Bladder cancer (BCa) is one of the most prevalent types of cancer and is the leading cause of death among patients with urinary tract disease [1
]. In 2016, the United States alone recorded more than 76,000 new cases of BCa and 16,000 deaths [2
]. Most BCa cases are diagnosed as non-muscle invasive tumors; however, 50–70% of these tumors recur frequently and approximately 15% eventually develop into muscle-invasive or metastatic BCa [3
]. Current treatment methods including radical cystectomy and systemic chemotherapy are effective in some muscle-invasive BCa patients, but 95% of metastatic BCa patients die within 5-years diagnosis, indicating the need for new therapeutic strategies [5
) is one of the major lipophilic compounds extracted from the root of a traditional Chinese medicine, Danshen (Salvia miltiorrhiza
], and has been used for the treatment of cardiovascular disease via its anti-oxidant and anti-inflammatory activity [8
]. In addition, Tan-IIA has been found to exert antitumor activity in various types of cancer including osteosarcoma [10
], gastric [11
], lung [12
], esophageal [13
], and prostate cancers [14
]. The antitumor activity of Tan-IIA mainly occurs through proliferation inhibition, apoptosis induction, and metastasis inhibition [15
]. For instance, Tan-IIA increased CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-4 expression, and induced apoptosis of human esophageal Ec-109 cells via the endoplasmic reticulum (ER) stress pathway [19
]. Tan-IIA induced cytochrome c-mediated caspase cascade apoptosis in A549 human lung cancer cells via the JNK pathway [20
]. Tan-IIA caused apoptosis in human oral cancer KB cells through a mitochondria-dependent pathway [21
]. However, Tan-IIA did not show significant cytotoxicity on human normal prostate epithelial cells (PrEC) and normal mammary epithelial cells (HMEC) at the concentrations high as 50 μM [22
]. Also, the toxicity in normal tissues was not observed in Tan-IIA treated mice [24
]. In our previous study, Tan-IIA was found to induce mitochondria-dependent apoptosis and suppress migration in BCa cells [25
]. However, the mechanism by which Tan-IIA inhibits the migration and invasion of BCa cells remains undetermined.
Previous reports found a correlation of urinary CCL2 levels with tumor stage, grade and metastasis in patients with BCa [26
], and patients with stages T2–T4 BCa were found to have a higher mean CCL2 concentration in their urine as compared to those with T1 stage tumors [27
]. Previous studies also showed that CCL2 can regulate tumor progression and metastasis by altering the tumor microenvironment [28
]. CCL2 induced epithelial mesenchymal transition (EMT) in order to promote tumor metastasis in various cancer types [31
]. Down-regulation of CCL2 expression by inhibiting phosphorylation of STAT3 led to the suppression of metastasis in breast and lung cancer [34
]. STAT3 signaling is an important pathway which is frequently activated in many tumors including BCa [35
]. The transcriptional activity of STAT3 is required for the phosphorylation at the tyrosine residue 705 (Tyr705) and has been demonstrated to be critical for BCa cell growth and survival [36
]. In addition, activation of STAT3 promoted migration and invasion of BCa cells [38
]. Thus, we seek to elucidate the role of STAT3-CCL2 signaling in Tan-IIA-induced EMT inhibition in BCa cells.
The results of the present study demonstrate that Tan-IIA inhibited the migration and invasion of human BCa cells. Tan-IIA inhibited EMT in BCa cells via the suppression of CCL2 expression which cannot be reversed by addition of CCL2 recombinant protein. In addition, Tan-IIA suppressed the phosphorylation of STAT3 (Tyr705), which cannot be restored by addition of CCL2 recombinant protein. Our data suggests that Tan-IIA might inhibit EMT in BCa cells through the STAT3-CCL2 signaling inhibition.
Our previous study reported that Tan-IIA could inhibit the proliferation and migration of human BCa cells [25
], but the underlying mechanism of Tan-IIA attenuating the migration and invasion of BCa cells remains unclear. EMT is a process by which epithelial cells gradually transform into mesenchymal-like cells to promote the migration and invasiveness of cancer cells [42
]. Our results showed that Tan-IIA treatment could inhibit the process of EMT as evidenced by increased level of the epithelial marker E-cadherin and decreased level of mesenchymal markers (N-cadherin and Vimentin). Activation of MMP proteins leads to cell migration and penetration to the basement membrane, playing an important role in EMT processes [43
]. In previous studies, Tan-IIA decreased migration or invasion through inhibiting MMP-9/-2 secretion in gastric cancer and osteosarcoma [11
]. Similar results observed in our study showed that Tan-IIA suppressed both the protein expression and enzymatic activity of MMP-9/-2 on human BCa cells (Figure 1
B). Together, these findings suggest that Tan-IIA inhibits EMT in human BCa cells.
