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Thymol inhibits bladder cancer cell proliferation via inducing cell cycle arrest and apoptosis
Accepted Manuscript Thymol inhibits bladder cancer cell proliferation via inducing cell cycle arrest and apoptosis Yi Li, Jia-ming Wen, Chuan-jun Du, Su-min Hu, Jia-xi Chen, Shi-geng Zhang, Nan Zhang, Feng Gao, Shao-jiang Li, Xia-wa Mao, Hiroshi Miyamoto, Ke-feng Ding PII:
S0006-291X(17)30666-6
DOI:
10.1016/j.bbrc.2017.04.009
Reference:
YBBRC 37564
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 12 March 2017 Accepted Date: 3 April 2017
Please cite this article as: Y. Li, J.-m. Wen, C.-j. Du, S.-m. Hu, J.-x. Chen, S.-g. Zhang, N. Zhang, F. Gao, S.-j. Li, X.-w. Mao, H. Miyamoto, K.-f. Ding, Thymol inhibits bladder cancer cell proliferation via inducing cell cycle arrest and apoptosis, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.04.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Thymol inhibits bladder cancer cell proliferation via inducing cell cycle arrest and apoptosis Yi Li1,2,3,4，Jia-ming Wen1, Chuan-jun Du1, Su-min Hu1, Jia-xi Chen1, Shi-geng
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Zhang1, Nan Zhang1, Feng Gao1, Shao-jiang Li1, Xia-wa Mao1, Hiroshi Miyamoto4 and Ke-feng Ding2,3 1
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Department of Urology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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Department of Surgical Oncology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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Cancer Institute, Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education, Key Laboratory of Molecular Biology in Medical Sciences, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York, USA
Correspondence to: Ke-feng Ding, email: [email protected]
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Funding source: This work was supported by grants from National Natural Science Foundation of China (No. 81402099 to Yi Li, No. 81300475 to Jiaming Wen, No. 81400756 to Nan Zhang) and Projects of medical and health technology development program in Zhejiang province ( No. 2016147031 to Xiawa Mao).
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ACCEPTED MANUSCRIPT Abstract Thymol is a phenolic compound with various pharmacological activities such as anti-inflammatory, anti-bacterial and anti-tumor effects. However, the effect of thymol
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on bladder cancer cell growth is still elusive. The purpose of this study is to investigate the efficacy of thymol in bladder cancer cells and its underlying mechanism. Thymol inhibited bladder cancer cell proliferation in a dose and
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time-dependent manner. We also observed cell cycle arrest at the G2/M phase after
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the treatment of thymol. Moreover, thymol could induce apoptosis in bladder cancer cells via the intrinsic pathway along with caspase-3/9 activation, release of cytochrome c and down-regulation of anti-apoptotic Bcl-2 family proteins. The activation of JNK and p38 was also critical for thymol-induced apoptosis since it was
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abrogated by the treatment of JNK inhibitor (SP600125), and p38 inhibitor (SB203580) but not ERK inhibitor (SCH772984). Furthermore, the generation of
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ROS (reactive oxygen species) was detected after the treatment of thymol. ROS scavenger NAC (N-acetyl cysteine) could block the thymol-triggered apoptosis and
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activation of MAPKs. These findings offer a novel therapeutic approach for bladder cancer.
Key words: Thymol, bladder cancer, apoptosis, ROS, MAPKs
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ACCEPTED MANUSCRIPT Introduction
Bladder cancer is one of the most common urinary tract carcinomas and ranks as the
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9th leading cause of death worldwide [1]. In middle-aged and elderly males, bladder cancer is the second most common cancer after prostate cancer [2]. At diagnosis,
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approximately 30% of bladder cancer patients have muscle-invasive disease and 10% of the patients have metastatic disease [3]. Various strategies, such as radical
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cystectomy, chemotherapy, radiotherapy, immunotherapy, and their combinations, are widely employed for the treatment of bladder cancer [4,5]. However, these therapeutic approaches often correlate with side effects as well as high cost, and the prognosis of patients with advanced disease remains unsatisfactory [6,7]. Therefore, novel
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effective drugs are urgently needed to treat bladder cancer. Thymol is a major natural terpenoid compound present in the essential oil fraction of
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various plants, such as Thymus vulgaris (Lamiaceae) and Carum copticum (Apiaceae)
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[8]. It has been reported that thymol possesses broad activities including anti-microbial, anti-oxidant and anti-inflammatory effects. However, little is known about the anti-tumor effects of thymol. In the present study, we investigated the effects of thymol on the growth of bladder cancer cells. We found that thymol suppressed cell viability and induced cell cycle arrest and apoptosis in bladder cancer lines. This was the first study to utilize thymol
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ACCEPTED MANUSCRIPT to treat human bladder cancer cells and illustrated the mechanism of anti-tumor activity of thymol.
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Materials and Methods
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Cell culture and chemicals
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Bladder cancer cell lines, T24, SW780, and J82, were obtained from the American Type Culture Collection (VA, USA). The non-malignant immortalized urothelial cell line SV-HUC-1 was obtained from Cell Bank, Type Culture Collection, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in PRMI1640 medium
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(Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone) in a humidified incubator with a 5% CO2 atmosphere at 37°C. Thymol,
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2’,7’-dichlorofluorescein-diacetate
(DCF-DA),
3-(4,5-dimethylthiazol-2yl)-2,
5-diphenyltetrazoli-um bromide (MTT), N-acetyl cysteine (NAC) and DMSO were from
Sigma
(MO,
USA).
Z-LEHD-FMK,
Z-DEVE-FMK
and
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purchased
Z-IETD-FMK were purchased from Abcam (CA, USA)
MTT assay Cell viability was evaluated by MTT assay. Briefly, cells were seeded in a 96-well
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ACCEPTED MANUSCRIPT plate (5 X 103 cells/well) and then treated for up to 24 h with or without thymol. A total of 50 µl MTT solution (5 mg/ml) was added, and the cells were incubated for another 4 h. The medium was then removed and 200 µl of DMSO was added to each
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well. The absorbance of the solutions was measured on a BioTek microplate reader at 595 nm. The relative cell viability was measured by comparing with vehicle control
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which was treated with 0.1% DMSO.
