total protein lysates from matched normal and ovarian tumor tissues [stage I (n = 7), stage II (n = 10), stage III (n = 12), and stage IV (n = 15)] were subjected western blotting analyses using hPON2 antibody. Representative blots (two sets from each stage) are shown in c. The data were quantified using Image J software and presented in d
Fig. 2
Fig. 2. Characterization of ID8EV and ID8hPON2 cells.
a) Expression of hPON2 protein was analyzed by western blotting in ID8EV and ID8hPON2 stable cell lines. b) ID8EV and ID8hPON2 cell fractions were subjected to western blotting for PON2 and COX IV proteins. c) Colocalization of mitochondrial marker and PON2. ID8EV and ID8hPON2 cell were fixed and stained with Mito Tracker® Red (a mitochondrial marker, red) and PON2 antibody (green). The merged image of hPON2 and mitochondria (yellow) is shown in the bottom panel. d) Colocalization of ER marker and PON2. ID8EV and ID8hPON2 cell were fixed and stained for Calnexin (green), an ER marker, and hPON2 (red). The merged image of hPON2 and Calnexin is shown in the bottom panel. e) Mitochondria were isolated from ID8EV and ID8hPON2 cells and respiratory complex II + III assay was carried out as described in Materials and methods section. The results are expressed as Cytochrome c reduced/min/mg protein. f) mitochondrial superoxide levels were measured as described in the methods  and values were expressed as fold change over the ID8EV. *p < 0.05 compared to IDEV cells. g) ID8EV and ID8hPON2 cells were cultured for 48 h, serum starved for 12 h, and cell viability was measured as described in the method section. h) 5 × 103 ID8EV and ID8hPON2 cells were cultured for 48 h, serum starved for 12 h and cell proliferation was assessed using BrdU by spectrophotometry as described in the Materials and methods section
Fig. 3
Fig. 3. ID8hPON2 cell derived flank tumors have reduced tumor weight and volume.
ID8EV or ID8hPON2 cells (5 × 106per mouse) were injected subcutaneously in the right flank of C57BL/6J (n = 15 per group). After 5 weeks, mice were sacrificed and tumors were resected and analyzed. a) Tumor weight was measured and expressed in mg. b) Tumor volumes were determined by Vernier calipers measurements (using the formula 1/2 × L × W2) and expressed in mm3. c) Representative tumors from the two groups of mice. Four animals did not develop visible tumors in ID8hPON2 group. *p < 0.05 compared to ID8EV-derived tumors
Fig. 4
Fig. 4. PON2 overexpression reduces IGF-1 expression and inhibits cell proliferation in ID8 cells.
a) 3 × 105 ID8EV cells or ID8hPON2 cells were cultured on six-well plates in DMEM medium containing high glucose and l-glutamine (2 mM) and supplemented with 4% fetal bovine serum (FBS), penicillin (100 U ml−1), streptomycin (100 μg ml−1), and 1× ITS liquid media supplement (10 μg ml−1 insulin, 5 μg ml−1 transferin, and 5 ng ml−1 sodium selenite). Cells were incubated at 37 °C for 24 h in 5% CO2 and then starved for 12 h in serum free media. Total RNA was isolated and cDNA was prepared as described under materials and methods. IGF-1 mRNA was quantified by qPCR and normalized to cyclophilin. b) 3 × 105 ID8EV and ID8hPON2cells were grown 1–3 days and each day serum starved for 12 h, then cell culture supernatants were collected and IGF-1 protein levels were quantified by ELISA as described under Materials and methods. hPON2 siRNA or scrambled siRNA was transfected into SKOV3, HeLa, and A549 cells at 70% confluence. PON2 protein expression was quantified after 2 days as described under the Materials and methods (c). Total RNA was isolated and IGF-1 was quantified by qPCR (d). e) 5 × 103 ID8EV and ID8hPON2 cells were grown as indicated in b and cell proliferation was assessed using Brdu by spectrophotometry as described in the Materials and methods. f) ID8EV and ID8hPON2 cells were grown and serum starved as in b and each day conditioned media were collected. Subsequently, the media were incubated with ID8 cells for 48 h, and cell proliferation was assessed as described in the Materials and methods section. g) ID8EV and ID8hPON2 cells were grown for 2 days and serum starved for 12 h with 500 nM IGF-1 antibody (# ab9572, Abcam) or without IGF-1 antibody and cell proliferation was assessed as described in the materials and methods section. h) Following hPON2 siRNA transfection with SKOV3, HeLa, and A549 cells, cell proliferation was assessed using FITC Brdu. Brdu-positive cells were quantified using flow cytometer. *p < 0.05, compared to ID8EV. Values were expressed as fold (n = 3)
Fig. 5
Fig. 5. PON2 reduces the IGF-1 level via C-Jun transcription factor in ID8 cells.
