Metallothionein 3 Is a Hypoxia-Upregulated Oncogene Enhancing Cell Invasion and Tumorigenesis in Human Bladder Carcinoma Cells

Metallothioneins have been viewed as modulators in a number of biological regulations regarding cancerous development; however, the function of metallothionein 3 (MT3) in bladder cancer is unexplored. We determined the regulatory mechanisms and potential function of MT3 in bladder carcinoma cells. Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-qPCR) assays revealed that TSGH-8301 cells expressed more MT3 levels than RT-4, HT1376, and T24 cells. Immunoblot and RT-qPCR assays showed that arsenic (AS2O3) treatments enhanced the gene expression of MT3. Hypoxia induced HIF-1α, HIF-2α, and MT3 expression; furthermore, HIF-2α-knockdown attenuated hypoxic activation on MT3 expression. Ectopic overexpression of MT3 increased cell proliferation, invasion, and tumorigenesis significantly in T24 and HT1376 cells in vitro and in vivo; however, MT3-knockdown in TSGH-8301 cells had the reverse effect. Moreover, knockdown of MT3 enhanced arsenic-induced apoptosis determined by the Annexin V-FITC apoptosis assay. MT3-overexpression downregulated the gene expressions of N-myc downstream regulated gene 1 (NDRG1), N-myc downstream regulated gene 2 (NDRG2), and the mammary serine protease inhibitor (MASPIN) in HT1376 and T24 cells, whereas MT3-knockdown in TSGH-8301 cells had the opposite effect. The experiments indicated that MT3 is an arsenic- and hypoxia-upregulated oncogene that promotes cell growth and invasion of bladder carcinoma cells via downregulation of NDRG1, NDRG2, and MASPIN expressions.


Introduction
Bladder cancer has become the ninth most common worldwide cancer and the sixth most seen malignancy in the United States, according to an epidemiologic statistics report published in 2017 [1,2].

Arsenic and Hypoxia Upregulate Metallothionein 3 (MT3) Expression in Bladder Carcinoma Cells
The MT3 mRNA levels in several lines of cultured bladder cells (RT4, HT1376, T24, and TSGH-8301) were compared. Results of RT-qPCR assays revealed that TSGH-8301 cells had the highest levels of MT3 among the four bladder carcinoma cell lines ( Figure 1A). Results of immunoblot assays showed that arsenic upregulated HO-1, MT3, and NDRG1 protein levels in T24 cells ( Figure 1B). Results of quantitative analyses from three independent experiments are present in Figure 1C. Results of RT-qPCR revealed that arsenic treatment-induced MT3 and NDRG1 gene expressions were dosage-dependent ( Figure 1D). Further immunoblot assays indicated that 17 h of hypoxia upregulated HIF-1α, HIF-2α, and MT3 protein levels in TSGH-8301 cells ( Figure 1E); moreover, HIF-2α-knockdown in TSGH-8301 cells blocked HIF-2α and MT3 expressions under the hypoxic condition determined by immunoblotting ( Figure 1F) and RT-qPCR ( Figure 1G) assays. Results of reporter assays showed that transient overexpression of HIF-1α and HIF-2α induced promoter activity of the human MT3 gene ( Figure 1H); in addition, 5 -delation report assays showed that HIF-1α and HIF-2α induced MT3 promoter activity was dependent on the 5 -flanking DNA fragment (−1 to −480) ( Figure 1I).  , and β-actin were determined by immunoblotting; (C) quantitative analysis was done by determining the intensity of each band of the target gene and β-actin from three independent experiments. Data are presented as the foldinduction of the relative density of the target gene/β-actin (±SE, n = 3) in relation to the control solventtreated group (* p < 0.05, ** p < 0.01); (D) T24 cells were treated with various concentrations of As2O3 for 24 h. Total RNA was extracted for RT-qPCR (** p < 0.01); (E) TSGH-8301 cells were cultured at a hypoxic condition in different periods. Cells were lysed, and MT3, HIF-1α, HIF-2α, and β-actin were determined by immunoblotting; (F) HIF-2α-knockdown TSGH-8301 (8301-shHIF2α) and mockknockdown (8301-shCOL) cells were cultured at hypoxic or normoxic conditions for 24 h. Cells were lysed and MT3, HIF-2α, and β-actin were determined by immunoblotting; (G) HIF-2α-knockdown TSGH-8301 (8301-shHIF-2α) and mock-knockdown (8301-shCOL) cells were cultured at normoxic (black bars) or hypoxic (white bars) conditions for 16 h. Total RNA was extracted for RT-qPCR. Data are presented as the fold-induction of the mRNA levels of MT3/β-actin (±SE, n = 3) in relation to the mRNA levels of 8301-shCOL cells cultured at normoxic conditions (* p < 0.05, ** p < 0.01); (H) TSGH-8301 cells were cotransfected with an MT3 reporter vector and various concentrations of HIF-1α (black bars) or HIF-2α (white bars) expression vectors as indicated. Data are presented as the mean percentage ±SE (n = 6) of luciferase activity in relation to the control group (* p < 0.05, ** p < 0.01); (I) relative luciferase activity of reporter vectors containing different fragments from the MT3 promoter, as shown. The MT3 reporter vector-transfected HT1376 cells were cotransfected with the HIF-1α (white bars) or HIF-2α (black bars) expression vectors for 72 h. Luciferase activity was fold-induced (±SE, n = 6) in relation to the cotransfected pcDNA3 expression vector group.