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Cell cycle analysis
Cells were harvested after treatment with thymol and fixed in ethanol. The cells were washed with PBS and stained with propidium iodide (BD Bioscience, NJ, USA) for
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30 min in PBS supplemented with RNase at room temperature in the dark. The cell cycle distribution was evaluated using FACSVerseTM (Beckman Coulter Fullerton,
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CA, USA), and data were analyzed using FlowJo V10 (Flowjo, OH, USA).
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Apoptosis assay
Cells were harvested after treated with thymol and then double stained with an Annexin V-FITC/PI Apoptosis Detection Kit (BD Bioscience). The cell apoptosis was evaluated using FACSVerseTM, and data were analyzed using FlowJo V10.
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ACCEPTED MANUSCRIPT ROS detection To measure ROS generation, DCFH-DA was applied as described before with minor modifications [28]. After treatment with thymol, cells were collected and stained with
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FACSVerseTM. Data were analyzed using FlowJo V10.
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15 µM DCFH-DA for 30 min at 37°C, washed with PBS and then evaluated using
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Western blotting
After treatment with thymol, cells were lysed with RIPA buffer (Beyotime, Shanghai, China). The lysates (40 µg) were resolved by 12% SDS-PAGE and transferred to PVDF membrane. Primary antibodies against the following were used at
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4°Covernight: cyclin A (Abcam); cyclin B1 (Abcam); CDK2 (Abcam); and GAPDH (Sigma). Other primary antibodies, including p21, caspase-3, caspase-8, caspase-9, Bcl-2, Mcl-1, Bcl-xl, Bax, cytochrome c, Smac/DIABLO, p-JNK, JNK, p-Akt, Akt,
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p-ERK, ERK, p-p38, and p38, were purchased from Cellular Signaling Technology
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(MA, USA). Anti-rabbit and anti-mouse secondary antibodies (Sigma) were used. Signals were visualized by ECL reagent (Pierce, Rockford, IL).
Subcellular fraction The cytosolic and mitochondrial fractions were isolated using a special cytosolic and mitochondrial fraction kits (Beyotime, Shanghai, China), according to the 6
ACCEPTED MANUSCRIPT manufacturer’s guide. Briefly, cells were collected and isolation reagents were added incubated on ice followed by centrifugation at 1,500 rpm for 10 min. Supernatants were further centrifuged at 12,000 rpm for 15 min. The mitochondrial fraction was
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contained in the pellets, while the cytosolic fraction was contained in the supernatant.
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Statistical analyses
All the experiments were carried out at least three times. The data were analyzed
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using GraphPad Prism 6.0 software. The results were shown as the mean ± standard deviation (SD), and the differences were measured using Student’s t-test. Statistical
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Results
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significance was considered at p<0.05
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Thymol reduces cell viability and induces cell cycle arrest in bladder cancer cells To investigate the effects of thymol on bladder cancer cell growth, we treated bladder cancer cells T24, SW780, J82 and non-malignant urothelial cells SV-HUC-1 with different concentrations of thymol (0, 25, 50, 100, 150 µM) for 24 h or a certain dose (100 µM) of thymol for different times (0, 6, 12, 24, 36 h). The changes in cell viability were measured by MTT assays. As shown in Figure 1A and 1B, thymol
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ACCEPTED MANUSCRIPT significantly reduced the viability of human bladder cancer T24, SW780 and J82 cells in a dose- and time-dependent manner. Meanwhile, thymol decreased proliferation of SV-HUC-1 only at much higher concentrations (150µM) (Figure 1 A).
The IC50 of
selective anti-tumor activity in bladder cancer cells.
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thymol in different cells were shown in Table 1. These results suggest that thymol has
To unveil the mechanisms of the inhibition by thymol, distribution of the cell cycle in
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T24 which was more sensitive to thymol than other lines was analyzed by PI staining
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and flow cytometry (Figure 1C). We observed that thymol treatment could induce cell cycle arrest at G2/M phase in a dose-dependent manner (Figure 1D). To identify the regulatory proteins associated with cell cycle arrest, we carried out western blotting assay. After the treatment with thymol, considerable decreases in the expression of
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cyclin A, cyclin B1, and CDK2, as well as an increase in p21 expression, were
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observed in a dose-dependent manner (Figure 1E).
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Thymol induces apoptosis via the intrinsic pathway We then tested whether thymol could induce apoptosis in human bladder cancer cells. T24 cells were exposed to different concentrations (0, 25, 50, 100 µM) of thymol for 24 h, and apoptotic cells were evaluated using Annexin V-FITC/PI staining. As shown in Figure 2A and 2B, thymol triggered apoptosis in a dose-dependent manner. There are two known pathways leading to the apoptosis, namely intrinsic pathway and
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ACCEPTED MANUSCRIPT extrinsic pathway [18]. In order to unveil which pathway was responsible for the apoptosis induced by thymol, we performed western blotting to detect the caspases. We found increases in the expression of cleaved caspase-3 and caspase-9, but not
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caspase-8, after thymol treatment (Figure 2C), suggesting induction of apoptosis via the intrinsic pathway. To further confirm it, different specific caspase inhibitors were applied. As indicated in MTT assay and apoptosis assay, a caspase-3 inhibitor
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(Z-LEHD-FMK) and a caspase-9 inhibitor (Z-DEVE-FMK), but not a caspase-8
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inhibitor (Z-IETD-FMK), could significantly block the thymol-mediated inhibition of the viability of T24 cells (Figure 2D, left) and the thymol-induced apoptosis in T24 cells (Figure 2D, right). Bcl-2 family proteins are well known for regulation of the intrinsic apoptotic pathway [18]. Therefore, we tested whether Bcl-2 family proteins
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were involved in the apoptosis induced by thymol. As shown in Figure 2E, thymol reduced the expression of Bcl-2, Bcl-xl and Mcl-1 in T24 cells in a dose-dependent manner and augmented Bax expression. We also observed the release of cytochrome c
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and Smac/DIABLO from mitochondria into cytosol after the treatment of thymol
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(Figure 2F). These data further suggested that thymol induced apoptosis via the intrinsic pathway in human bladder cancer cells.
