Bladder Cancer Chemosensitivity Is Affected by Paraoxonase-2 Expression

The goal of the current study was to identify potential roles of paraoxonase-2 in bladder carcinogenesis. T24 bladder cancer cells were transfected with plasmids inducing paraoxonase-2 silencing or overexpression. Upon the selection of clones stably down- or upregulating paraoxonase-2, cell proliferation, migration, and the production of reactive oxygen species were evaluated, before and after treatment with cisplatin and gemcitabine, used alone or in combination. The activity levels of both caspase-3 and caspase-8 were also analyzed. shRNA-mediated gene silencing and the overexpression of paraoxonase-2 revealed that the enzyme was able to promote both the proliferation and migration of T24 cells. Moreover, the knockdown of paraoxonase-2 was significantly associated with a reduced cell viability of T24 cells treated with chemotherapeutic drugs and led to both an increase of reactive oxygen species production and caspase-3 and caspase-8 activation. Conversely, under treatment with anti-neoplastic compounds, a higher proliferative capacity was found in T24 cells overexpressing paraoxonase-2 compared with controls. In addition, upon enzyme upregulation, both the production of reactive oxygen species and activation of caspase-3 and caspase-8 were reduced. Although further analyses will be required to fully understand the involvement of paraoxonase-2 in bladder tumorigenesis and in mechanisms leading to the development of chemoresistance, the data reported in this study seem to demonstrate that the enzyme could exert a great impact on tumor progression and susceptibility to chemotherapy, thus suggesting paraoxonase-2 as a novel and interesting molecular target for effective bladder cancer treatment.


Introduction
Bladder cancer (BC) is the ninth most common cancer in the world. The incidence of this neoplasm is gender-related, meaning that its frequency is 3-4 times higher in men than in women [1]. Moreover, BC is more frequent at an advanced age and the greatest number of cases occurs in people over 75 years old [1]. Smoking habits and exposure to some chemical compounds also increase the risk of developing BC [1]. Approximately 25% of patients diagnosed with BC display muscle-invasive disease, which is including vascular cells [22]. Upon translation, protein is incorporated into the lipid bilayer thanks to its transmembrane domain. PON2 is located in the cell plasma membrane, the endoplasmic reticulum (ER), and mitochondria. Nevertheless, data on the predominant distribution of PON2 inside the cell are rather controversial because of its dynamic translocation from the cytosol to the plasma membrane in response to oxidative stress. Oxidative stress is responsible for intracellular calcium release and subsequent peroxidation of the lipid bilayer at the plasma membrane.
Hagmann et al. demonstrated that the calcium signal triggers the translocation of PON2 to the plasma membrane [23]. The main function exerted by the enzyme within cells is mainly related to its antioxidant activity. Indeed, Ng et al. demonstrated that PON2 contributes, together with other intracellular enzymes and systems, to protecting cells from oxidative stress [24]. Due to its intracellular localization, as well as its antioxidant function, PON2 was reported to display an anti-apoptotic role, with potential consequences for tumor cell behavior [25]. Over the past years, many studies have described the involvement of PON2 in cancer. In particular, PON2 expression was shown to be increased in some solid tumors, including pancreatic cancer [26], glioblastoma multiforme [27], and recently BC [28]. Concerning bladder cancer, in our previous study, we demonstrated that the enzyme levels were significantly higher in tumors compared with adjacent normal looking tissue samples from BC patients. Moreover, preliminary results obtained from analyses performed on bladder cancer cell lines seemed to suggest that PON2 is able to promote cell proliferation and resistance to oxidative stress [28].
The analyses carried out in the present study aimed to further investigate the role of PON2 in BC. Enzyme silencing and overexpression were induced in the T24 bladder cancer cell line. Subsequently, T24 cell proliferation, migration, and susceptibility to oxidative stress were evaluated, before and after treatment with cisplatin and gemcitabine. In addition, the activity levels of both caspase-3 and caspase-8, as key regulators of the apoptotic response, were investigated.