Effects of Ectopic Overexpression of MT3 on Proliferation and Invasion of Bladder Carcinoma HT1376 Cells
A human MT3 expression vector was transfected into bladder carcinoma HT1376 cells to investigate the role of MT3 in proliferation and invasion. Results of the immunoblot assay confirmed the ectopic overexpression of MT3 in HT1376 (HT−MT3) cells (Figure 2A). Matrigel invasion assays revealed that HT−MT3 cells expressed markedly a higher invasive capacity than HT−DNA cells ( Figure 2B). [ 3 H]thymidine incorporation assays revealed that the numbers of HT−MT3 cells increased 2.82 folds after five days of incubation. However, HT−DNA cells increased only by 1.45folds ( Figure 2C). Furthermore, [ 3 H]thymidine incorporation assays revealed that MT3overexpressed HT1376 cells attenuated the effect of doxorubicin on cell proliferation. Doxorubicin (0.4 μg/mL) blocked 93% of proliferation of HT−DNA cells, whereas proliferation of HT−MT3 cells was decreased only by 49% after 48 h of treatment ( Figure 2D). (C) quantitative analysis was done by determining the intensity of each band of the target gene and β-actin from three independent experiments. Data are presented as the fold-induction of the relative density of the target gene/β-actin (±SE, n = 3) in relation to the control solvent-treated group (* p < 0.05, ** p < 0.01); (D) T24 cells were treated with various concentrations of As 2 O 3 for 24 h. Total RNA was extracted for RT-qPCR (** p < 0.01); (E) TSGH-8301 cells were cultured at a hypoxic condition in different periods. Cells were lysed, and MT3, HIF-1α, HIF-2α, and β-actin were determined by immunoblotting; (F) HIF-2α-knockdown TSGH-8301 (8301-shHIF2α) and mock-knockdown (8301-shCOL) cells were cultured at hypoxic or normoxic conditions for 24 h. Cells were lysed and MT3, HIF-2α, and β-actin were determined by immunoblotting; (G) HIF-2α-knockdown TSGH-8301 (8301-shHIF-2α) and mock-knockdown (8301-shCOL) cells were cultured at normoxic (black bars) or hypoxic (white bars) conditions for 16 h. Total RNA was extracted for RT-qPCR. Data are presented as the fold-induction of the mRNA levels of MT3/β-actin (±SE, n = 3) in relation to the mRNA levels of 8301-shCOL cells cultured at normoxic conditions (* p < 0.05, ** p < 0.01); (H) TSGH-8301 cells were cotransfected with an MT3 reporter vector and various concentrations of HIF-1α (black bars) or HIF-2α (white bars) expression vectors as indicated. Data are presented as the mean percentage ±SE (n = 6) of luciferase activity in relation to the control group (* p < 0.05, ** p < 0.01); (I) relative luciferase activity of reporter vectors containing different fragments from the MT3 promoter, as shown. The MT3 reporter vector-transfected HT1376 cells were cotransfected with the HIF-1α (white bars) or HIF-2α (black bars) expression vectors for 72 h. Luciferase activity was fold-induced (±SE, n = 6) in relation to the cotransfected pcDNA3 expression vector group.

