Treatment of Human Placental Choriocarcinoma Cells with Formaldehyde and Benzene Induced Growth and Epithelial Mesenchymal Transition via Induction of an Antioxidant Effect

Cigarette smoke (CS) causes about 480,000 deaths each year worldwide, and it is well-known to have harmful effects on the human body, leading to heart disease, stroke, lung cancer, and cardiovascular problems. In this study, the effects of formaldehyde (FA) and benzene (Bz), the main components of CS, on cell proliferation and epithelial mesenchymal transition (EMT) of JEG-3 human choriocarcinoma cells were examined to confirm the relationship between CS components and placenta carcinoma. Upon MTT assay, FA (10−8 M to 10−5 M) and Bz (10−11 M to 10−8 M) increased JEG-3 cell proliferation. Western blot assay revealed that the protein expression of cyclin D1 and E1 increased, while the levels of p21 and p27 were reduced following treatment. In Scratch assay, FA (10−8 M and 10−5 M) and Bz (10−11 M and 10−8 M) increased migration of JEG-3 cells at 24 h and 48 h compared with that at 0 h. In addition, the expression of the epithelial marker, E-cadherin, was significantly decreased, while the expression of the mesenchymal marker, N-cadherin, was significantly increased by FA (10−8 M and 10−5 M) and Bz (10−11 M and 10−8 M). snail and slug transcriptional factors were associated with EMT, which were also up-regulated by FA and Bz, indicating that FA and Bz lead to an increase in the EMT process in JEG-3 choriocarcinoma cells. We further evaluated reactive oxygen species (ROS) and activation of antioxidant effect using dichlorofluorescin diacetate (DCFH-DA) and Western blot assay. FA and Bz increased the ROS production and an antioxidant related marker, Nrf2, in JEG-3 cells. However, eIF2α levels were reduced by FA and Bz via activation of the antioxidant reaction. Taken together, these results indicated that FA and Bz induce the growth and migration of human choriocarcinoma cells via regulation of the cell cycle and EMT and activation of ROS and antioxidant related markers.


Introduction
Cigarette smoke (CS) causes about 480,000 deaths each year worldwide, and is well known to be harmful to humans, being responsible for heart disease, stroke, lung cancer, and cardiovascular problems. Moreover, CS is known to be one of the largest risk factors of cancer via a variety of bio-mechanisms [1,2]. In addition, previous studies reported that CS can cause death or survival of different cancer cells at the same time [3,4].
Many previous studies have investigated the relationship between lung cancer and smoking [5], and recent studies have focused on women as the proportion of female smokers has increased worldwide [6]. This causes an increase in female diseases associated with CS. In addition, maternal smoking during pregnancy also influences the health of the fetus and fertility, leading to low birth weight, premature delivery, spontaneous abortion, placental abruption, and ectopic pregnancy [7].
Based on these biological processes, we specifically investigated the effects of FA and Bz on the proliferation and metastasis of JEG-3 choriocarcinoma cells to provide their roles in the induction of cancer progression via ROS production and antioxidation regulated by Nrf2 when FA and Bz are exposed.

MTT Assay
JEG-3 cells were seeded at a 5 × 10 3 cells per well in 96-well plates (SPL Life Science, Seoul, Korea) in a humidified atmosphere of 5% CO 2 at 37 • C. After the cells were incubated with phenol red-free DMEM containing 5% charcoal dextran fetal bovine serum (CD-FBS) medium for 24 h, they were treated with various concentrations of FA and Bz (FA: 10 −8 to 10 −5 M and Bz: 10 −11 to 10 −8 M) in phenol red-free DMEM with 5% CD-FBS supplemented with 0.1% DMSO for 9 days. During this period, the media were changed to the same new media every third day. DMSO was used as a vehicle to carry the chemicals to the media. 3-(4-5-dimethylthiazol-2-yl)-2.5-dyphenyltetrazolium bromide (MTT; Sigma-Aldrich) solution was used to confirm increased cell proliferation. Each well of the 96-well plates was treated with 10 µL (5 mg/mL solution) and the plates were incubated for 3 h at 37 • C in a humidified atmosphere of 5% CO 2 . Supernatants were removed and 100 µL DMSO was added to each well to dissolve resultant formazan crystals. Each well was measured using an ELISA reader (VERSA man, Corp., Molecular Devices, Sunnyvale, CA, USA) at an optical density (OD) value of 540 nm and then used to calculate the number of viable cells.

