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RESEARCH ARTICLE

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Peroxiredoxin 2 Controls Proliferation in Extracellular Matrix-Detached SKOV3 Ovarian Cancer Cells

Kassidy M. Jungles, Darya Bondarenko, Marianna Sanchez, Kailey A. Schramm, and Calli A. Davison-Versagli 

Department of Biology, Saint Mary’s College, Notre Dame, IN 46556, USA 

Correspondence: cversagli@saintmarys.edu (C.A.D-V.) 

Jungles KM et al. Reactive Oxygen Species 9(27):109–117, 2020; ©2020 Cell Med Press

http://dx.doi.org/10.20455/ros.2020.817

(Received: July 27, 2019; Revised: January 13, 2020; Accepted: January 19, 2020) 

ABSTRACT | Ovarian carcinoma is one of the leading causes of cancer death for women living in the United States, where approximately 53% of individuals diagnosed succumb to this disease. This is primarily due to lack of early detection and the ability of ovarian cancer cells to spread, or metastasize, thus making the disease more difficult to treat. Non-tumorigenic epithelial cells rely on attachment to the extracellular matrix (ECM) to thrive; however, cancer cells must be able to survive detached from the ECM in order to metastasize to other areas of the body. Studies have examined how cancer cells can metastasize and survive detachment, and it is evident that these cancer cells must contend with increased reactive oxygen species (ROS). This study focuses on the antioxidant enzyme peroxiredoxin 2 (PRDX2) and its role in proliferation and cell survival in metastatic ovarian carcinoma. Here, we report that PRDX2 deficiency severely compromises growth in anchorage independence, and that PRDX2 deficiency uniquely abrogates proliferation in ECM detachment. Consequently, PRDX2 should be further explored as a therapeutic target for late-stage ovarian carcinoma. 

KEYWORDS | ECM-detachment; Metastasis; Ovarian cancer; Peroxiredoxin 2; Reactive oxygen species; SKOV3 

ABBREVIATIONS | ECM, extracellular matrix; PRDX2, peroxiredoxin 2; ROS, reactive oxygen species; shRNA, short hairpin RNA 

CONTENTS 

  1. Introduction
  2. Materials and Methods

2.1. Cell Culture

2.2. Peroxiredoxin 2 Elimination Using shRNA Techniques

2.3. Western Blotting

2.4. Soft Agar Assays

2.5. Cell Viability and Proliferation Assays

2.6. Statistical Analysis

  1. Results

3.1. PRDX2 Deficiency in SKOV3 Cells Compromises Cell Growth in Anchorage Independence

3.2. Elimination of PRDX2 Results in Abrogated Cell Proliferation in ECM Detachment

3.3. PRDX2 Knockdown Has no Effect on Proliferation or Viability of ECM Attached SKOV3 Cells

  1. Discussion
  2. Conclusion

1. INTRODUCTION 

Ovarian cancer is the fifth leading cause of cancer death for women in the U.S. [1]. Ovarian carcinoma, the most common and aggressive subtype, is difficult to diagnose due to mild symptoms and poor diagnostic tools [1, 2]. Thus, many patients are diagnosed after the disease has already spread (metastasized) throughout the abdominal cavity (stage III) or distant sites (stage IV) making the disease more difficult to treat [1, 2]. The development of alternative therapeutic approaches is imperative to successfully treat patients with the progressed form of the disease. 

Uniquely, ovarian carcinoma cells metastasize by utilizing the peritoneal fluid to spread to organs throughout the abdominal cavity [3]. To complete this task, ovarian carcinoma cells must overcome effects caused by detachment from the extracellular matrix (ECM), including coping with increases in cellular reactive oxygen species (ROS) [4, 5]. ROS influence many cellular activities, including cell death and proliferation [4, 6]. It is recognized that ECM-detached breast cancer cells rely on antioxidant enzymes to abrogate ECM-detachment-induced ROS [4]. Furthermore, we recently reported that the ROS-controlling enzymes, catalase and sestrin 2, are critical in the survival of ECM-detached ovarian carcinoma cells [7, 8]. In aggregate, antioxidant enzymes may be viable therapeutic targets for ovarian carcinoma cells, but the role of many other antioxidant enzymes in ECM-detached metastatic ovarian carcinoma cells has yet to be unveiled. 

Peroxiredoxin 2 (PRDX2) is a member of a family of antioxidant enzymes made up of six known isoforms (PRDX1–6) that eliminate ROS. PRDX2 is highly expressed in colon and prostate cancer and is associated with a poor prognosis and a negative outcome in the cancer patients [9, 10]. Furthermore, PRDX2 is known to play an important role in the formation of lung metastases in metastatic breast cancer [11], and PRDX2 overexpression contributes to the development of cisplatin-resistance in human breast and ovarian carcinoma cells [12]. However, the role of PRDX2 in the survival and proliferation of ECM-detached metastatic ovarian carcinoma cells has yet to be explored. 

