Artesunate Induces Apoptosis of Bladder Cancer Cells by miR-16 Regulation of COX-2 Expression

Bladder cancer is the most common malignant tumor of the urinary tract and remains one of the major causes of cancer death worldwide. In this study, we investigated the effect and mechanism of Artesunate (ART), a traditional Chinese medicine, on inducing apoptosis of human bladder cancer cells. In vivo antitumor activity was investigated in bladder cancer in rat by subcutaneous injection of different concentration of ART. The effect of ART on growth inhibition and apoptosis of bladder cancer cells was evaluated using dimethylthiazoly-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis, respectively. Cyclooxygenase-2 (COX-2) and miR-16 expression levels were determined with real-time PCR. The concentrations of prostaglandin E2 (PGE2) in the supernatants of bladder cancer cells were measured with an ELISA kit. The miR-16 inhibitor or mimic were transfected into cells to up- or down-regulate miR-16 expression. ART efficiently inhibited orthotopic tumor growth in the bladder cancer rat, which is accompanied with an increase of miR-16 expression and a decrease of COX-2 expression. In vitro, ART could induce cytotoxicity and apoptosis in bladder cancer cells, but presented a much lighter toxicity effect against normal human urothelial cells. ART significantly increased miR-16 expression and decreased the expression of COX-2 and the production of PGE2. More importantly, down-regulation of miR-16 expression could reverse the effect of ART on apoptosis and COX-2 expression in bladder cells. Moreover, exogenous PGE2 could inhibit apoptosis of bladder cancer cells treated with ART. In conclusion, ART can elicit an anti-tumor effect against bladder cancer by up-regulation of miR-16 expression, which resulted in the decrease of COX-2 expression and PGE2 production. Hence, ART might be an effective drug for the treatment of bladder cancer.

demonstrates the finding that miR-16 inhibits COX-2 expression leading to ART-induced apoptosis of bladder cancer cells.

Artesunate (ART) Inhibited Tumor Growth in the Bladder Cancer Rat
No relevant changes were obtained between the groups during the study concerning body weight and beverage consumption (data not shown). To evaluate the feasibility of ART therapy for bladder cancer, the efficacy of ART in inhibiting tumor growth was measured in the bladder cancer rat. In group 1, the percentage of rats with bladder cancer was 70.0% (7 in 10), with a mean of 1.1 ± 0.4 tumors per rat with tumors. A similar profile was found in groups 2, 3 and 4. However, The sizes of tumors were significantly decreased after treatment with ART (20, 100, 200 mg/kg) in a dose-dependent manner ( Figure 1A).
In the group 1, there was evident malignant transformation including aggressive bladder cancers of squamous cell phenotypes. ART treatment could reduce malignant lesions. Rats in groups 3 and 4 showed no infiltrative bladder cancers and had a lower incidence of high grade tumors than those in group 1 ( Figure 1B).

Figure 1.
Chemical structure of Artesunate (ART) and ART inhibited tumor growth in the bladder cancer rat. Chemical structure of ART (A); Rats were given 0.05% of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in drinking water. Ten weeks later, the rats were treated with ART (0, 20, 100, 200 mg/kg) by subcutaneous injection. Ten rats were used for each treatment group. Ten weeks after treatment with ART, all rats were sacrificed and the tumor size was measured (B); Hematoxylin and Eosin (HE) stained cancer tissues showed ART treatment could reduce malignant lesions in a dose-dependent manner. Scale bar is equal to 20 μm (C); * p < 0.05, indicate significant differences from those treated with 0 mg/kg ART. To explore the molecular mechanism of ART inhibition of tumor growth, we measured the level of miR-16 and COX-2 in the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced tumor tissue. Compared with the rats treated with solvent control, the level of miR-16 in tumor tissue was significantly increased after treatment with ART ( Figure 2A). However, the expression of COX-2 was dose-dependently decreased in BBN-induced bladder cancer rats treated with ART ( Figure 2B,C). The level of miR-16 and COX-2 mRNA level in tumors from the cervical cancer rat after treatment with ART was detected (A,B); The protein level of COX-2 was measured in tumors from the cervical cancer rat after treatment with ART. Fold changes of COX-2 protein levels were also determined (C). * p < 0.05, indicates significant differences from those treated with 0 mg/kg ART.