Effects of Ectopic Overexpression of MT3 on Proliferation and Invasion of Bladder Carcinoma HT1376 Cells
A human MT3 expression vector was transfected into bladder carcinoma HT1376 cells to investigate the role of MT3 in proliferation and invasion. Results of the immunoblot assay confirmed the ectopic overexpression of MT3 in HT1376 (HT−MT3) cells ( Figure 2A). Matrigel invasion assays revealed that HT−MT3 cells expressed markedly a higher invasive capacity than HT−DNA cells ( Figure 2B

Effect of MT3-Knockdown on Proliferation and Invasion of Bladder Carcinoma TSGH-8301 Cells
Using immunoblot assays, we confirmed that the expression of MT3 was only 10% in MT3-knockdown TSGH-8301 (8301-shMT3) cells compared to mock-knockdown (8301-shCOL) cells ( Figure 3A). [ 3 H]thymidine incorporation assays revealed a 2.30-fold increase in the number of 8301-shCOL cells after five days of incubation. However, the number of 8301-shMT3 cells increased only by 1.54 folds ( Figure 3B). Matrigel assays indicated that knockdown of MT3 resulted in a 64% decrease in invasion capacity compared with 8301-shCOL cells ( Figure 3C). These results suggested that knockdown of MT3 in bladder carcinoma TSGH-8301 cells, which have higher endogenous MT3 levels, blocked cell proliferation and invasion. Further studies of immunoblot ( Figure 3D) and RT-qPCR ( Figure 3E) assays showed that MT3-knockdown (8301-shMT3) cells exhibited markedly higher levels of NDRG1 and MASPIN gene expressions than mock-knockdown (8301-shCOL) cells. The expresion of NDRG2 was induced a little but significantly after knockdown of MT3. However, MT3-knockdown did not significantly affect NDRG3 gene expression in TSGH-8301 cells. NDRG2, and MASPIN gene expressions than mock-overexpression (HT-DNA) cells. HT-MT3 and HT-DNA cells did not display significant differences in the expressions of the NDRG3 gene.

Effect of MT3-Knockdown on Proliferation and Invasion of Bladder Carcinoma TSGH-8301 Cells
Using immunoblot assays, we confirmed that the expression of MT3 was only 10% in MT3knockdown TSGH-8301 (8301-shMT3) cells compared to mock-knockdown (8301-shCOL) cells ( Figure 3A). [ 3 H]thymidine incorporation assays revealed a 2.30-fold increase in the number of 8301-shCOL cells after five days of incubation. However, the number of 8301-shMT3 cells increased only by 1.54 folds ( Figure 3B). Matrigel assays indicated that knockdown of MT3 resulted in a 64% decrease in invasion capacity compared with 8301-shCOL cells ( Figure 3C). These results suggested that knockdown of MT3 in bladder carcinoma TSGH-8301 cells, which have higher endogenous MT3 levels, blocked cell proliferation and invasion. Further studies of immunoblot ( Figure 3D) and RT-qPCR ( Figure 3E) assays showed that MT3-knockdown (8301-shMT3) cells exhibited markedly higher levels of NDRG1 and MASPIN gene expressions than mock-knockdown (8301-shCOL) cells. The expresion of NDRG2 was induced a little but significantly after knockdown of MT3. However, MT3-knockdown did not significantly affect NDRG3 gene expression in TSGH-8301 cells.