The purpose of this study is to explore the role of PRDX2 in the proliferation and survival of ECM-detached metastatic ovarian carcinoma cells by utilizing a widely used model system for ovarian cancer: the SKOV3 human ovarian carcinoma cell line. Here, we report that PRDX2 deficiency in SKOV3 cells results in compromised anchorage-independent growth. Furthermore, we find that elimination of PRDX2 uniquely abrogates cell proliferation in anchorage-independent SKOV3 cells, a phenomenon not observed in ECM-attached PRDX2-deficient SKOV3 cells. These data suggest that PRDX2 may have a unique role in controlling proliferation of ECM-detached ovarian carcinoma cells. Thus, PRDX2 may be a potential therapeutic target for treating metastatic ovarian carcinoma.

2. MATERIALS AND METHODS 

2.1. Cell Culture 

Cells were cultured as described previously [7]. SKOV3 cells (ATCC, Manassas, VA, USA), an ovarian adenocarcinoma cell line, were cultured in McCoy’s media (GIBCO, Waltham, MA, USA), 10% fetal bovine serum (FBS) (Alkali Scientific, Fort Lauderdale, FL, USA) to provide a myriad of proteins to the cultures, and 1% penicillin/streptomycin (GIBCO). All cells studied were maintained within a 37⁰C, 5% CO2 incubator. 

2.2. Peroxiredoxin 2 Elimination Using shRNA Techniques 

MISSION shRNA to target PRDX2 and an empty vector control were obtained from Sigma-Aldrich (Saint Louis, MO, USA).A control cell line with a PRDX2-deficient cell line was created following the protocol described previously [7]. 

2.3. Western Blotting 

To confirm PRDX2 knockdown, SKOV3 parental and SKOV3 PRDX2-deficient cells were harvested, washed once with cold phosphate-buffered saline (PBS), and lysed for 20 min in 1% Nonidet P-40 (Sigma-Aldrich) supplemented with aprotinin (1 mg/ml) (GIBCO) and leupeptin (5 mg/ml) on ice. Lysates were collected after spinning for 30 min at 4°C at 18,188 g and normalized by utilizing a BCA assay (Pierce, Rockford, IL, USA). Lysates were run on a 12% SDS-PAGE gel (Bio-Rad, Hercules, CA, USA) before being transferred to PVDF membrane (Millipore, St. Louis, MO, USA). The membrane was blocked in 5% milk in 1× TBST for 1 h (Bio-Rad). The following antibodies were utilized: PRDX2 rabbit polyclonal antibody (cat# 10545-2-AP, Proteintech, Rosemont, IL, USA), b actin mouse monoclonal antibody (cat # 66009-1-Ig, Proteintech), goat anti-rabbit HRP-conjugated antibody (cat # SA00001-2, Proteintech), and goat anti-mouse HRP-conjugated antibody (cat# SA00001-1, Proteintech). The blot was developed using chemiluminescence (Alkali Scientific, Ocala, FL, USA). This protocol was described in [7]. 

2.4. Soft Agar Assays 

To recreate the anchorage-independent metastatic process, SKOV3 cells were grown in soft agar assays. A total of 20,000 SKOV3 parental cells and SKOV3 PRDX2 deficient cells were added to 1.5 ml of McCoy’s media plus 0.4% low-melt agarose (Sigma-Aldrich) and plated on top of a 2 ml bed of 0.5% low-melt agarose/media mixture (Sigma-Aldrich) in a 6-well plate (Cytoone, Ocala, FL, USA). For each individual experiment, cells were grown in triplicate, supplemented with 1 ml of media every other day for 21 days, and viable colonies were stained with iodonitrotetrazolium chloride (INT) (Sigma-Aldrich). Colony counts were determined using ImageJ software (Bethesda, MD, USA). In total, 10 individual experiments of triplicate-plated SKOV3 parental and SKOV3 PRDX2 deficient cells were completed and analyzed. This protocol was described in [7]. 

2.5. Cell Viability and Proliferation Assays 

A total of 50,000 SKOV3 parental cells and SKOV3 PRDX2 deficient cells were plated in triplicate on poly-HEMA plates (non-adherent plates) 6 well plates or normal adherent 6 well plates. After 24, 48, and 72 h, cells were removed from plates, stained with trypan blue, and counted using a hemocytometer. Total proliferation and percent viability were calculated. Experiments on poly-HEMA coated plates and normal adherent plates were repeated three times for each time point. This protocol was described before [7]. 