ART Induced Cytotoxicity and Apoptosis in Bladder Cancer Cells
It has been reported that ART could inhibit growth and induce apoptosis of cancer cells [21]. We detected the inhibitory effects of ART on the growth of bladder cancer cells and normal human urothelial cells using dimethylthiazoly-2,5-diphenyltetrazolium bromide (MTT) assays. ART treatment significantly decreased the growth of T24 and RT4 cells in a dose-dependent manner ( Figure 3A). For instance, when T24 cells were treated with 20, 100 and 200 μM of ART for 48 h, the inhibition rate of cell viability was 3.35%, 44.48% and 60.06%, respectively. And the IC 50 value at 48 h was 129.7 μM. The similar results were obtained in RT4 cells after treatment with ART and the IC 50 value at 48 h was 103.2 μM. ART exerts weak cytotoxicity effects on normal human urothelial cells. And the IC 50 value at 48 h was 1149.6 μM for SV-HUC-1 cells after treatment with ART.
Activation of caspases plays an important role in the execution of apoptosis [22]. In order to determine whether ART can induce apoptosis of bladder cancer cells, we evaluated caspase-3 activity. As shown in Figure 3B, ART could significantly enhance caspase-3 activity both in T24 and RT4 cells, but could not increase that in SV-HUC-1 cells. Flow cytometry analysis further confirmed that ART could induce apoptosis of bladder cancer cells ( Figure 3C). To identify whether ART could affect miR-16 expression levels in bladder cancer cells, we performed real-time PCR to detect levels of miR-16 expression. As shown in Figure 4A, the level of miR-16 was lower in bladder cancer cells (T24 and RT4 cells) than that in normal human urothelial cells (SV-HUC-1 cells). ART treatment significantly increased the expression of miR-16 in T24 and RT4 cells but did not change that in SV-HUC-1 cells ( Figure 4B).

T24
In order to assess the role of miR-16 in the effect of ART on apoptosis of bladder cancer cells, we added ART on cells after transfection with a miR-16 inhibitor. As shown in Figure 4C, the miR-16 inhibitor can significantly decrease the expression of miR-16 in T24 and RT4 cells. Interestingly, ART alone can increase the caspase-3 level, but miR-16 inhibitor with ART causes a decrease in the caspase-3 level ( Figure 4D). After transfection with miR-16 inhibitor for 24 h, T24 and RT4 cells were added with ART for an additional 24 h, and then the level of miR-16 was detected. * p < 0.05, compared to negative control (C); After transfection with miR-16 inhibitor for 24 h, T24 and RT4 cells were added with ART for an additional 48 h, and then caspase-3 activity was measured. * p < 0.05, compared to vehicle controls group. # p < 0.05, compared to negative control. $ p < 0.05, compared to negative control +ART (100 μM) treated group (D).

ART Decreases COX-2 Expression and Prostaglandin E2 (PGE2) Production in Bladder Cancer Cells
It has been reported that COX-2 was involved in growth inhibition and apoptosis of bladder cancer cells [23]. We also examined the effect of ART on the expression of COX-2 using real time PCR and western blot. Treatment of T24 and RT4 cells with ART for 24 h led to a dose-dependent decrease in the mRNA level of COX-2 ( Figure 5A). Furthermore, ART decreased COX-2 protein expression as shown in Figure 5B. A significant decrease in release of prostaglandin E2 (PGE2) in response to ART treatment was observed in T24 and RT4 cells ( Figure 5C). We first investigated whether miR-16 could regulate COX-2 expression and transfected miR-16 mimic into T24 and RT4 cells. The result of real-time PCR revealed that miR-16 mimic could significantly increase the expression of miR-16 in T24 and RT4 cells ( Figure 6A). Furthermore, miR-16 mimic decreased the expression of COX-2 and the concentration of PGE2 as shown in Figure 6B,C.
Next, we explored the role of miR-16 in the effect of ART on the regulation of COX-2 expression; ART was added to cells after transfection with the miR-16 inhibitor. As shown in Figure 6, miR-16 inhibitor could increase COX-2 expression and PGE2 levels in bladder cancer treated with ART. Figure 6. miR-16 is involved in ART regulation of COX-2 expression in bladder cancer cells. The level of miR-16 was detected in T24 and RT4 cells transfected with miR-16 mimic for 24 h. * p < 0.05, compared to negative control (A); COX-2 protein expression (B) in T24 and RT4 cells and PGE2 levels (C) in the supernatants of T24 and RT4 cells transfected with miR-16 mimic for 24 h. * p < 0.05, compared to negative control; After transfection with miR-16 inhibitor for 24 h, T24 and RT4 cells were added with ART for an additional 24 h, and then COX-2 mRNA and protein level was measured (D,E); After transfected with miR-16 inhibitor for 24 h, T24 and RT4 cells were added with ART for an additional 24 h, and then PGE2 levels in the supernatants were determined (F). * p < 0.05, compared to vehicle controls group. # p < 0.05, compared to negative control. $ p < 0.05, compared to negative control +ART (100 μM) treated group.