Thymol induces activation of MAPKs and represses Akt Mounting evidence suggests that PI3K/Akt signaling pathway is involved in the progression of bladder cancer [7,9]. Therefore, we assessed the change in the 9
ACCEPTED MANUSCRIPT phosphorylation of Akt (p-Akt), a downstream of PI3K, after the treatment of thymol. Western blotting assay indicated that incubation with thymol reduced the levels of p-Akt in a dose-dependent manner, but the levels of total Akt protein remained
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unchanged (Figure 3A, top). The endotoxin LPS is a potent activator of the PI3K/Akt signaling pathway [10]. To further confirm the ability of thymol to repress the p-Akt, we tested whether thymol could regulate LPS-induced up-regulation of p-Akt. As
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shown in Figure 3A (bottom), stimulation with LPS significantly induced Akt
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phosphorylation; however, pretreatment with thymol abrogated this effect. We next investigated whether MAPKs were also involved in the anti-tumor effects of thymol. As indicated in Figure 3B, thymol significantly induced phosphorylation of JNK, ERK and p38 in T24 cells in a dose-dependent manner. To further elucidate
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whether activation of MAPKs contributed to the anti-tumor effects of thymol, SP600125 (a pharmacological inhibitor of JNK), SCH772984 (a pharmacological
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inhibitor of ERK) and SB203580 (a pharmacological inhibitor of p38) were applied. As shown in Figure 3C, pretreatment of SP600125 and SB203580, but not
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SCH772984, significantly attenuated the decrease in cell viability and inhibited the apoptosis induced by thymol. Therefore, activation of JNK and p38 appeared to associate with the anti-tumor effects of thymol. Taken together, these data suggest that thymol may exert anti-tumor effects through inhibition of PI3K/Akt and activation of MAPKs.
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ACCEPTED MANUSCRIPT ROS generation is required for the anti-tumor effects of thymol Next we assessed whether ROS contributed to thymol-induced apoptosis and activation of MAPKs in human bladder cancer cells. As indicated in Figure 4A,
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thymol induced ROS generation in a dose-dependent manner. Pretreatment with N-acetyl cysteine (NAC), a ROS scavenger, could also abrogated the activation of MAPKs as well as the cleavage of caspase-3 and caspase-9 (Figure 4B and 4C). In
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addition, NAC abolished the effects of thymol on the cell viability (Figure 4D, upper)
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and apoptosis (Figure 4D, bottom). Thus, ROS generation appears to play an important role in the anti-tumor effects of thymol.
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Discussion
Thymol, a natural monoterpene phenol, is known to possess a variety of pharmacological properties. Our goals were to determine whether thymol had
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anti-tumor activities in human bladder cancer cells and to establish a potential
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rationale for its clinical application. Our results suggest that thymol is an active agent against human bladder cancer cells. Thymol and its essential oils are widely employed as a general antiseptic in different aspects including cosmetics, agriculture, food industry and medical practices [12]. Interestingly, thymol also possesses an anticancer activity. For example, several studies have proved that thymol inhibits cell proliferation and induces apoptosis in 11
ACCEPTED MANUSCRIPT several types of malignancies [12,13,14]. Consistent with these findings, thymol is shown to exert an anti-tumor activity in bladder cancer cells, including repression of cell viability and induction of cell cycle arrest and apoptosis.
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The cell cycle progression regulates the growth and proliferation of normal cells, however, carcinoma cells are lack of this regulation [15]. We observed an arrest at the G2/M phase accompanied by down-regulation of the expression of cell cycle
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regulator proteins, cyclin A, cyclin B1 and CDK2, and up-regulation of p21
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expression. Our findings in bladder cancer cells are inconsistent with those in a previous study showing that thymol induces cell cycle arrest at the G1 phase in gastric cancer cells [12]. There may be a cell type-specific difference and further investigation is needed.
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Apoptosis is a well-organized program which can be initiated by either extrinsic pathway or intrinsic pathway [9]. Many natural compounds have been found to trigger
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apoptosis via the intrinsic pathway [9,16]. We found activation of caspase-9 and caspase-3, but not caspase-8, after thymol treatment. Moreover, inhibitors of
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caspase-9 and caspase-3, but not caspase-8, could abrogate the apoptosis induced by thymol. These findings indicate that thymol-induced apoptosis is dependent on the activation of caspase-9/3. It is well documented that mitochondria plays a critical role in regulating the intrinsic pathway. The mechanism of mitochondria-regulated apoptosis relies on the dysfunction of mitochondria including release of cytochrome c and Smac/DIABLO into cytosol, which subsequently activates caspase cascade 12
ACCEPTED MANUSCRIPT [17,18]. In our study, the release of cytochrome c and Smac/DIABLO from mitochondria into cytosol was detected after treatment of thymol. Bcl-2 proteins are well-characterized regulators of intrinsic apoptosis [19]. Based on their roles in the
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process of apoptosis, Bcl-2 family proteins can be classified into two types: anti-apoptotic and pro-apoptotic. The ratio between pro-apoptotic and anti-apoptotic Bcl-2 family members is considered as a critical factor whether cells will undergo
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apoptosis [20]. Our results showed that up-regulation of pro-apoptotic protein Bax
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and down-regulation of anti-apoptotic proteins, Bcl-2, Bcl-xl and Mcl-1, in thymol-treated T24 cells. Taken together, these findings confirmed that thymol induced apoptosis via the intrinsic apoptotic pathway.