Several reports have demonstrated the importance of CCL2 and EMT signals in BCa progression. Chiu et al. reported that blocking the CCL2/CCR2 pathway could decrease the migration and invasion of BCa cells [39
]. Additional reports show that CCL2 signals promote EMT in various tumors including BCa [32
]. The present study provides evidence that Tan-IIA decreased CCL2 expression in a dose-dependent manner by qRT-PCR and ELISA analysis (Figure 3
). The addition of CCL2 recombinant protein resulted in a partial reversal of EMT markers, and attenuated the Tan-IIA-induced migration and invasion inhibition in BCa cells. Our results show that Tan-IIA inhibits the EMT in BCa cells via the suppression of CCL2 expression.
Additional reported data suggested that CCL2 induced EMT through the activation of STAT3 signals [33
] and inhibited STAT3 signaling to reduce the invasiveness of tumor cells [41
]. Our data showed that Tan-IIA could inhibit the p-STAT3 (Tyr705) in a time- and dose-dependent manner. Besides, inhibition of STAT3 expression by STAT3 siRNA transfection attenuated the expression of CCL2. The phosphorylation of STAT3, inhibited by Tan-IIA, cannot be restored by CCL2 recombinant protein addition. These data suggested that Tan-IIA inhibits EMT of human BCa cells via modulation of STAT3-CCL2 signaling (Figure 5
). Several effects of Tan-IIA on human cancer were also integrated to get a better view of possible anti-cancerogenic effects of Tan-IIA [47
]. In addition, since the results from this study were based on in vitro assays of human BCa cells, the in vivo experiments are necessary for future study.
EMT is orchestrated by several signaling pathways, including JAK/STAT3 and TGF-β/Smad signaling. Recent studies have demonstrated that TGF-β-mediated cancer metastasis was associated with the activation of STAT3 pathway in colorectal and lung cancer [51
]. STAT3 activation can increase smad7 expression and form an inhibitory complex with smad3 which eventually suppress EMT [53
]. Recent study elucidated the mechanisms behind the tumoricidal activity of TCM clinical prescription Jianpi Huayu Decoction (JHD) in Hepatocellular carcinoma (HCC) treatment. Their results indicated that Tan-IIA might be the one of crucial components of the JHD that targets on the TGF-β/Smad3 pathway and inhibits EMT [50
]. Taken together, the targeted blockade of the STAT3/smad3 axis in tumor cells may be an effective therapeutic strategy against tumor metastatic progression and worth for further investigation.
4. Materials and Methods
4.1. Chemicals and Antibodies
Tanshinone IIA (C19H18O3, >97% HPLC), Dimethyl sulfoxide (DMSO), [3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), Tween-20, methanol, and horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The antibodies against p-STAT3 (Tyr705), STAT3, E-cadherin, N-cadherin, Vimentin, Slug, Snail, MMP-2, MMP-9 and β-actin were all purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). The human CCL2 recombinant protein was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Polyvinyldenefluoride (PVDF) membranes, BSA protein assay kit and western blot chemiluminescence reagent were purchased from Amersham Biosciences (Arlington Heights, IL, USA).
4.2. Cell Culture
The human BCa cell lines 5637 (grade II carcinoma), BFTC (BFTC 905, papillary transitional cell carcinoma), and T24 (transitional cell carcinoma) were purchased from BCRC (Bioresource Collection and Research Center, Hsinchu, Taiwan). Cells were cultured in appropriate medium supplemented with 10% FBS, 100 U/mL penicillin and 100 U/mL streptomycin (all from Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere with 5% CO2.
4.3. Western Blot Analysis
Five hundred thousand cells per 6-cm plate were lysed with 200 µL M-PER mammalian protein extraction reagent containing protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA) and centrifuged at 13,000× g at 4 °C for 10 min. The protein concentration in the supernatants was quantified using a BSA Protein Assay Kit. Electrophoresis was performed on a NuPAGE Bis-Tris Electrophoresis System using 20 µg of reduced protein extract per lane. Resolved proteins were transferred to PVDF membranes, blocked with 5% skim milk for 1 h at room temperature, finally probed with the specific primary antibodies at 4 °C overnight. After the PVDF membrane was washed three times with TBS/0.2% Tween-20 at room temperature, it was incubated with appropriate secondary antibody labeled with horseradish peroxidase (Sigma Chemical, St. Louis, MO, USA) for 1 h at room temperature. All resolved proteins bands were detected using Western Lightning™ Chemiluminescence Reagent Plus (Amersham Biosciences, Arlington Heights, IL, USA).