2.6. Statistical Analysis 

For soft agar assays, cell proliferation, and cell viability, results were statistically compared using a two-tailed ttest where p < 0.05 was considered significant. Error bars represent the standard error of the mean (SEM).

3. RESULTS 

3.1. PRDX2 Deficiency in SKOV3 Cells Compromises Cell Growth in Anchorage Independence 

During metastasis, ovarian carcinoma cells must be able to grow in ECM detachment to travel to secondary sites. To determine if PRDX2 is important in the growth of anchorage-independent SKOV3 cells, shRNA techniques were utilized to stably eliminate PRDX2 expression. PRDX2 elimination was confirmed through western blotting (Figure 1A), and SKOV3 Parental cells and SKOV3 PRDX2 deficient cells were grown in soft agar. SKOV3 PRDX2-deficient cells had abrogated growth in soft agar compared to SKOV3 parental cells (Figure 1B and 1C), suggesting that PRDX2-deficient SKOV3 cells are at a disadvantage for growth in anchorage independence.

 

FIGURE 1. PRDX2 deficiency severely compromises cell growth in soft agar. PRDX2 deficiency was confirmed through western blotting (panel A). To measure ability to grow in anchorage-independence, SKOV3 parental cells and SKOV3 PRDX2 deficient cells were grown in triplicate for 21 days in soft agar, stained with iodonitrotetrazolium chloride (INT), and colonies were counted using ImageJ. This process was completed 10 times. Representative soft agar images are in panel B. Quantitation of one representative triplicate-plated experiment is shown in panel C. *, p < 0.05 as determined using a two-tailed t test. Error bars represent SEM. 

3.2. Elimination of PRDX2 Results in Abrogated Cell Proliferation in ECM Detachment 

Changes in cell growth in soft agar can be a result of alterations in proliferation or cell viability. To determine which cellular process was being altered by PRDX2 deficiency in ECM detachment, PRDX2-deficient SKOV3 cells and SKOV3 parental cells were grown on non-adherent poly-HEMA coated 6-wellplates. Percentcellviability(Figure2A)and proliferation (Figure 2B) were calculated at 24, 48, and 72 h. No changes in cell viability between ECM-detached SKOV3 PRDX2 deficient cells and ECM-detached SKOV3 parental cells were seen at any time point (Figure 2A). However, ECM-detached SKOV3 PRDX2 deficient cells showed compromised proliferation at 48 and 72 h compared to ECM-detached SKOV3 parental cells (Figure 2B). Given that PRDX2 deficiency did not compromise cell viability in ECM detachment, the abrogated colony formation visualized in PRDX2-deficient SKOV3 cells in soft agar was due to compromised cell proliferation.

 

FIGURE 2. Elimination of PRDX2 abrogates cell proliferation in ECM-detached SKOV3 cells. Approximately 50,000 parental SKOV3 cells and PRDX2-deficient SKOV3 cells were plated per well of a non-adherent (poly-HEMA-coated) 6-well plate in triplicate. At 24, 48, and 72 h, cells from each well were removed, dead cells were stained from trypan blue, and cells were counted using a hemocytometer. This experiment was completed three times. From these counts, percent cell viability (panel A) and cell proliferation (panel B) from all experiments were calculated. *, p < 0.05 as determined using a two-tailed t test. Error bars represent SEM. 

3.3. PRDX2 Knockdown Has no Effect on Proliferation or Viability of ECM Attached SKOV3 Cells 

Given that PRDX2 deficiency affected cell proliferation of ECM-detached SKOV3 cells, PRDX2 deficiency and its effects on proliferation and viability of ECM-attachedcellswerestudied.SKOV3parental and SKOV3 PRDX2-deficient cells were grown on adherent 6-well plates and analyzed after 24, 48, and 72 h. PRDX2 deficiency had no effect on proliferation (Figure 3A) or cell viability (Figure 3B) in ECM-attached cells. In aggregate, these data suggest that PRDX2 deficiency does not impact ECM-attached cells, indicating that PRDX2 deficiency does not play a large role in cell proliferation or viability of cells at the primary tumor.

 

FIGURE 3. PRDX2 deficiency does not affect cell proliferation or cell viability in ECM-attached SKOV3 cells. SKOV3 Parental and SKOV3 PRDX2 deficient cells were plated on adherent 6-well plates in triplicate, stained with trypan blue at 24, 48, and 72 h, and counted using a hemocytometer. This experiment was completed three times, and total proliferation (panel A) and cell viability (panel B) were calculated. *, p < 0.05 as determined using a two-tailed t test. Error bars represent SEM.