MAPK signaling cascade has been shown to involve response to extra-cellular stimuli
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and mediate the cellular signals [21]. Among MAPKs, ERK is usually associated with cell proliferation while JNK and p38 are closely associated with cell death [22]. We
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further elucidated the mechanisms underlying anti-tumor effects of thymol in bladder cancer cells and observed activation of MAPKs after treatment of thymol. These
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findings are contradictory to those in a study in which thymol is shown to inhibit MAPKs in non-neoplastic mouse mammary epithelial cells [23]. In addition, either a p38 inhibitor or a JNK inhibitor, but not an ERK inhibitor, could block the anti-tumor effects of thymol in bladder cancer cells. Therefore, activation of JNK and p38 is likely required for the cytotoxicity of thymol. The activation of Akt supports cells with a survival signal, and thereby allows cells to escape from apoptosis [24]. Various 13
ACCEPTED MANUSCRIPT studies have shown significance of the Akt pathway in inhibition of carcinoma cell growth [9,25,26]. In bladder cancer cells, phosphorylation of Akt was reduced by thymol treatment. We also observed that thymol abrogated the phosphorylation of Akt
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induced by LPS, which further confirmed that thymol could inhibit the Akt signaling pathway.
ROS is well known to regulate intracellular signal cascades, such as MAPKs, and
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excessive ROS generation can lead to mitochondria dysfunction and apoptosis [27,28].
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Previous studies have shown that polyphenolic compounds can increase ROS levels in human cancer cells [29,30]. Similar to previous results [13], we detected the generation of ROS after the treatment of thymol. Moreover, ROS scavenger NAC significantly abolished the apoptosis and activation of MAPKs induced by thymol.
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These results suggest that ROS plays a critical role in thymol-induced apoptosis and activation of MAPKs. Given that many anti-tumor agents exert anti-tumor effects via
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inducing ROS generation, thymol may in some extent exhibit cytotoxicity against bladder cancer cells by providing oxidative stress.
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In summary, we demonstrated that thymol was able to inhibit cell viability and trigger cell cycle arrest and mitochondria-related apoptosis in bladder cancer lines via the ROS-JNK/p38 pathway. Thymol also inhibited the Akt pathway in bladder cancer cells. These findings illustrated a detailed mechanism of the anticancer activity of thymol. Therefore, thymol may be utilized as a promising anticancer agent against bladder cancer. 14
ACCEPTED MANUSCRIPT Figure Legends
Figure 1. Thymol inhibits cell viability and induces cell cycle arrest in bladder cancer
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lines.
(A) Human bladder cancer cells, T24, SW780 J82, and non-malignant urothelial cells
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SV-HUC-1, were treated with various concentrations of thymol for 24 h, and cell viability was measured by MTT assays. (B) Human bladder cancer cells, T24, SW780
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J82, and non-malignant urothelial cells SV-HUC-1 were treated with 100 µM thymol for up to 36 h as indicated, and cell viability was measured by MTT assays. (C) Representative flow cytometry analysis in T24 cells after treatment with the indicated
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doses of thymol for 24 h. (D) Cell cycle populations after the treatment of thymol were estimated. (E) Western blotting analysis of indicated proteins after the treatment of 0-100 µM thymol for 24 h. Data represent mean ± SD of three indepednent
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vs control.
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experiments with triplicate sets in each assay. *P< 0.05, **P<0.01, and ***P<0.001
Figure 2. Thymol induces cell apoptosis via the intrinsic pathway. (A) After treatment with 0-100 µM thymol for 24 h, apoptosis of T24 cells was evaluated with Annexin V-FITC/PI staining and flow cytometry. (B) The quantification of apoptosis. (C) Western blotting analysis of indicated proteins after 15
ACCEPTED MANUSCRIPT treatment with 0-100 µM thymol for 24 h. (D) T24 cells were treated with 100 µM thymol, 25 µM Z-LEHD-FMK, 25 µM Z-DEVE-FMK, 25 µM Z-IETD-FMK, or their combinations for 24 h. Cell viability was evaluated using MTT assay (right) and the
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cell apoptosis was measured using Annexin V-FITC/PI staining (left). (E) T24 cells were treated with 0-100 µM thymol for 24 h, and the expression of the indicated proteins were evaluated by western blotting. (F) T24 cells treated with 0-100 µM
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thymol for 24 h were harvested and separated into cytosolic and mitochondrial
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fractions. The level of cytochrome c and Smac/DIABLO in cytosol and mitochondria were separately evaluated by western blotting. Each value shown represents mean ± SD of triplicate measurements. **P<0.01 and ***P<0.001 vs control.
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Figure 3. Thymol induces activation of MAPKs and inhibits phosphorylation of Akt. (A) T24 cells were treated with 0-100 µM thymol for 24 h (upper) or treated with
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100 µM thymol, LPS (2 µg/ml) and their combination for 24 h (bottom). Cell lysates
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were subjected to western blotting analysis with indicated antibodies. (B) T24 cells were treated with 0-100 µM thymol for 24 h, and cell lysates were subjected to western blotting analysis with indicated antibodies. (C) T24 cells were pre-treated with SP600125 (40 µM), SCH772984 (0.1 µM), and SB203580 (10 µM) for 1 h and treated with 100 µM thymol for 24 h. Then, cell viability and apoptosis were analyzed by MTT assay and PI/Annexin-V FITC staining assay, respectively. Each value shown represents mean ± SD of triplicate measurements. *P<0.05 and **P<0.01 vs control. 16
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Figure 4. Thymol induces apoptosis and MAPK activation dependent on the generation of ROS.
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(A) T24 cells were treated with 0-100 µM thymol for 24 h, and the intracellular ROS generation was measured using the DCFH-DA probe by flow cytometry. M reflects
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the positive DCF fluorescence. (B and C) T24 cells were pretreated with 5 mM NAC for 1 h and treated with 100 µM thymol for 24 h. Then, cell lysates were subjected to
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western blotting analysis with indicated antibodies. (D) T24 cells were pretreated with 5 mM NAC for 1 h and treated with 100 µM thymol for 24 h. Then, cell viability and apoptosis rate were evaluated using MTT assay and PI/Annexin-V FITC staining,
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**P<0.01 vs control.
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respectively. Each value shown represents mean ± SD of triplicate measurements.