Effect of Overexpression of MT3 on Proliferation and Invasion of Bladder Carcinoma T24 Cells
The ectopic expression of MT3 in T24 cells was confirmed using immunoblot assays ( Figure S1A) and Matrigel invasion assays indicated that the invasive ability of T24−MT3 cells was about 3.78-fold higher than that of T24−DNA cells ( Figure S1B). [ 3 H]thymidine incorporation assays revealed a 5.06-fold increase in the number of T24−MT3 cells after five days of incubation. However, the number of T24−DNA cells increased only by 3.32 folds ( Figure S1C). The results of immunoblot ( Figure S1D), RT-qPCR ( Figure S1E), and reporter ( Figure S1F) assays showed that MT3-overexpression (T24−MT3) cells had markedly lower levels of NDRG1, NDRG2, and MASPIN gene expressions than mock-overexpression (T24−DNA) cells.

Effects of Ectopic Overexpression of MT3 on Tumorigenesis of Bladder Carcinoma T24 cells
The effect of MT3 on tumor growth in vivo was evaluated using xenografts in BALB/cAnN-Foxn1 NU mice. Tumors generated from T24−DNA cells grew slower than those derived from T24−MT3 cells. Additionally, tumors generated from T24−DNA cells were approximately 32% smaller than tumors generated from T24−MT3 cells (18.01 ± 7.38 vs. 57.30 ± 14.53 mm 3 ) after 7 weeks of growth ( Figure 4A). Thus, ectopic overexpression of MT3 enhanced cell proliferation, invasion, and tumorigenesis of T24 cells. The results of immunoblot assays confirmed that MT3 was overexpressed in the xenograft tumors, which were inoculated by T24−MT3 cells ( Figure 4B). Moreover, the results of RT-qPCR assays revealed that overexpression of MT3 blocked NDRG1, NDRG2, and MASPIN mRNA levels in xenograft tumors ( Figure 4C).

Effect of Overexpression of MT3 on Proliferation and Invasion of Bladder Carcinoma T24 Cells
The ectopic expression of MT3 in T24 cells was confirmed using immunoblot assays ( Figure S1A) and Matrigel invasion assays indicated that the invasive ability of T24−MT3 cells was about 3.78-fold higher than that of T24−DNA cells ( Figure S1B). [ 3 H]thymidine incorporation assays revealed a 5.06fold increase in the number of T24−MT3 cells after five days of incubation. However, the number of T24−DNA cells increased only by 3.32 folds ( Figure S1C).

Effects of Ectopic Overexpression of MT3 on Tumorigenesis of Bladder Carcinoma T24 cells
The effect of MT3 on tumor growth in vivo was evaluated using xenografts in BALB/cAnN-Foxn1 NU mice. Tumors generated from T24−DNA cells grew slower than those derived from T24−MT3 cells. Additionally, tumors generated from T24−DNA cells were approximately 32% smaller than tumors generated from T24−MT3 cells (18.01 ± 7.38 vs. 57.30 ± 14.53 mm 3 ) after 7 weeks of growth ( Figure 4A). Thus, ectopic overexpression of MT3 enhanced cell proliferation, invasion, and tumorigenesis of T24 cells. The results of immunoblot assays confirmed that MT3 was overexpressed in the xenograft tumors, which were inoculated by T24−MT3 cells ( Figure 4B). Moreover, the results of RT-qPCR assays revealed that overexpression of MT3 blocked NDRG1, NDRG2, and MASPIN mRNA levels in xenograft tumors ( Figure 4C).