Cell Lines and Culture Conditions
The human bladder cancer cell line T24, obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA), was maintained in DMEM/F12 medium, as previously described [28].
To induce the overexpression of PON2, cells were seeded in 6-well plates (2.4 x 10 5 cells/well) the day before transfection and were then transfected with the pcDNA3-PON2 plasmid vector (3µg/well). Control cells were transfected with the empty vector (pcDNA3) or treated with transfection reagent only (mock).
Both procedures were performed using FuGENE HD Transfection Reagent (Promega, Madison, WI, USA), following the manufacturer's instructions. Forty-eight hours from the beginning of the transfection, culture medium was discarded and replaced with complete medium containing puromycin (1 µg/mL) or geneticin (800 µg/mL), in order to select cellular clones downregulating or overexpressing PON2, respectively. For all subsequent experiments, puromycin-and geneticin-resistant cells were maintained in complete selection medium. The efficiency of PON2 silencing and overexpression in T24 cells were evaluated by Real-Time PCR and Western blot analysis.

Real-Time PCR
Quantitative Real-Time PCR was performed as reported elsewhere [28]. The relative expression of PON2 was calculated by the 2 −∆∆Ct method. Each experiment was performed in triplicate and independently repeated three times.

Western Blot Analysis
A Western blot assay was set up to evaluate PON2 protein levels, as previously reported [28]. Each experiment was performed in triplicate and independently repeated three times.

Monolayer Wound Healing Assay
To evaluate the migration capacity, T24 cells were seeded in 6-well plates (6 × 10 5 cells/well) and allowed to attach and grow up to 90-100% confluency. Cell monolayers were scratched using a sterile 200 µL pipette tip to make a vertical wound. Wounded monolayers were then washed three times to remove cell debris and incubated in medium containing 0.5% FBS (Fetal Bovine Serum). Upon medium replacement, cells were monitored under a microscope equipped with a camera (Deiss) and photographed at 6, 12, and 24 h. Each experiment was performed in triplicate and independently repeated three times.

Detection of Intracellular Oxidative Stress
To evaluate the intracellular oxidative stress, the oxidation of H 2 DCF-DA (Sigma-Aldrich, St. Louis, MO, USA) was measured as previously reported [31]. ROS production was evaluated in untreated cells (0 h) and at different time points (12,24,36,48, and 72 h) after starting the treatment with drugs. Each experiment was performed in triplicate and independently repeated three times.
Each experiment was performed in triplicate and independently repeated three times.

Statistical Analysis
Data were analysed using GraphPad Prism software version 8.00 for Windows (GraphPad Software, San Diego, CA, USA). Differences between groups were determined using one-way analysis of variance (ANOVA). A p-value < 0.05 was accepted as statistically significant.

Efficiency of PON2 shRNA-Mediated Knockdown and Overexpression in T24 Cells
T24 cells were transfected as reported in the materials and methods section. To evaluate the efficiency of transfection, PON2 mRNA and protein levels were evaluated by Real-Time PCR and Western blot analysis, respectively.

Effect of PON2 Knockdown and Overexpression on T24 Cell Proliferation and Migration
To examine the role of PON2 in tumor cell metabolism and analyze the biological effect associated with enzyme knockdown or overexpression, the cell viability and migration capacity were analyzed at different time points in T24 cells.
The effect of PON2 knockdown and overexpression on cell proliferation was evaluated by the MTT assay. As shown in Figure 2A, enzyme knockdown led to a significant (p < 0.05) decrease in cell growth of T24 cells starting from the 24 h time-point with respect to control cells (mock), as well as those treated with an empty vector (pLKO.1-puro). On the other side, enzyme upregulation was associated with a significant (p < 0.05) increase in cell proliferation of T24 cells starting from the 48h time-point ( Figure 2B). To assess the biological influence of PON2 on cell migration, T24 cells transfected with pLKO.1-647 or pcDNA3-PON2 plasmids were subjected to a wound healing assay. Compared with the mock-treated cells, the migration ability of T24 cells significantly (p < 0.05) decreased at 6h (32% reduction) and 12 h (50% reduction) time-points after PON2 silencing ( Figure 3A-C). On the contrary, the overexpression of PON2 led to a promotion of the migration ability (p < 0.05) of T24 cells at 12 h (13% increase) and 24h (25% increase) time-points with respect to the control ( Figure 3B-D).