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ACCEPTED MANUSCRIPT Table 1. IC50 of thymol in bladder cancer cell lines and SV-HUC-1 for 24 h. IC50 Values (µM) 90.1±7.6 108.6±11.3 130.5±10.8 >200
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Cell lines T24 SW280 J28 SV-HUC-1 Data represent mean ± S.D (n=3)
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ACCEPTED MANUSCRIPT Highlights
1. We demonstrate for the first time that thymol, a phenolic compound, possesses anti-tumor effects against bladder cancer cells.
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2. Thymol is able to repress the PI3K/Akt signaling pathway in bladder cancer cells.
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3. Generation of reactive oxygen species is vital for the anti-tumor effects of thymol.
S0006-291X(17)30666-6
DOI:
10.1016/j.bbrc.2017.04.009
Reference:
YBBRC 37564
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 12 March 2017 Accepted Date: 3 April 2017
Please cite this article as: Y. Li, J.-m. Wen, C.-j. Du, S.-m. Hu, J.-x. Chen, S.-g. Zhang, N. Zhang, F. Gao, S.-j. Li, X.-w. Mao, H. Miyamoto, K.-f. Ding, Thymol inhibits bladder cancer cell proliferation via inducing cell cycle arrest and apoptosis, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.04.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Thymol inhibits bladder cancer cell proliferation via inducing cell cycle arrest and apoptosis Yi Li1,2,3,4，Jia-ming Wen1, Chuan-jun Du1, Su-min Hu1, Jia-xi Chen1, Shi-geng
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Zhang1, Nan Zhang1, Feng Gao1, Shao-jiang Li1, Xia-wa Mao1, Hiroshi Miyamoto4 and Ke-feng Ding2,3 1
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Department of Urology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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Department of Surgical Oncology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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4
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Cancer Institute, Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education, Key Laboratory of Molecular Biology in Medical Sciences, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York, USA
Correspondence to: Ke-feng Ding, email: [email protected]
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Funding source: This work was supported by grants from National Natural Science Foundation of China (No. 81402099 to Yi Li, No. 81300475 to Jiaming Wen, No. 81400756 to Nan Zhang) and Projects of medical and health technology development program in Zhejiang province ( No. 2016147031 to Xiawa Mao).
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ACCEPTED MANUSCRIPT Abstract Thymol is a phenolic compound with various pharmacological activities such as anti-inflammatory, anti-bacterial and anti-tumor effects. However, the effect of thymol
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on bladder cancer cell growth is still elusive. The purpose of this study is to investigate the efficacy of thymol in bladder cancer cells and its underlying mechanism. Thymol inhibited bladder cancer cell proliferation in a dose and
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time-dependent manner. We also observed cell cycle arrest at the G2/M phase after
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the treatment of thymol. Moreover, thymol could induce apoptosis in bladder cancer cells via the intrinsic pathway along with caspase-3/9 activation, release of cytochrome c and down-regulation of anti-apoptotic Bcl-2 family proteins. The activation of JNK and p38 was also critical for thymol-induced apoptosis since it was
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abrogated by the treatment of JNK inhibitor (SP600125), and p38 inhibitor (SB203580) but not ERK inhibitor (SCH772984). Furthermore, the generation of
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ROS (reactive oxygen species) was detected after the treatment of thymol. ROS scavenger NAC (N-acetyl cysteine) could block the thymol-triggered apoptosis and
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activation of MAPKs. These findings offer a novel therapeutic approach for bladder cancer.
Key words: Thymol, bladder cancer, apoptosis, ROS, MAPKs
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ACCEPTED MANUSCRIPT Introduction
Bladder cancer is one of the most common urinary tract carcinomas and ranks as the
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9th leading cause of death worldwide [1]. In middle-aged and elderly males, bladder cancer is the second most common cancer after prostate cancer [2]. At diagnosis,
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approximately 30% of bladder cancer patients have muscle-invasive disease and 10% of the patients have metastatic disease [3]. Various strategies, such as radical
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cystectomy, chemotherapy, radiotherapy, immunotherapy, and their combinations, are widely employed for the treatment of bladder cancer [4,5]. However, these therapeutic approaches often correlate with side effects as well as high cost, and the prognosis of patients with advanced disease remains unsatisfactory [6,7]. Therefore, novel
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effective drugs are urgently needed to treat bladder cancer. Thymol is a major natural terpenoid compound present in the essential oil fraction of
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various plants, such as Thymus vulgaris (Lamiaceae) and Carum copticum (Apiaceae)
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[8]. It has been reported that thymol possesses broad activities including anti-microbial, anti-oxidant and anti-inflammatory effects. However, little is known about the anti-tumor effects of thymol. In the present study, we investigated the effects of thymol on the growth of bladder cancer cells. We found that thymol suppressed cell viability and induced cell cycle arrest and apoptosis in bladder cancer lines. This was the first study to utilize thymol
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ACCEPTED MANUSCRIPT to treat human bladder cancer cells and illustrated the mechanism of anti-tumor activity of thymol.
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Materials and Methods
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Cell culture and chemicals
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Bladder cancer cell lines, T24, SW780, and J82, were obtained from the American Type Culture Collection (VA, USA). The non-malignant immortalized urothelial cell line SV-HUC-1 was obtained from Cell Bank, Type Culture Collection, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in PRMI1640 medium
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(Hyclone, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone) in a humidified incubator with a 5% CO2 atmosphere at 37°C. Thymol,
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2’,7’-dichlorofluorescein-diacetate
(DCF-DA),
3-(4,5-dimethylthiazol-2yl)-2,
5-diphenyltetrazoli-um bromide (MTT), N-acetyl cysteine (NAC) and DMSO were from
Sigma
(MO,
USA).
Z-LEHD-FMK,
Z-DEVE-FMK
and
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purchased
Z-IETD-FMK were purchased from Abcam (CA, USA)
MTT assay Cell viability was evaluated by MTT assay. Briefly, cells were seeded in a 96-well
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well. The absorbance of the solutions was measured on a BioTek microplate reader at 595 nm. The relative cell viability was measured by comparing with vehicle control
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which was treated with 0.1% DMSO.