Scratch Assay
JEG-3 cells were cultured to 80% of confluent growth in each well of 6-well plates (SPL Life Science, Seoul, Korea) at 37 • C in a humidified atmosphere of 95% and 5% CO 2 . To perform the scratch assay, a region with the same length and width was scratched, after which JEG-3 cells were treated with negative control (1% DMSO) or formaldehyde (10 −8 M and 10 −5 M) and Bz (10 −11 M and 10 −8 M) with medium containing 5% CD-FBS, then incubated for 48 h. Next, images were captured with a microscope under 40× magnification at 0 h, 24 h, and 48 h after treatment. The percentage of unrecovered scratched length was measured by dividing the uncovered area at 0 h, 24 h, and 48 h by the initial wound area at time zero. Migration was measured and quantitative analysis was conducted using an Olympus Cellsens dimension program.

Determination of ROS Production
Intracellular ROS in JEG-3 cells was observed as previously described using 20,70-dichlorofluorescein diacetate (DCF-DA) (22). JEG-3 cells were seeded at 2 × 10 4 cells per well in a 96-well plate with phenol-Red free DMEM media containing 5% CD-FBS. After 24 h, the medium was changed to a new medium with FA (10 −8 M and 10 −5 M) or Bz (10 −11 M and 10 −8 M). After 72 h incubation, the culture medium was removed, and each well was treated with new medium containing 200 µL DCF-DA solution (10 mM-in PBS) for 30 min. This plate was then placed on a shaker for 10 min in a dark room at room temperature, after which the fluorescence was measured using a fluorescence microscope (IX73 fluorescence microscope, Olympus, Japan).

Data Analysis
All experiments were conducted at least three times, and all data were analyzed with the Graph-pad Prism software (San Diego, CA, USA). Data were expressed as the means ± SD and analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. The p-values < 0.05 were considered to be statistically significant.

FA and Bz Induced Increased Cell Proliferation
The cell proliferation assay using MTT was conducted to investigate the effects of FA and Bz on proliferation of JEG-3 placenta carcinoma cells. As shown in Figure 1, FA and Bz induced increased cell proliferation within the range of concentrations tested (10 −11 M to 10 −5 M) relative to the control. Additionally, FA and Bz were shown to upregulate cell proliferation ( Figure 1A

Effects of CS Components on Protein Expression of Cell Cycle Regulatory Genes
Based on the results of the MTT assay, Western blot was performed to evaluate the effects of FA and Bz on the expression of cell cycle related genes such as cyclin D1, cyclin E1, p21, and p27.

Effects of CS Components on Protein Expression of Cell Cycle Regulatory Genes
Based on the results of the MTT assay, Western blot was performed to evaluate the effects of FA and Bz on the expression of cell cycle related genes such as cyclin D1, cyclin E1, p21, and p27. FA and Bz were observed to increase the protein expressions of cyclin D1 and cyclin E1 and decrease the protein expression of p21 and p27 in a dose dependent manner (Figure 2A Bz were observed to increase the protein expressions of cyclin D1 and cyclin E1 and decrease the protein expression of p21 and p27 in a dose dependent manner (Figure 2A,B). FA and Bz affect cancer cell proliferation through induction of the cell cycle progression, which corresponds to the induction of cell proliferation by the treatment of JEG-3 cells with FA and Bz.