ID8EV and ID8hPON2 cell culture lysates were prepared and utilized for chromatin immunoprecipitation assays with C-Jun (a) and p53 (b) antibodies. c) Equal number of ID8hPON2 cells were plated in six-well plates. At 60 % confluence level, ID8hPON2 cells were transfected with either hPON2 siRNA or scrambled siRNA, and PON2 expression analyzed by western blotting. d) Data in c are quantified using ImageJ software. e) Four six-well plates of cells were pooled from PON2-siRNA-treated ID8hPON2 or scrambled siRNA-treated ID8hPON2 cells. Chromatin immunoprecipitation assay was performed and C-Jun promoter was quantified (e) as described in the materials and methods section. Cell culture supernatants obtained from cells described under (e) and IGF-1 level was analyzed by ELISA (f), g) cell proliferation, and h) mitochondrial superoxide levels were measured as described in the method section. *p < 0.05, compared to ID8EV. Values were expressed as fold changes (n = 3)
Fig. 6
Fig. 6. Overexpression of hPON2 inhibits cell proliferation via IGF-1 signaling pathway by altering the caveolin-1 and IGF-1R interaction in ID8 cells.
a) 5 × 103 cells were plated in 96 well plates and were incubated at 37 °C in 5% CO2. After 24 h, cells were serum starved for 12 h. Following this, cells were treated with IGF-1 at the concentration of 500 nM or vehicle for 6 h, 12 h, and 24 h. Cell proliferation was measured as described above. b) ID8EV cells or ID8hPON2 cells were cultured in six-well plates as described above and treated with IGF-1 at the concentration of 1 µM for 30, 60, and 90 min. 50 µg of total protein (100 µg for ERK1/2) was subjected to western blotting and phosphorylated IGF-1R, total IGF-1R, phosphorylated ERK1/2, total ERK1/2, cyclin D1, and vinculin were detected using respective antibodies as described in the materials and methods section. c) Immunoprecipitation was performed with caveolin-1 antibody and IGF-1R was detected by western blotting. d) Lipids were extracted from ID8EV and ID8hPON2 cells using chloroform: isopropanol: NP (7:11:0.11), air dried at 50 °C to remove the chloroform and placed under vacuum for 30 min, dispersed with assay buffer and cholesterol was measured as described in the Materials and methods section. e) Mitochondria and cytosol were isolated as described in the materials and methods section and acetyl CoA was measured using a kit according to manufacturer’s protocol. Values were normalized based on protein concentration. Values were expressed as fold change (n = 3). f) Citrate lyase enzyme activity was measured as described in the Materials and methods section. (n = 3). *p < 0.05, compared to ID8EV
Fig. 7
Fig. 7. Schematic presentation of a working model for the role of PON2 in ovarian tumor growth.
ID8 cells form tumors in immunocompetent C57BL6/J mice. ID8hPON2 cells overexpressing human PON2 develop reduced tumors. Based on the data presented in this paper, PON2 overexpression regulates three anti-tumorigenic pathways. I). PON2 overexpression reduces mitochondrial superoxide levels, which regulate c-Jun activation. Reduced c-JUN binding to the IGF-1 promoter leads to decreased expression of IGF-1 protein. II). PON2 expression enhances electron transport chain activity leading to decreased cholesterol levels resulting in impaired caveolin-1 and IGF-1R interaction. I & II result in decreased cell proliferation and reduced tumor growth
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