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Cell cycle analysis
Cells were harvested after treatment with thymol and fixed in ethanol. The cells were washed with PBS and stained with propidium iodide (BD Bioscience, NJ, USA) for
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30 min in PBS supplemented with RNase at room temperature in the dark. The cell cycle distribution was evaluated using FACSVerseTM (Beckman Coulter Fullerton,
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CA, USA), and data were analyzed using FlowJo V10 (Flowjo, OH, USA).
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Apoptosis assay
Cells were harvested after treated with thymol and then double stained with an Annexin V-FITC/PI Apoptosis Detection Kit (BD Bioscience). The cell apoptosis was evaluated using FACSVerseTM, and data were analyzed using FlowJo V10.
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ACCEPTED MANUSCRIPT ROS detection To measure ROS generation, DCFH-DA was applied as described before with minor modifications [28]. After treatment with thymol, cells were collected and stained with
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FACSVerseTM. Data were analyzed using FlowJo V10.
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15 µM DCFH-DA for 30 min at 37°C, washed with PBS and then evaluated using
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Western blotting
After treatment with thymol, cells were lysed with RIPA buffer (Beyotime, Shanghai, China). The lysates (40 µg) were resolved by 12% SDS-PAGE and transferred to PVDF membrane. Primary antibodies against the following were used at
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4°Covernight: cyclin A (Abcam); cyclin B1 (Abcam); CDK2 (Abcam); and GAPDH (Sigma). Other primary antibodies, including p21, caspase-3, caspase-8, caspase-9, Bcl-2, Mcl-1, Bcl-xl, Bax, cytochrome c, Smac/DIABLO, p-JNK, JNK, p-Akt, Akt,
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p-ERK, ERK, p-p38, and p38, were purchased from Cellular Signaling Technology
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(MA, USA). Anti-rabbit and anti-mouse secondary antibodies (Sigma) were used. Signals were visualized by ECL reagent (Pierce, Rockford, IL).
Subcellular fraction The cytosolic and mitochondrial fractions were isolated using a special cytosolic and mitochondrial fraction kits (Beyotime, Shanghai, China), according to the 6
ACCEPTED MANUSCRIPT manufacturer’s guide. Briefly, cells were collected and isolation reagents were added incubated on ice followed by centrifugation at 1,500 rpm for 10 min. Supernatants were further centrifuged at 12,000 rpm for 15 min. The mitochondrial fraction was
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contained in the pellets, while the cytosolic fraction was contained in the supernatant.
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Statistical analyses
All the experiments were carried out at least three times. The data were analyzed
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using GraphPad Prism 6.0 software. The results were shown as the mean ± standard deviation (SD), and the differences were measured using Student’s t-test. Statistical
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Results
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significance was considered at p<0.05
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Thymol reduces cell viability and induces cell cycle arrest in bladder cancer cells To investigate the effects of thymol on bladder cancer cell growth, we treated bladder cancer cells T24, SW780, J82 and non-malignant urothelial cells SV-HUC-1 with different concentrations of thymol (0, 25, 50, 100, 150 µM) for 24 h or a certain dose (100 µM) of thymol for different times (0, 6, 12, 24, 36 h). The changes in cell viability were measured by MTT assays. As shown in Figure 1A and 1B, thymol
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ACCEPTED MANUSCRIPT significantly reduced the viability of human bladder cancer T24, SW780 and J82 cells in a dose- and time-dependent manner. Meanwhile, thymol decreased proliferation of SV-HUC-1 only at much higher concentrations (150µM) (Figure 1 A).
The IC50 of
selective anti-tumor activity in bladder cancer cells.
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thymol in different cells were shown in Table 1. These results suggest that thymol has
To unveil the mechanisms of the inhibition by thymol, distribution of the cell cycle in
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T24 which was more sensitive to thymol than other lines was analyzed by PI staining
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and flow cytometry (Figure 1C). We observed that thymol treatment could induce cell cycle arrest at G2/M phase in a dose-dependent manner (Figure 1D). To identify the regulatory proteins associated with cell cycle arrest, we carried out western blotting assay. After the treatment with thymol, considerable decreases in the expression of
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cyclin A, cyclin B1, and CDK2, as well as an increase in p21 expression, were
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observed in a dose-dependent manner (Figure 1E).
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Thymol induces apoptosis via the intrinsic pathway We then tested whether thymol could induce apoptosis in human bladder cancer cells. T24 cells were exposed to different concentrations (0, 25, 50, 100 µM) of thymol for 24 h, and apoptotic cells were evaluated using Annexin V-FITC/PI staining. As shown in Figure 2A and 2B, thymol triggered apoptosis in a dose-dependent manner. There are two known pathways leading to the apoptosis, namely intrinsic pathway and
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ACCEPTED MANUSCRIPT extrinsic pathway [18]. In order to unveil which pathway was responsible for the apoptosis induced by thymol, we performed western blotting to detect the caspases. We found increases in the expression of cleaved caspase-3 and caspase-9, but not
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caspase-8, after thymol treatment (Figure 2C), suggesting induction of apoptosis via the intrinsic pathway. To further confirm it, different specific caspase inhibitors were applied. As indicated in MTT assay and apoptosis assay, a caspase-3 inhibitor
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(Z-LEHD-FMK) and a caspase-9 inhibitor (Z-DEVE-FMK), but not a caspase-8
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inhibitor (Z-IETD-FMK), could significantly block the thymol-mediated inhibition of the viability of T24 cells (Figure 2D, left) and the thymol-induced apoptosis in T24 cells (Figure 2D, right). Bcl-2 family proteins are well known for regulation of the intrinsic apoptotic pathway [18]. Therefore, we tested whether Bcl-2 family proteins
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were involved in the apoptosis induced by thymol. As shown in Figure 2E, thymol reduced the expression of Bcl-2, Bcl-xl and Mcl-1 in T24 cells in a dose-dependent manner and augmented Bax expression. We also observed the release of cytochrome c
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and Smac/DIABLO from mitochondria into cytosol after the treatment of thymol
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(Figure 2F). These data further suggested that thymol induced apoptosis via the intrinsic pathway in human bladder cancer cells.