FA and Bz Induced Activation of Migration in JEG-3
A scratch assay was performed to investigate the effects of FA and Bz on the migration of JEG-3 placenta choriocarcinoma cells as seen in Figure 3. After 48 h of treatment with FA and Bz, the mobility of cancer cells through the uncovered area was measured to determine the change. The uncovered area decreased significantly in response to treatment with both FA and Bz relative to DMSO treated cells, and this decrease was shown in a dose-dependent manner ( Figure 3A,B). These results indicate that FA and Bz induce the ability of JEG-3 placenta carcinoma cells to migrate. After protein extraction, Western blot assay was conducted to conform to the protein expression of cell cycle related genes (cyclin D1, cyclin E1), cell cycle arrest genes (p21 and p27), and housekeeping genes (glyceraldehyde 3-phosphate dehydrogenase (GAPDH)). Quantification of cyclin D1, cyclin E1, p21, and p27 protein was conducted by measuring band densities using a CS analyzer 4 (ATTO, Corp., Japan), and their protein levels were normalized by the band value of GAPDH. Values shown are the means ± SD. * mean values were significantly different from 0.1% DMSO (control), p < 0.05. (Dunnett's multiple comparison test).

FA and Bz Induced Activation of Migration in JEG-3
A scratch assay was performed to investigate the effects of FA and Bz on the migration of JEG-3 placenta choriocarcinoma cells as seen in Figure 3. After 48 h of treatment with FA and Bz, the mobility of cancer cells through the uncovered area was measured to determine the change. The uncovered area decreased significantly in response to treatment with both FA and Bz relative to DMSO treated cells, and this decrease was shown in a dose-dependent manner ( Figure 3A,B). These results indicate that FA and Bz induce the ability of JEG-3 placenta carcinoma cells to migrate.

Effects of CS Components on Protein Expression of EMT Progress Genes
Western blot assays were conducted using E-cadherin, N-cadherin, Snail, and Slug antibodies, which are considered important EMT markers, to identify the effect of FA and Bz on protein expression [33]. As shown in Figure 4A, FA significantly decreased the protein expression of Ecadherin, but increased that of N-cadherin, Snail, and slug. Bz also significantly decreased the expression of E-cadherin, but increased that of N-cadherin, Snail, and slug ( Figure 4B). Moreover, the

Effects of CS Components on Protein Expression of EMT Progress Genes
Western blot assays were conducted using E-cadherin, N-cadherin, Snail, and Slug antibodies, which are considered important EMT markers, to identify the effect of FA and Bz on protein expression [33]. As shown in Figure 4A, FA significantly decreased the protein expression of E-cadherin, but increased that of N-cadherin, Snail, and slug. Bz also significantly decreased the expression of E-cadherin, but increased that of N-cadherin, Snail, and slug ( Figure 4B). Moreover, the changes in the expression of EMT markers were dose dependent ( Figure 4A,B). These results indicate that FA and Bz induced EMT ability though regulation of the transcription factors Snail and slug, which also are associated with alterations in expression of the cell surface markers E-cadherin and N-cadherin. changes in the expression of EMT markers were dose dependent ( Figure 4A,B). These results indicate that FA and Bz induced EMT ability though regulation of the transcription factors Snail and slug, which also are associated with alterations in expression of the cell surface markers E-cadherin and N-cadherin.

FA and Bz Activated ROS Synthesis and Increased Antioxidant Factor
Because up-regulation of cell proliferation and EMT by FA and Bz was observed (Figures 1 and 3), we further evaluated their effects on ROS activation and protein expression of the anti-oxidant factor Nrf2 and the apoptosis related gene p-eIF2α. Fluorescence microscopy analysis using DCF-DA

FA and Bz Activated ROS Synthesis and Increased Antioxidant Factor
Because up-regulation of cell proliferation and EMT by FA and Bz was observed (Figures 1 and 3), we further evaluated their effects on ROS activation and protein expression of the anti-oxidant factor Nrf2 and the apoptosis related gene p-eIF2α. Fluorescence microscopy analysis using DCF-DA solution revealed that FA and Bz significantly increased activation of ROS in JEG-3 cells compared to that of the control as shown in Figure 5. Western blot assay revealed that FA and Bz increased the protein expression of anti-oxidant factors, but decreased the protein expression of the ER stress-apoptosis related gene p-eIF2α ( Figure 6A,B). These findings indicated that FA and Bz may induce cancer cell survival and migration of JEG-3 cells via activation of antioxidation. Health 2017, 14, 854 10 of 16 solution revealed that FA and Bz significantly increased activation of ROS in JEG-3 cells compared to that of the control as shown in Figure 5. Western blot assay revealed that FA and Bz increased the protein expression of anti-oxidant factors, but decreased the protein expression of the ER stress-apoptosis related gene p-eIF2α ( Figure 6A,B). These findings indicated that FA and Bz may induce cancer cell survival and migration of JEG-3 cells via activation of antioxidation.