PON2 Influence on the Sensitivity of T24 Cells to Treatment with Chemotherapeutic Drugs
The MTT colorimetric assay was used to evaluate the effect of chemotherapeutic drugs administrated as reported in the materials and methods section on cell viability. In agreement with previously reported results [29], chemotherapeutic drugs significantly reduced the cell viability of T24 control cells (Figure 4). As demonstrated in Figure 4A, treatment with cisplatin alone led to a significant (p < 0.05) decrease in the proliferative capacity of cells transfected with pLKO.1-647 compared with that of the mock, after 36h (15% reduction) and 48h (25% reduction). Treatment with gemcitabine alone or with both drugs was significantly (p < 0.05) associated with a reduction in the viability of PON2 downregulated cells (pLKO.1-647) with respect to that of the mock at 24 h (14% reduction for gemcitabine and 16% reduction for both drugs), 36 h (27% reduction for gemcitabine and 37% reduction for both drugs), and 48 h (28% reduction for gemcitabine and 59% reduction for both drugs) time points ( Figure 4A).
Conversely, the induction of PON2 overexpression led to a significant (p < 0.05) enhancement in the proliferative capacity of T24 cells (pcDNA3-PON2) compared with controls (mock) at the 24 h time-point (21% increase for cisplatin, 28% increase for gemcitabine, and 36% reduction for both drugs). However, this effect seemed to be lost at higher time points ( Figure 4B).

Effect of PON2 Expression on the ROS Production of T24 Cells Treated with Chemotherapeutic Drugs
To assess the effect of the induction of PON2 knockdown and overexpression on oxidative stress, intracellular Reactive Oxygen Species (ROS) levels were evaluated in T24 cells after incubation with chemotherapeutic drugs, as well as in untreated cells. Chemotherapeutic treatment led to a significant increase of ROS production in T24 control cells ( Figure 5). As shown in Figure 5A, upon treatment with cisplatin, ROS production was significantly (p < 0.05) higher in PON2 downregulated cells (pLKO.1-647) compared with mock-treated cells at the 48h time point only (1.56-fold increase). Incubation with gemcitabine alone or with the cisplatin/gemcitabine combination led to significantly (p < 0.05) higher ROS levels in T24 cells transfected with pLKO.1-647 compared with the mock, both at 36h (1.30-fold increase for gemcitabine and 1.61-fold increase for both drugs) and 48h (1.85-fold increase for gemcitabine and 2.55-fold increase for both drugs) time points ( Figure 5A).
On the contrary, upon treatment with cisplatin and gemcitabine, used alone or in combination, intracellular ROS production was significantly (p < 0.05) lower in PON2 overexpressing cells compared with the mock at 48h (1.37-fold decrease for cisplatin, 1.96-fold decrease for gemcitabine, and 3.03-fold decrease for both drugs) and 72 h (2.00-fold decrease for cisplatin, 2.78-fold decrease for gemcitabine, and 4.00-fold decrease for both drugs) time points, while cisplatin alone had no significant effect at 24 h (1.26-fold decrease for gemcitabine and 1.69-fold decrease for both drugs) ( Figure 5B).