MT3 Modulates Cell Apoptosis Induced by As 2 O 3 in Bladder Carcinoma Cells
To distinguish among early apoptotic, late apoptotic, and necrotic cells of bladder carcinoma, we used Annexin V-FITC with propidium iodide (PI) staining. The flow cytometry of fluorescence intensity for Annexin V-FITC and PI staining in 8301-shCOL and 8301-shMT3 cells after treatments with various concentrations of As 2 O 3 for 24 h revealed that MT3-knockdown TSGH-8301 (8301-shMT3) cells significantly enhanced cell apoptosis induced by As 2 O 3 in comparison to mock-knockdown TSGH-8301 (8301-shCOL) cells ( Figure 5A). On the contrary, ectopic overexpression of MT3 in T24 (T24−MT3) cells significantly attenuated cell apoptosis induced by As 2 O 3 in comparison to mock-overexpression T24 (T24−DNA) cells ( Figure 5B).

MT3 Modulates Cell Apoptosis Induced by As2O3 in Bladder Carcinoma Cells
To distinguish among early apoptotic, late apoptotic, and necrotic cells of bladder carcinoma, we used Annexin V-FITC with propidium iodide (PI) staining. The flow cytometry of fluorescence intensity for Annexin V-FITC and PI staining in 8301-shCOL and 8301-shMT3 cells after treatments with various concentrations of As2O3 for 24 h revealed that MT3-knockdown TSGH-8301 (8301-shMT3) cells significantly enhanced cell apoptosis induced by As2O3 in comparison to mockknockdown TSGH-8301 (8301-shCOL) cells ( Figure 5A). On the contrary, ectopic overexpression of MT3 in T24 (T24−MT3) cells significantly attenuated cell apoptosis induced by As2O3 in comparison to mock-overexpression T24 (T24−DNA) cells ( Figure 5B).