Discussion
A previous study reported that maternal cigarette smoking or exposure to cigarette smoke (CS) alters human placental development via dysregulation of cytotrophoblast proliferation or cellular responses to oxygen [34,35], indicating that cigarette smoke was associate with harmful effects on human placenta or human choriocarcinoma. Accordingly, recent studies have focused on the effects of components of cigarette smoke on the human body.

Discussion
A previous study reported that maternal cigarette smoking or exposure to cigarette smoke (CS) alters human placental development via dysregulation of cytotrophoblast proliferation or cellular responses to oxygen [34,35], indicating that cigarette smoke was associate with harmful effects on human placenta or human choriocarcinoma. Accordingly, recent studies have focused on the effects of components of cigarette smoke on the human body.
Nicotine, which is the main constituent among about 5000 toxic components in CS, is a contributing factor to cancer onset, growth, and migration [36]. Moreover, nicotine has potential effects on the endometrium. A previous study showed that nicotine induced endometrial decidualization by decreasing the weight of the uterus after mechanically induced decidualization [37]. Another group also recently observed decreased endometrial proliferation though nitric oxide (NO)-mediated pathways [38]. Moreover, another compound of CS, benzo a pyrene (BaP), is also known to alter cell proliferation of endometrial stroma and epithelial cells via arylhydrocarbon receptor AhR [39].
In the present study, we investigated the effects of FA and Bz, which are the principal components of CS, on cell proliferation and epithelial mesenchymal transition (EMT) of JEG-3 human placenta choriocarcinoma cells. FA significantly increased cell proliferation at a low concentration of 10 −8 M, while Bz significantly increased cell proliferation at the considerably low concentration of 10 −11 M. Therefore, we confirmed via Western blot that there were changes in expression of proteins related to the cell cycle such as cyclin D1, cyclin E1, p21, and p27. The cell cycle plays an important role in cells, leading to division, and many proteins are involved in cell cycle checkpoints, including cyclin-dependent kinases (CDKs) and cell cycle arrest genes. Moreover, cyclin D1 and cyclin E1 are known to be major inducers of the cell cycle [40,41]. In a previous study, CS induced survival or migration of cancer cell, while it repressed CDK inhibitors such as p21 and p27. A previous study reported that CS induces the growth of cancer cells by accelerating the G1 phase of the cell cycle [42,43]. In the present study, FA and Bz were found to increase cell proliferation through increased expression of cyclin D1 and E1 in a dose dependent manner. Moreover, these compounds decreased the expression of the cell cycle arrest proteins p21 and p27 in a dose dependent manner.
EMT plays an important role in cancer metastasis and aggravated statuses of cancer patients [44]. Through this process, cells acquire the capacity of motility., which leads to decreased adhesive ability for embryonic development in various tissues or organs [45]. A recent study reported that nicotine and CS induced growth and metastasis [19]. Therefore, we hypothesized that EMT increased in response to FA and Bz in JEG-3. In a scratch assay, two components of CS significantly induced JEG-3 cell migration. Cell migration occurs via regulation of the expression of intracellular protein, which changes the expression of E-cadherin, an epithelial cell marker, and N-cadherin, a mesenchymal marker. Western blot assay revealed that the expression of E-cadherin was reduced in response to treatment with FA and Bz, while the expression of its reverse transition marker, N-cadherin, increased. Moreover, FA and Bz upregulated the EMT associated transcriptional factors, Snail and Slug. Zinc-finger transcription factors play an important role in EMT [46], and Snail not only suppresses epithelial genes, but also promotes mesenchymal gene transcription.
We also focused on the mediation of ROS, antioxidants and ER stress pathways. We previously observed that FA and Bz induced an increase of apoptosis in the human colon cancer cell, SW620 [47]. However, in the present study, FA and Bz induced an increase in cell proliferation and EMT in JEG-3. Therefore, in this study, we focused on changes in the protein level of Nrf2 activated by ROS. ROS are known to induce apoptosis through the C/EBP-homologous protein (CHOP) pathway of ER-stress [48], as well as to activate Nrf2 [49,50]. Nrf2 prevents apoptosis through inactivation of ER stress [51,52]. Although Nrf2 has been known as a transcription factor that regulates the expression of antioxidants and cytoprotective genes under oxidative stress, recent data has revealed that Nrf2 activity is also associated with oncogenic function and survival and metastasis of cancer cells [53], and blockage of Nrf2 inhibited the metastatic abilities such as migration and invasion of esophageal squamous cell carcinoma cells [54]. The overexpression of Nrf2 was identified in a variety of tumors [55], and, especially, it was found that Nrf2 promoted cell proliferation and metastasis by increasing RhoA protein stability and expression in MCF-7 and MDA-MB-231 breast cancer cells [56]. In addition, in pancreatic ductal adenocarcinoma cells, interleukin-6-mediated Stat3 activation induced Nrf2 signaling to promote EMT [57].) Therefore, we assumed that Nrf2 interferes with activation of the ER-stress apoptosis pathway or directly induces increased proliferation of cancer and increased EMT by Nrf2. Activation of intracellular ROS by FA and Bz was observed through DCFH-DA. In the DCFH-DA assay, activation of ROS was significantly increased in JEG-3 cells treated with FA and Bz relative to DMSO, and FA and Bz induced an increase in protein expression of Nrf2. Moreover, the expression of eIF2α was decreased.