Thymol induces activation of MAPKs and represses Akt Mounting evidence suggests that PI3K/Akt signaling pathway is involved in the progression of bladder cancer [7,9]. Therefore, we assessed the change in the 9
ACCEPTED MANUSCRIPT phosphorylation of Akt (p-Akt), a downstream of PI3K, after the treatment of thymol. Western blotting assay indicated that incubation with thymol reduced the levels of p-Akt in a dose-dependent manner, but the levels of total Akt protein remained
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unchanged (Figure 3A, top). The endotoxin LPS is a potent activator of the PI3K/Akt signaling pathway [10]. To further confirm the ability of thymol to repress the p-Akt, we tested whether thymol could regulate LPS-induced up-regulation of p-Akt. As
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shown in Figure 3A (bottom), stimulation with LPS significantly induced Akt
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phosphorylation; however, pretreatment with thymol abrogated this effect. We next investigated whether MAPKs were also involved in the anti-tumor effects of thymol. As indicated in Figure 3B, thymol significantly induced phosphorylation of JNK, ERK and p38 in T24 cells in a dose-dependent manner. To further elucidate
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whether activation of MAPKs contributed to the anti-tumor effects of thymol, SP600125 (a pharmacological inhibitor of JNK), SCH772984 (a pharmacological
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inhibitor of ERK) and SB203580 (a pharmacological inhibitor of p38) were applied. As shown in Figure 3C, pretreatment of SP600125 and SB203580, but not
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SCH772984, significantly attenuated the decrease in cell viability and inhibited the apoptosis induced by thymol. Therefore, activation of JNK and p38 appeared to associate with the anti-tumor effects of thymol. Taken together, these data suggest that thymol may exert anti-tumor effects through inhibition of PI3K/Akt and activation of MAPKs.
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ACCEPTED MANUSCRIPT ROS generation is required for the anti-tumor effects of thymol Next we assessed whether ROS contributed to thymol-induced apoptosis and activation of MAPKs in human bladder cancer cells. As indicated in Figure 4A,
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thymol induced ROS generation in a dose-dependent manner. Pretreatment with N-acetyl cysteine (NAC), a ROS scavenger, could also abrogated the activation of MAPKs as well as the cleavage of caspase-3 and caspase-9 (Figure 4B and 4C). In
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addition, NAC abolished the effects of thymol on the cell viability (Figure 4D, upper)
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and apoptosis (Figure 4D, bottom). Thus, ROS generation appears to play an important role in the anti-tumor effects of thymol.
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Discussion
Thymol, a natural monoterpene phenol, is known to possess a variety of pharmacological properties. Our goals were to determine whether thymol had
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anti-tumor activities in human bladder cancer cells and to establish a potential
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rationale for its clinical application. Our results suggest that thymol is an active agent against human bladder cancer cells. Thymol and its essential oils are widely employed as a general antiseptic in different aspects including cosmetics, agriculture, food industry and medical practices [12]. Interestingly, thymol also possesses an anticancer activity. For example, several studies have proved that thymol inhibits cell proliferation and induces apoptosis in 11
ACCEPTED MANUSCRIPT several types of malignancies [12,13,14]. Consistent with these findings, thymol is shown to exert an anti-tumor activity in bladder cancer cells, including repression of cell viability and induction of cell cycle arrest and apoptosis.
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The cell cycle progression regulates the growth and proliferation of normal cells, however, carcinoma cells are lack of this regulation [15]. We observed an arrest at the G2/M phase accompanied by down-regulation of the expression of cell cycle
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regulator proteins, cyclin A, cyclin B1 and CDK2, and up-regulation of p21
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expression. Our findings in bladder cancer cells are inconsistent with those in a previous study showing that thymol induces cell cycle arrest at the G1 phase in gastric cancer cells [12]. There may be a cell type-specific difference and further investigation is needed.
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Apoptosis is a well-organized program which can be initiated by either extrinsic pathway or intrinsic pathway [9]. Many natural compounds have been found to trigger
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apoptosis via the intrinsic pathway [9,16]. We found activation of caspase-9 and caspase-3, but not caspase-8, after thymol treatment. Moreover, inhibitors of
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caspase-9 and caspase-3, but not caspase-8, could abrogate the apoptosis induced by thymol. These findings indicate that thymol-induced apoptosis is dependent on the activation of caspase-9/3. It is well documented that mitochondria plays a critical role in regulating the intrinsic pathway. The mechanism of mitochondria-regulated apoptosis relies on the dysfunction of mitochondria including release of cytochrome c and Smac/DIABLO into cytosol, which subsequently activates caspase cascade 12
ACCEPTED MANUSCRIPT [17,18]. In our study, the release of cytochrome c and Smac/DIABLO from mitochondria into cytosol was detected after treatment of thymol. Bcl-2 proteins are well-characterized regulators of intrinsic apoptosis [19]. Based on their roles in the
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process of apoptosis, Bcl-2 family proteins can be classified into two types: anti-apoptotic and pro-apoptotic. The ratio between pro-apoptotic and anti-apoptotic Bcl-2 family members is considered as a critical factor whether cells will undergo
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apoptosis [20]. Our results showed that up-regulation of pro-apoptotic protein Bax
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and down-regulation of anti-apoptotic proteins, Bcl-2, Bcl-xl and Mcl-1, in thymol-treated T24 cells. Taken together, these findings confirmed that thymol induced apoptosis via the intrinsic apoptotic pathway.
MAPK signaling cascade has been shown to involve response to extra-cellular stimuli
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and mediate the cellular signals [21]. Among MAPKs, ERK is usually associated with cell proliferation while JNK and p38 are closely associated with cell death [22]. We
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further elucidated the mechanisms underlying anti-tumor effects of thymol in bladder cancer cells and observed activation of MAPKs after treatment of thymol. These
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findings are contradictory to those in a study in which thymol is shown to inhibit MAPKs in non-neoplastic mouse mammary epithelial cells [23]. In addition, either a p38 inhibitor or a JNK inhibitor, but not an ERK inhibitor, could block the anti-tumor effects of thymol in bladder cancer cells. Therefore, activation of JNK and p38 is likely required for the cytotoxicity of thymol. The activation of Akt supports cells with a survival signal, and thereby allows cells to escape from apoptosis [24]. Various 13
ACCEPTED MANUSCRIPT studies have shown significance of the Akt pathway in inhibition of carcinoma cell growth [9,25,26]. In bladder cancer cells, phosphorylation of Akt was reduced by thymol treatment. We also observed that thymol abrogated the phosphorylation of Akt
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induced by LPS, which further confirmed that thymol could inhibit the Akt signaling pathway.