PON2 Protects T24 Cells against Apoptosis through Caspase-3 and Caspase-8 Activation
In order to investigate the contribution of PON2 to BC tumorigenesis by promoting apoptotic escape, caspase-3 and caspase-8 activities were evaluated in T24 cells expressing either reduced or elevated PON2 levels, as well as corresponding controls, upon treatment for 24 h with chemotherapeutic drugs.
Treatment was able to induce a significant (p < 0.05) activation of both caspase-3 and caspase-8 in T24 cells transfected with pLKO.1-647 compared with the mock. In particular, caspase-3 activation was higher in PON2 downregulated cells treated with both compounds (1.41-fold increase) than in T24 cells upon incubation with cisplatin (1.20-fold increase) or gemcitabine (1.21-fold increase) used alone. On the other hand, the activation of caspase-8 did not seem to be significantly different (pLKO.1-647 versus mock) among treatments (1.28-fold increase for cisplatin, 1.26-fold increase for gemcitabine, and 1.23-fold increase for both drugs) ( Figure 6A,B). On the other hand, T24 cells overexpressing PON2 showed significantly (p < 0.05) lower levels of caspase-3 and caspase-8 activity than those determined in the mock. Concerning caspase-3, the reduction of activity levels was enhanced in PON2 overexpressing cells that underwent combined treatment (1.35-fold reduction) compared with that observed after incubation with cisplatin (1.18-fold reduction) or gemcitabine (1.19-fold reduction) alone. Conversely, the decrease of caspase-8 activity detected in T24 cells upon enzyme overexpression (pcDNA3-PON2) did not seem to be significantly altered based on treatment with different compounds or the drug combination (1.25-fold reduction for cisplatin, 1.30-fold decrease for gemcitabine, and 1.28-fold decrease for both drugs) ( Figure 6C,D).