Conclusions
The results of the present study showed that FA and Bz induced an increase in proliferation by inducing expression of the cell cycle related proteins, cyclin D1 and cyclin E1, or by reducing expression of the cell cycle arrest proteins, p21 and p27. These chemicals also promoted the EMT process thought up-and down-regulation of EMT related genes such as E-cadherin, N-cadherin, snail, and slug. Moreover, FA and Bz induce the activation of ROS, which activates oxidative stress and increases the expression of Nrf2, an antioxidant factor, thereby blocking ROS activation and preventing normal cells from going to abnormal cell states such as apoptosis as demonstrated in Figure 7. Overall, the results of the present study suggest that FA and Bz may affect cell proliferation and migration of human choriocarcinoma cells JEG-3 through inhibition of ER stress activity and increased activity of Nrf2. relative to DMSO, and FA and Bz induced an increase in protein expression of Nrf2. Moreover, the expression of eIF2α was decreased.

Conclusion
The results of the present study showed that FA and Bz induced an increase in proliferation by inducing expression of the cell cycle related proteins, cyclin D1 and cyclin E1, or by reducing expression of the cell cycle arrest proteins, p21 and p27. These chemicals also promoted the EMT process thought up-and down-regulation of EMT related genes such as E-cadherin, N-cadherin, snail, and slug. Moreover, FA and Bz induce the activation of ROS, which activates oxidative stress and increases the expression of Nrf2, an antioxidant factor, thereby blocking ROS activation and preventing normal cells from going to abnormal cell states such as apoptosis as demonstrated in Figure 7. Overall, the results of the present study suggest that FA and Bz may affect cell proliferation and migration of human choriocarcinoma cells JEG-3 through inhibition of ER stress activity and increased activity of Nrf2.   3 This activates apoptosis by activating apoptosis-related eIF2, or by activating the antioxidant factor, Nrf2, 4 to regulate the activation of ROS by inducing an antioxidant effect through the increase of Nrf2 which blocks the activation of ROS. However, FA and Bz not only activate ROS, but also increase the activity of Nrf2, which prevents the activation of oxidative stress and apoptosis.