4.4. Cell Migration and Invasion Assay
The trans-well assay was performed using Hanging inserts (Millipore Co., Billerica, MA, USA) with 8 μm polycarbonate membrane in a 24-well plate. Cells were seeded in 6 well plates and treated without or with 4 μg/mL Tan-IIA with or without CCL2 for 24 h. Cells were then detached and seeded (5 × 104) to the upper chamber of the transwell plates. Upper chambers were filled with serum free medium and lower chambers were filled with cultured medium containing 10% FBS as a chemo-attractant. Incubation was carried out at 37 °C for the indicated 24 h. The hanging inserts were washed with PBS, and cells on the upper filter surface were wiped away with a cotton swab. The inserts were subsequently fixed with 10% formalin for 10 min at room temperature, stained with 0.2% w/v crystal violet, washed with PBS, the remaining cells were counted on the opposite site of the filter under a light microscope operating at 200× magnification. The migration cell numbers of control group were considered as 100%. For the invasion assay, a Matrigel basement membrane matrix (BD Biosciences, San Jose, CA, USA) was coated to the upper side of the hanging inserts at a concentration of 2 mg/mL. Cells were seeded onto the coated hanging inserts and followed by migration assay protocol.
4.5. RNA Extraction and Real-Time RT-PCR
Total RNA was extracted from cell lines using RNeasy Mini Kit®
(Qiagen, Valencia, CA, USA) and reverse transcribed at 37 °C for 60 min with Omniscript RT Kit®
(Qiagen) according to the manufacturer’s instructions. Real-time RT-PCR analysis was performed in triplicate in a Step One Plus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with Power SYBR®
Green PCR Master Mix (Applied Biosystems) in a final volume of 20 μL/reaction. Threshold cycle (Ct
) value of each tested gene was normalized to the Ct
value of the GAPDH control from the same RNA preparation. The ratio of transcription of each gene was calculated as 2–(ΔCt)
, where ΔCt
is the difference Ct
(GAPDH). Real-time RT-PCR primer sequences used in this study are listed in Table 1
4.6. Enzyme-Linked Immunosorbent Assay (ELISA)
Human MCP-1/CCL2 ELISA kit was purchased from R&D Systems. BCa cells were cultured in serum-free medium with or without Tan-IIA for 72 h. The medium were collected (400 μL/sample in 96-well) for ELISA assay according to manufacturer’s instructions.
4.7. Gelatin Zymography
The BCa cells were cultured in serum-free medium containing Tan-IIA (0, 1, 2, 4 μg/mL) for 48 h and the supernatant was collected. The supernatant was mixed with non-reducing SDS gel sample buffer. Electrophoresis was carried out using 10% native polyacrylamide gel containing 0.1% gelatin (Sigma, St. Louis, MO, USA) on a NuPAGE Bis-Tris Electrophoresis System. After electrophoresis, the gels were washed in wash buffer containing 2.5% Triton X-100 at room temperature, and then incubated with the reaction buffer containing l M CaC12, 2% NaN3, 1 M Tris-HCl (pH 8.0) at 37 °C overnight. Gels were stained by Coomassie Brilliant Blue R-250 solution and gelatinolytic activity was shown as clear areas in the gel.
4.8. Small Interfering RNA (siRNA) Transfection
STAT3 siRNA (#6582) was purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). Non-targeting siRNA (ON-TARGET plus non-targeting pool) were purchased from Dharmacon RNAi Technologies (Lafayette, CO, USA). Non-targeting control sequences were not provided. BFTC cells at 50–60% confluence were transfected with siRNA (40 or 80 nM) using the DharmaFECT 4 transfection reagents (GE Healthcare Dharmacon, Lafayette, CO, USA) according to the manufacturer’s protocol. Cells were cultured for 24 h, and then treated with Tan-IIA or vehicle for an additional 48 h. Proteins were then isolated for western blotting.
4.9. Statistical Analysis
All data were shown as mean ± S.D. Statistical differences were analyzed using the Student’s t-test for normally distributed values.