4. DISCUSSION 

Delineating cellular mechanisms involved in mediating cell survival and proliferation in metastatic ovarian cancer is critical in the development of therapeutics for this deadly disease. The present study works to unravel the importance of PRDX2 in the survival and proliferation of ECM-detached SKOV3 ovarian carcinoma cells. Here, we report that PRDX2 plays a critical role in the growth of SKOV3 cells in soft agar through compromising cellular proliferation. While PRDX2 deficiency severely abrogates proliferation in ECM-detached SKOV3 cells, elimination of PRDX2 has no impact on proliferation of ECM-attached SKOV3 cells. Interestingly, it appears that PRDX2 is not critical in maintaining cell viability in either ECM-attached or ECM-detached SKOV3 cells (Figure 4).

 

FIGURE 4. Effects of PRDX2 deficiency on ECM-detached metastatic ovarian carcinoma working model. PRDX2 deficiency does not impact the proliferation or viability of ECM-attached SKOV3 cells. However, elimination of PRDX2 severely compromises proliferation thus anchorage-independent growth in ECM-detached SKOV3 cells. Thus, PRDX2 elimination may be an interesting therapeutic target to decrease proliferation of ECM-detached metastatic ovarian carcinoma cells. 

This is the first (to our knowledge) to study the importance of PRDX2 in the proliferation and cell viability of ECM-detached metastatic ovarian carcinoma cells and adds to a growing body of literature that expression of peroxiredoxins are critical in tumor development, metastasis, and drug resistance in many tumor types and contexts [13–27]. Known to be highly efficient in eliminating hydrogen peroxide [13], our studies suggest that PRDX2 plays a unique role in promoting metastasis of epithelial ovarian cancer cells compared to other hydrogen peroxide-eradicating antioxidant enzymes. Most recently, we reported that elimination of catalase specifically abrogates cell viability in ECM-detached metastatic ovariancarcinomacells[7].Furthermore,recent studies unveiled that elimination of peroxiredoxin 1 (PRDX1) severely compromises both proliferation and viability of ECM-detached metastatic ovarian carcinoma cells [28]. Here, we report that PRDX2 deficiency uniquely abrogates proliferation in ECM-detached metastatic ovarian carcinoma cells. In aggregate, hydrogen peroxide-eliminating enzymes play distinctive and non-redundant roles in ovarian cancer metastasis, which underscores the need to study other antioxidant enzymes and their role in this process. 

Collectively, these data highlight the potential success of other already discovered inhibitors targeting antioxidant enzymes for the treatment of metastatic ovarian cancer. In particular, the inhibitor Conoidin A, is found to specifically inhibit PRDX2 in human epithelial cells [29]. Investigation of this inhibitor and its potential to treat and prevent metastatic ovarian carcinoma could lead to new treatment options for this deadly disease. Additionally, therapies known to serve as ROS inducers, such as histone deacetylase inhibitors, could also be potential candidates for treating late-stage ovarian carcinoma patients [30]. However, further exploration into the potential utility of these therapies is still needed. 

In summary, we find that PRDX2 plays a novel role in proliferation of ECM-detached metastatic ovarian carcinoma cells. Furthermore, these data highlight PRDX2 as a potential therapeutic target for metastatic ovarian carcinoma. Given the low 5-year survival rate associated with late-stage diagnoses [1, 2], it is evident that new and improved therapies to treat these patients are needed. Future studies aimed at accessing PRDX2 as a therapeutic target for ECM-detached metastatic cancer cells may be critical in the development of therapies designed to eradicate metastatic ovarian carcinoma.

5. CONCLUSION 

Here, we investigated the role of PRDX2 in the growth of ECM-detached metastatic ovarian carcinoma cells. PRDX2 deficiency resulted in abrogated growth of anchorage-independent SKOV3 cells in soft agar assays. Furthermore, PRDX2 deficiency compromised cell proliferation of ECM-detached cells. Interestingly, PRDX2 deficiency did not affect proliferation or cell viability of ECM-attached cells, suggestingthatPRDX2deficiencydoesnotimpact cell growth at the primary tumor. In conclusion, therapies targeting PRDX2 may have potential in treating metastatic ovarian carcinoma. 

ACKNOWLEDGMENTS 

The authors would like to acknowledge the funding support awarded through the Helen Kuhn Carey research fund (CADV), Beta BetaBeta Biological Honors Society (KMJ), and the Biology Department at Saint Mary’s College. The authors declare no conflicts of interest. 

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