Exogenous PGE2 Can Reverse the Effect of ART on Inhibition of Bladder Cancer Cell Growth
To identify whether ART inhibits growth and induces apoptosis in a COX-2-dependent pathway, we used exogenous PGE2 to treat bladder cancer cells. As shown in Figure 7A, pretreatment of PGE2 significantly increased growth of cancer cells exposed to ART. Meanwhile, PGE2 can reverse the effect of ART on apoptosis of T24 and RT4 cells (Figure 7).

Figure 7.
Exogenous PGE2 can reverse the effect of ART on inhibition of bladder cancer cells growth. After pretreated with exogenous PGE2 (10 μM) for 2 h, T24 and RT4 cells were added with ART (100 μM) for an additional 48 h, and then cell viability and caspase-3 activity were determined (A,B). * p < 0.05, compared to vehicle controls group. # p < 0.05, compared to only ART (100 μM) treated group.

Discussion
ART, as a potential cancer chemoprevention agent, has a potential anti-cancer effect on several kinds of tumors such as Kaposi's sarcoma, hepatocellular carcinoma, and non-small cell lung cancer [17][18][19][20] and ART is recognized as a safe compound to treat malaria [24] Our experimental design provides a useful model for studying the effect and molecular mechanism of ART on bladder cancer and to our knowledge, this is the first time the therapeutic effect of ART on bladder cancer has been reported. There was no difference in weight among rats in each group (data not shown), which indicated that ART could inhibit tumor growth in the bladder cancer rat without any side effects on weight maintenance. When they were tested on T24 and RT4 cell line, the IC 50 value at 48 h after treatment was 129.7 and 103.2 μM, respectively. But the IC 50 value for SV-HUC-1 cells was very obviously higher than that for bladder cancer cells. These results indicated that ART could specifically kill bladder cancer cells without destruction to normal human urothelial cell.
The recommended daily dose of ART is 4 mg/kg and is administered for 3 days to treat malaria in humans. However, in the present study, we used 20, 100, and 200 mg/kg of ART to treat rats with bladder cancer. Moreover, Zhang et al. reported that ART at the dose of 50 and 100 mg/kg could inhibit the growth of cervical cancer, which resulted from inhibiting activity of regulatory T cells through PGE2 [25]. We speculate that the difference in the concentrations levels of ART necessary for malaria treatment compared to the treatment of tumors is due to ART not being attracted specifically to tumor sites. As a result, it is important in the future to find efficient drug carriers to deliver ART specifically to tumors.
It has been demonstrated that miR-16 could regulate proliferation and apoptosis in many types of cancers including bladder cancer [26]. The tumor suppressor function of miR-16 has been addressed both in vivo and in vitro [27]. As a result, miR-16 is a promising target for treatment of cancer. Here, we found that up-regulation of miR-16 expression was associated with ART inhibition of tumor growth (Figures 2 and 4B). In addition, the expression of miR-16 was found to be down-regulated in bladder cancer cells in comparison with normal urothelial cells (Figure 4). These findings suggest that up-regulation of miR-16 may be a therapeutic target for ART to treat bladder cancer. In agreement with this hypothesis, down-regulation of miR-16 expression could reverse the effect of ART on apoptosis of bladder cancer cells.
Young et al. reported that miR-16 could bind the COX-2 3'-UTR and inhibit COX-2 expression in colorectal cancer cells [28]. We also found that over-expression of miR-16 could down-regulate COX-2 expression in bladder cancer cells ( Figure 6B). Accumulating evidence suggests an important role for COX-2 in induction of cell proliferation and reduction of apoptosis of bladder cancer cells [10][11][12]. Inhibition of COX-2 activity and/or expression caused reduced cell proliferation and increased apoptosis [29]. In the present study, we found that ART inhibited the COX-2 expression of bladder cancer cells and PGE2 production in a dose-dependent manner, which could be restored by miR-16 inhibitor ( Figure 6C). Furthermore, reduction in growth was observed in T24 and RT4 cells treated with ART, which was then rescued by exogenous PGE2 (Figure 7), implicating a COX-2-dependent pathway in bladder cancer cells. According to these results, we draw a conclusion that ART suppresses COX-2 expression and PGE2 production by enhancing miR-16 expression (Figure 8).