Discussion
Metallothioneins (MTs), a class of metal-binding proteins characterized by high-cysteine content and low-molecular weight, may provide protection against metal toxicity and oxidative stress [13,21]. Early reports found that an increased expression of MTs was related to cancer in the breast, colon, kidney, liver, skin, lung, nasopharynx, ovary, prostate, thyroid, and urinary bladder, whereas hepatocellular carcinoma and liver adenocarcinoma produced a lower level of MT expression [6]. Furthermore, defects in the MT function or expression may lead to a malignant transformation of cancer, including bladder cancer, because MTs play an important role in transcription factor regulation [22][23][24]. The MT3 isoform possesses a unique sequence of eight amino acids which is not present in any other members of the MT gene family [25]. In addition, in nerve-derived cell cultures, MT3 possesses an inhibitory activity for neural cell growth, which is not duplicated by other MT isoforms [26]. However, some studies revealed that MT3 was found in normal prostate and renal tissues with an altered expression in organ-derived malignancies [27,28]. A prior study suggested that MT3 might be an effective biomarker for bladder cancer although the biologic functions of MT3 have not been fully understood [14]. Our study suggests that MT3 expression in the bladder cell lines could be dependent on the cell type but not relevant to the extent of neoplasia in vitro. A similar result was also found in a previous study using prostate carcinoma cells [15].
An epidemiologic study indicated that arsenic pollution in water is associated significantly with the incidence of bladder cancer in Taiwan [29]. Animal studies found that MT3 was overexpressed in tumor heterotransplants derived from arsenic-transformed human urothelial cells [30]. Ours has been the first study to reveal that arsenic can induce HO-1, NDRG1 and MT3 gene expressions in bladder carcinoma T24 cells. The results of upregulation of MT3 by arsenic are consistent with those of a previous study on prostate carcinoma LNCaP cells [15]. Although early studies indicated that arsenic-induced metal-responsive transcription factor-1 (MTF-1) binds to the metal-response element (MRE) of the promoter MT3 and NDRG1 genes [18,31], the molecular mechanisms of arsenic on gene expressions of MT3 and NDRG1 in bladder carcinoma cells are still undefined. Expression of HO-1, a gene known to be induced in response to sodium arsenite, was used as positive control [32]. The HO-1 expression increased as the dosage of As 2 O 3 increased, demonstrating the effectiveness of treatment.
Studies in mouse MEF cells and primary culture chondrocytes indicated that hypoxia induced both MT1 and MT2 expressions through cooperative interactions between transcription factors and HIF-1α or HIF-2α, respectively [33,34]. Although studies suggested that hypoxia upregulated MT3 in human prostate carcinoma PC-3 cells and adipocytes, no mechanisms were illustrated in their reports [11,35]. As shown in Figure 1, the present study has been the first to indicate that upregulation of MT3 by hypoxia in human bladder carcinoma cells is dependent on HIF-1α and HIF-2α, which are well-known to be overexpressed in bladder cancer in vivo [36].
Our findings indicated that MT3 affects cell proliferation and invasion in bladder carcinoma cells. Results of 3 H-thymidine incorporation assays, Matrigel invasion assays, and xenografts in mice showed that mock-transfected bladder carcinoma cells grew slower in vitro and in vivo than the MT3-overexpressed bladder carcinoma cells, whereas MT3-knockdown attenuated cell invasion of TSGH-8301 cells. These results are similar to our earlier study using prostate carcinoma cells, which showed that mock-transfected PC-3 cells grew slower in vitro and in vivo than PC-3 cells overexpressing MT3 [15]. To the best of our knowledge, our study has been the first to provide laboratory evidence that MT3 plays a tumor inductive role in human bladder carcinoma cells. Our study also indicated that MT3 overexpression increased resistance to doxorubicin in HT1376 cells (Figure 2). This finding of MT3 overexpression increasing chemotherapeutic drug resistance is in agreement with a previous study in prostate carcinoma cells [15,37]. It is possible that overexpression of MT3 is one of the mechanisms of bladder tumor cell resistance to cancer treatment.
Studies suggested that metallothioneins could be involved in protection against toxicity, and regulate the interactive effects of metals and metalloids including arsenic [11]. This study showed that As 2 O 3 induced more apoptosis in MT3-knockdown TSGH-8301 cells than mock-transfected TSGH-8301 cells. On the contrary, ectopic overexpression of MT3 in T24 cells significantly attenuated cell apoptosis induced by As 2 O 3 in comparison to mock-overexpression of T24 cells ( Figure 5). Our results demonstrated that MT3 might be involved in the protection against arsenic toxicity in bladder carcinoma cells. Results also suggested that MT3 was similar to other metallothioneins involved in the intracellular defense mechanism against arsenic cytotoxicity [38].
In the present study, we found that ectopic MT3 overexpression in HT1376 and T24 cells blocked gene expressions of NDRG1, NDRG2, and MASPIN, but not NDRG3. In vitro and in vivo studies have shown that NDRG1-induced expression downregulated the growth of bladder carcinoma cells [39]. Overexpression of NDRG2 in bladder carcinoma cells inhibited cell proliferation, invasion, and tumorigenesis in vitro and in vivo; moreover, the expression of NDRG2 correlated negatively with the tumor grade and pathologic stage of bladder cancer [40]. Mammary serine protease inhibitor (MASPIN), a member of the serine protease inhibitor family, inhibited cell proliferation, migration, and invasion of bladder carcinoma cells [41]. Our results suggest that decreased expressions of NDRG1, NDRG2, and MASPIN genes may account for the increased cell proliferation and invasiveness in bladder carcinoma cells with MT3 stably overexpressed. A prior study identified 43 MT3-target genes after ectopic overexpression of MT3 in HL-60 cells [42] although signal pathways of MT3 on its downstream genes still need to be explored further.

Cell Cultures and Chemicals
The bladder transitional carcinoma cell lines, RT-4, HT1376, TSGH-8301, and T24 cells, were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) as described previously [43]. The RT-4 cell line was obtained from the explant of a recurrent papillary bladder tumor [44]. The HT1376 cell line, generated from a Caucasian woman with grade 3 transitional cell bladder cancer, contained the well-differentiated human bladder carcinoma cells with high tumorigenic capability [45]. The TSGH-8301 cell line was taken from a Taiwanese well-differentiated transitional cell carcinoma [46]. The T24 cell line was poorly differentiated transitional carcinoma cells with low tumorigenic capability [47]. DAPI (4,6-diamino-2-phenylindole), bovine serum albumin (BSA), humic acid (HA), and As 2 O 3 were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). The As 2 O 3 stock solution was dissolved in 1 mM humic acid solution at a concentration of 100 mM, as modified from a previous study [48]. We purchased fetal calf serum (FCS) from HyClone (Logan, UT, USA), RPMI 1640 media from Invitrogen (Carlsbad, CA, USA), and Matrigel from BD Biosciences (Bedford, MA, USA).