ROS is well known to regulate intracellular signal cascades, such as MAPKs, and
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excessive ROS generation can lead to mitochondria dysfunction and apoptosis [27,28].
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Previous studies have shown that polyphenolic compounds can increase ROS levels in human cancer cells [29,30]. Similar to previous results [13], we detected the generation of ROS after the treatment of thymol. Moreover, ROS scavenger NAC significantly abolished the apoptosis and activation of MAPKs induced by thymol.
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These results suggest that ROS plays a critical role in thymol-induced apoptosis and activation of MAPKs. Given that many anti-tumor agents exert anti-tumor effects via
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inducing ROS generation, thymol may in some extent exhibit cytotoxicity against bladder cancer cells by providing oxidative stress.
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In summary, we demonstrated that thymol was able to inhibit cell viability and trigger cell cycle arrest and mitochondria-related apoptosis in bladder cancer lines via the ROS-JNK/p38 pathway. Thymol also inhibited the Akt pathway in bladder cancer cells. These findings illustrated a detailed mechanism of the anticancer activity of thymol. Therefore, thymol may be utilized as a promising anticancer agent against bladder cancer. 14
ACCEPTED MANUSCRIPT Figure Legends
Figure 1. Thymol inhibits cell viability and induces cell cycle arrest in bladder cancer
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lines.
(A) Human bladder cancer cells, T24, SW780 J82, and non-malignant urothelial cells
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SV-HUC-1, were treated with various concentrations of thymol for 24 h, and cell viability was measured by MTT assays. (B) Human bladder cancer cells, T24, SW780
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J82, and non-malignant urothelial cells SV-HUC-1 were treated with 100 µM thymol for up to 36 h as indicated, and cell viability was measured by MTT assays. (C) Representative flow cytometry analysis in T24 cells after treatment with the indicated
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doses of thymol for 24 h. (D) Cell cycle populations after the treatment of thymol were estimated. (E) Western blotting analysis of indicated proteins after the treatment of 0-100 µM thymol for 24 h. Data represent mean ± SD of three indepednent
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vs control.
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experiments with triplicate sets in each assay. *P< 0.05, **P<0.01, and ***P<0.001
Figure 2. Thymol induces cell apoptosis via the intrinsic pathway. (A) After treatment with 0-100 µM thymol for 24 h, apoptosis of T24 cells was evaluated with Annexin V-FITC/PI staining and flow cytometry. (B) The quantification of apoptosis. (C) Western blotting analysis of indicated proteins after 15
ACCEPTED MANUSCRIPT treatment with 0-100 µM thymol for 24 h. (D) T24 cells were treated with 100 µM thymol, 25 µM Z-LEHD-FMK, 25 µM Z-DEVE-FMK, 25 µM Z-IETD-FMK, or their combinations for 24 h. Cell viability was evaluated using MTT assay (right) and the
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cell apoptosis was measured using Annexin V-FITC/PI staining (left). (E) T24 cells were treated with 0-100 µM thymol for 24 h, and the expression of the indicated proteins were evaluated by western blotting. (F) T24 cells treated with 0-100 µM
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thymol for 24 h were harvested and separated into cytosolic and mitochondrial
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fractions. The level of cytochrome c and Smac/DIABLO in cytosol and mitochondria were separately evaluated by western blotting. Each value shown represents mean ± SD of triplicate measurements. **P<0.01 and ***P<0.001 vs control.
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Figure 3. Thymol induces activation of MAPKs and inhibits phosphorylation of Akt. (A) T24 cells were treated with 0-100 µM thymol for 24 h (upper) or treated with
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100 µM thymol, LPS (2 µg/ml) and their combination for 24 h (bottom). Cell lysates
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were subjected to western blotting analysis with indicated antibodies. (B) T24 cells were treated with 0-100 µM thymol for 24 h, and cell lysates were subjected to western blotting analysis with indicated antibodies. (C) T24 cells were pre-treated with SP600125 (40 µM), SCH772984 (0.1 µM), and SB203580 (10 µM) for 1 h and treated with 100 µM thymol for 24 h. Then, cell viability and apoptosis were analyzed by MTT assay and PI/Annexin-V FITC staining assay, respectively. Each value shown represents mean ± SD of triplicate measurements. *P<0.05 and **P<0.01 vs control. 16
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Figure 4. Thymol induces apoptosis and MAPK activation dependent on the generation of ROS.
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(A) T24 cells were treated with 0-100 µM thymol for 24 h, and the intracellular ROS generation was measured using the DCFH-DA probe by flow cytometry. M reflects
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the positive DCF fluorescence. (B and C) T24 cells were pretreated with 5 mM NAC for 1 h and treated with 100 µM thymol for 24 h. Then, cell lysates were subjected to
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western blotting analysis with indicated antibodies. (D) T24 cells were pretreated with 5 mM NAC for 1 h and treated with 100 µM thymol for 24 h. Then, cell viability and apoptosis rate were evaluated using MTT assay and PI/Annexin-V FITC staining,
References:
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**P<0.01 vs control.
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respectively. Each value shown represents mean ± SD of triplicate measurements.
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ACCEPTED MANUSCRIPT Table 1. IC50 of thymol in bladder cancer cell lines and SV-HUC-1 for 24 h. IC50 Values (µM) 90.1±7.6 108.6±11.3 130.5±10.8 >200
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1. We demonstrate for the first time that thymol, a phenolic compound, possesses anti-tumor effects against bladder cancer cells.
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2. Thymol is able to repress the PI3K/Akt signaling pathway in bladder cancer cells.
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3. Generation of reactive oxygen species is vital for the anti-tumor effects of thymol.