Figure 8.
Diagram depicting the mechanism of ART induced bladder cancer cells apoptosis. The arrow of "↑" means increase and the arrow of "↓" means decrease.

Cell Culture
The immortalized normal human urothelial cell line SV-HUC-1 and bladder cancer cell lines T24 and RT4 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). These cells were cultured in DMEM supplemented with 10% FBS, 10 mM HEPES, 100 U penicillin/mL and 10 μg streptomycin/mL at 37 °C in a humidified atmosphere containing 95% air and 5% CO 2 . ART and PGE2 were dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO in the culture medium was kept less than 0.1%. Vehicle controls were prepared for all treatments.

Animals
All animal studies were performed according to guidelines established by the Research Animal Care Committee of Nanjing Medical University, China (Permit Number: NJMU-ERLAUA-20120507). Male Wistar rats (grade SPF; 200g) were purchased from Shanghai Laboratory Animal Centre (Chinese Academy of Sciences, Shanghai, China). The urinary bladder cancer model was performed according to a previously reported method [30]. Briefly, rats were given 0.05% of BBN in drinking water for several weeks. Then, rats (n = 40) were randomly and equally divided into 4 groups 10 weeks after drinking water containing 0.05% of BBN. Group 1 rats received corn oil by subcutaneous injection, once per day. Groups 2, 3 and 4 rats were treated with 20, 100 and 200 mg/kg of ART, respectively. Ten weeks after treatment with ART, all rats were sacrificed and the tumor size was measured. Tumor volume was calculated assuming a spherical shape, with the average tumor diameter being the square root of the product of cross-sectional diameters.

Flow Cytometry Analysis
Flow cytometry analysis is based on the translocation of phosphatidylserine from the inner leaflet of the plasma membrane to the cell surface in early apoptotic cells [31]. Briefly, cells were resuspended in a binding buffer. Next, annexin V-EGFP and PI were added and the solution was incubated at room temperature for 15 min in the dark, followed by assay on FACScan (Becton Dickinson, East Rutherford, NJ, USA). The percentage of apoptosis was computed using Cell-Quest software (Becton Dickinson).

Caspase-3 Activity Assays
Caspase-3 activity was analyzed using the caspase-3 activity assay kit according to the manufacturer's protocol. Cells were lysed and total cellular protein extracts were quantified using a protein-assay kit. Next, an equal amount of total protein extract was incubated at 37 °C overnight with either Ac-IETD-pNA for caspase-3 assay. The release of pNA was estimated by determining the absorbance at 405 nm on a microplate ELISA reader (Bio-Rad Laboratories, Hercules, CA, USA). The relative activity of caspase-3 was calculated as follows: caspase-3 activity = (mean experimental absorbance/mean control absorbance) × 100 (%).

ELISA
Analysis of PGE2 levels in the culture medium was performed using an ELISA kit according to the manufacturer's protocol. The corresponding cells were lysed, and the protein concentration was determined to normalize the PGE2 levels measured in the medium samples.

Western Blot Analysis
T24 and RT4 cells were lysed with ice-cold lysis buffer containing: 50 mmol/L Tris-HCl, pH 7.4; 1% NP-40; 150 mmol/L NaCl; 1 mmol/L EDTA; 1 mmol/L phenylmethylsulfonyl fluoride; and complete proteinase inhibitor mixture (one tablet per 10 mL; Roche Molecular Biochemicals, Indianapolis, IN, USA). Protein concentration in the cell lysate was quantified using the DC protein assay kit (Bio-Rad Laboratories). After protein content determination using a DC Protein Assay kit, western blot analysis was performed.

Statistical Analysis
Statistical analysis was performed with statistical analysis software SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). Comparisons between groups were made using one-way ANOVA, followed by Student's t-test. Results are presented as means ± SEM. Values of p < 0.05 were considered to be significant.

Conclusions
We found that a reduction in tumor cell proliferation accounted for the therapeutic effect of ART on bladder cancer. This is in line with the finding that increased miR-16 levels correlated with decreased expression of COX-2 in bladder cancer cells. We have established miR-16 was involved in ART mediating cytotoxicity and apoptosis of bladder cancer cells. Our data support the hypothesis that ART stimulates miR-16 expression, leading to the reduction of COX-2 expression levels, which resulted in a decrease of PGE2 production. These data indicate ART is an effective medicine to treat human bladder cancer.

Author Contributions
W.Z. designed research, analyzed data and wrote the paper. W.Z., Z.W. and J.X. performed research.

Conflicts of Interest
The authors declare no conflict of interest.