Immunoblot Assays
For nuclear and cytoplasmic extraction, cells were cultured in an RPMI-1640 medium with 10% FCS for 48 h, and then harvested with trypsin, and washed twice with PBS. Nuclear and cytoplasmic fractions were separated using the NE-PER TM Nuclear and cytoplasmic extraction kit (Thermo, Rockford, NJ, USA) as described by the manufacturer. Equal amounts of whole cell, nuclear, or cytoplasmic lysis were loaded onto a 10% SDS-polyacrylamide gel and assayed by

[ 3 H]thymidine Incorporation Assay
The [ 3 H]thymidine incorporation assay was used to measure cell proliferation as described previously [49].

Matrigel Invasion Assay
Cells were suspended at a density of 1 × 10 5 /100 µL in a serum-free medium and seeded into a 24-well transwell chamber with an 8-µm pore membrane. The 4% (w/v) paraformaldehyde was used to fix the cells that migrated into the Matrigel-coated transmembrane; these were then stained with a 0.1% (w/v) crystal violet solution. In order to capture images, a digital camera connected to an inverted microscope (IX71, Olympus, Tokyo, Japan) was used. By using PAX-it image analysis software, we analyzed the images following standardization of light intensity as described previously [50].

Annexin V-FITC Apoptosis Detection
Cell pellets were harvested after cells were treated with or without arsenic (AS 2 O 3 ) for 24 h. Apoptosis detection and quantification were performed after treatments with Annexin V-FITC (BioVision Inc, Milpitas, CA, USA) and propidium iodide (PI) for 1 h using the FACSCalibur E6147 Cytometer (BD Biosciences) as described previously [51].

Tumor Xenograft Study
This animal study was approved by the Institutional Animal Care and Use Committee of the Chang Gang University (IACUC Approval No.: CGU14-075, 9 September 2014). In this study, we purchased 4-week-old male BALB/cAnN-Foxn1 NU mice from the National Laboratory Animal Center, Taipei, Taiwan. These mice were under anesthesia during surgical procedures and all effort was made to minimize distress and pain. Each mouse was under intraperitoneal anesthesia when the pre-prepared T24−DNA and T24−MT3 cells were mixed (1:1) with Matrigel and injected subcutaneously into one side of the back near the shoulder of each mouse (n = 6). The mice were kept in a barrier facility under HEPA filtration and their health was monitored weekly during the experiment. Xenograft growth was measured by Vernier calipers at intervals as indicated, and tumor volume was calculated as volume = [π/6 × largest diameter × (smallest diameter) 2 ] as described previously [43].

Statistical Analysis
Results are expressed as the mean ± (SE) of at least three independent replications of each experiment. Statistical significance was determined by one-way ANOVA and Student's t test using the SigmaStat program for Window version 2.03 (SPSS Inc, Chicago, IL, USA). The * represents p < 0.05 and the ** represents p < 0.01.

Conclusion
Our experiments provided evidence indicating that arsenic and hypoxia upregulated MT3 expression. Ectopic overexpression of MT3 enhanced tumorigenesis of bladder carcinoma T24 cells in vivo. The downregulation of NDRG1, NDRG2, and MASPIN gene expressions could account for the enhancement of proliferative and invasive functions of MT3 in bladder carcinoma cells. It was found that MT3 might also participate in the protection against arsenic toxicity in bladder carcinoma cells. The results suggested that MT3 is the oncogene in bladder cancer and ectopic overexpression of MT3 enhances tumorigenesis of human bladder carcinoma cells.