Discussion
In recent years, great efforts have been made to identify molecules that are involved in BC tumorigenesis and/or are able to reflect changes in neoplastic tissue, thus acting as potential biomarkers for early and noninvasive BC detection [32]. Moreover, chemoresistance remains a critical problem in patients with BC due to the lack of effective second-line therapies, and therefore, a better understanding of chemoresistance mechanisms in BC is urgently required [33].
The known mechanisms by which BC becomes resistant to platinum drugs are multifactorial, including: (1) Reduced intracellular drug uptake consequent to a downregulation of CTR1; (2) promoted drug efflux by increasing cellular glutathione (GSH), as shown by Kotoh et al., who demonstrated that the content of GSH was significantly increased in the cisplatin-resistant BC cell line (T24/DDP7), compared with the level of GSH in the T24 parental line; 3) enhanced DNA damage repair through the increased activity of nucleotide excision repair (NER) and homologous recombination repair (HR); 4) impairments of the apoptotic pathways, such as the loss of expression of p53 [34,35].
However, chemotherapeutic drugs can increase ROS levels, and most cancer cells treated with chemotherapeutics suffer from ROS-mediated apoptosis [36].
Some cancer cells can evolve and develop mechanisms to escape ROS-mediated apoptosis and, thus, acquire tolerance to anti-cancer drugs [37]. The ROS system has a dual function that can either induce apoptosis or allow cells to adapt to various environments; thus ROS regulation has been proposed as a critical target for developing anticancer drugs [38].
There is a growing scientific consensus that recognizes a possible role of PON2 in the physiopathology of cancer. In fact, several studies have reported the upregulation of PON2 in different human cancers [25,39], including BC [28].
In this work, the potential role of PON2 in the in vitro tumorigenicity of BC cells was investigated. PON2 knockdown and overexpression were induced in T24 human bladder cancer cells further treated with chemotherapeutic drugs. Subsequent analyses were carried out to evaluate the effect of the modulation of PON2 expression on cell proliferation, migration, response to oxidative stress, and apoptosis.
The reported data demonstrated that PON2 expression exerts a positive influence on T24 cell proliferation and migration, thus highlighting its potential role in promoting bladder tumorigenesis. In particular, PON2 overexpression led to an increase in the cell viability of T24 cells treated with chemotherapeutic drugs, while PON2 downregulation was associated with a significant reduction of their proliferative capacity. As reported in the literature, these results confirmed that PON2 is involved in the survival and proliferation of cancer cells [40].
Moreover, ROS production, known to be induced upon treatment with cisplatin and gemcitabine [31,41], was significantly affected by PON2 dysregulation. Indeed, PON2 downregulation led to an increase of ROS levels in T24 bladder cancer cells treated with chemotherapeutic agents. On the contrary, PON2 upregulation significantly counteracted the increase in cellular ROS production in response to oxidative stress triggered by chemotherapeutic compounds. This evidence is in accordance with data presented in the literature showing that PON2 enzyme activity protected macrophages, vascular, and other cells against oxidative stress, whereas PON2 downregulation reversed these effects [24,42].
The results obtained from further in vitro experiments demonstrated the influence of PON2 expression on drug-induced caspase activation. After treatment with chemotherapeutic drugs, we observed a comparative increase in caspase activity upon PON2 silencing in T24 cells, and a reduction in PON2-overexpressing cells.
Our results are consistent with data presented in the literature coming from other studies which have investigated the involvement of PON2 in cancer. In particular, the role played by PON2 as a metabolic regulator has been confirmed in a study carried out by Nagarajan et al. In pancreatic ductal adenocarcinoma (PDAC) cell lines, the authors demonstrated that PON2 transcriptional repression is responsible for affecting the transport activity of glucose transporter 1 and subsequently, for an inhibition of PDAC tumor growth and metastasis [25]. Similarly, Tseng et al. demonstrated that valproic acid, decreasing the PON2 expression, made glioblastoma multiforme-derived cell lines more sensitive to oxidative damage and cell death [27].
It is well-known that cancer cell responses to apoptotic insults are significantly affected by the cellular redox status. On the other hand, it is also well-established that oxidative stress is closely linked to cell death and cancer [43][44][45]. The anti-apoptotic effect exerted by PON2 is reasonably strictly related to its intracellular localization, mainly within the membranous systems of the ER and mitochondria. Altenhofer et al. recognized the ability of PON2 to prevent mitochondria-derived superoxide formation, which is responsible for the activation of the apoptotic pathway in response to cardiolipin peroxidation and subsequent cytochrome c release [46].
Many studies have reported results that have facilitated understanding of the anti-apoptotic roles of PON2. The activation of executioner caspase-3 and subsequent induction of apoptosis can arise from longer-lasting ER stress, which triggers the unfolded protein response pathway [47]. Horke et al. demonstrated that human umbilical vein endothelial cells EA.hy 926, stably overexpressing PON2, displayed a significant reduction of caspase 3/7 activation, although treated with the unfolded protein response (UPR)-inducing reagent tunicamycin. Conversely, in PON2-silenced EA.hy 926 cells, treatment with tunicamycin led to an opposite effect and an increase of the caspase 3/7 activation status [26]. Further results demonstrated that PON2 could confer protection against ER-stress-induced caspase-3 activation, by the modulation of calcium homeostasis [48].
The data reported in the present in vitro study demonstrate that PON2 contributes to bladder tumorigenesis by promoting both cell proliferation and migration. Moreover, the enzyme was found to play a significant role in BC cell resistance to chemotherapeutic treatment with cisplatin and gemcitabine.

Conclusions
In this light, PON2 overexpression in BC could represent an adaptive mechanism of tumor cells to escape/survive cell death and apoptosis induced by chemotherapy.
Although further analyses will be required to deeply investigate the molecular mechanisms by which PON2 could participate in bladder carcinogenesis, our study clearly demonstrates, for the first time, the impact of the enzyme on tumor progression and the susceptibility of tumor cells to chemotherapeutics, thus suggesting a potential use of PON2 as an interesting molecular target for BC therapy. Further studies are required to solve the PON2 protein structure in order to develop selective inhibitors of the enzyme, which could be combined with chemotherapeutic drugs to improve the BC outcome. However, given the wide expression of PON2, it is necessary to design a proper drug-delivery strategy in order to selectively address these molecules to the BC cells.

Conflicts of Interest:
The authors declare no conflicts of interest.