Abstract
Background/Aim: Honokiol is a biphenolic component of the bark of Magnolia, and has been shown to exert several activities, including anti-depressant, anti-emetic, anti-oxidative, anti-thrombotic, anti-angiogenesis, anti-anxiolytic, anti-inflammatory and anti-tumor effects. Materials and Methods: The anti-tumor activities of honokiol and its synergistic effect with 5-fluorouracil (5-FU) in human urothelial cell carcinoma (UCC) cells were investigated. Results: Honokiol significantly suppressed the proliferation of UCC cells in a dose- and time-dependent manner. Moreover, honokiol inhibited the tumorigenesis of UCC cells in vitro. In addition, honokiol induced cell cycle arrest at G0/G1 phase and caused apoptosis of UCC cells through the intrinsic pathway. Importantly, we demonstrated that honokiol potentiated the cytotoxic effect of 5-FU, and displayed a synergistic effect with 5-FU in UCC cells. Conclusion: Honokiol causes growth inhibition, tumorigenesis suppression, cell cycle arrest, apoptosis, and importantly has a synergistic effect with 5-FU in human UCC cells. Therefore, this agent displays a therapeutic potential for treating human UCC.
Human bladder cancer is the second most common cancer of the genitourinary tract (1). The incidence ratio between males and females is approximately 3:1. The mortality of bladder cancer has increased in recent decades (1). More than 90% of bladder cancers are epithelial malignancies, with the majority being transitional cell carcinomas and about 5% are adenocarcinomas or squamous cell carcinomas (2). Urothelial cell carcinoma (UCC) is a heterogeneous disease, and 70% of patients present with non-muscle invasive disease (Ta, T1 and carcinoma in situ), which tends to recur but is generally not life-threatening, whereas 30% of patients present with muscle-invasive disease, which is associated with a high mortality risk from distant metastases (3). Although transurethral resection by itself can eradicate a Ta, T1 tumor completely, these tumors commonly recur and can progress to muscle-invasive bladder cancers. The risk of progression of UCC patients after 5 years is 45% (4). Tumor metastasis is the primary cause of death in UCC, and is associated with a 5-year survival rate of approximately 8.1% (1). Once the cancer progresses to locally invasive disease (T2-T4), and/or regional or distant metastasis, survival is limited without treatment (5). To improve these unsatisfactory results, chemotherapy has been used to prevent local recurrence. The most commonly used approach is intravesical instillation, whereby the agents are instilled into the bladder to delay and prevent tumor recurrence (6, 7). However, current treatments have limited effect on human UCCs. In addition, although the United States Food and Drug Administration (FDA) has approved 126 oncological medications from 2006 to 2015, none has been approved for the treatment of UCCs (8). Therefore, an effective and novel treatment modality is urgently needed for UCCs.
Chinese herbal medicines have been extensively used in clinical practice for thousands of years in Asian countries. Magnolia officinalis, also named Houpo in Chinese or Saiboku-tu in Japanese, is an important herb in traditional Chinese and Japanese medicine for treating conditions such as neuronal disorders, anxiety, gastrointestinal disorders, thrombotic stroke, typhoid fever, and allergic disease (9). M. officinalis has been reported to contain a diverse group of biologically active compounds, including magnolol, honokiol, 4-O-methylhonokiol, obovatol, and other neolignan compounds (10). Magnolol and honokiol are the two major secondary metabolites found in ethanolic extracts of Chinese M. officinalis (9). Honokiol, a biphenolic bioactive component, has exhibited multiple biological activities, including anti-oxidant, anti-inflammatory, anti-bacterial, anti-fungal, anti-virus, anti-cancer, pro-apoptotic, and anti-osteoclastogenic activities (11-15). The anti-tumor activity of honokiol has been shown in various tumors in humans, including neuroblastoma, glioblastoma, thyroid cancer, lung cancer, breast cancer, hepatoma, colon cancer, prostate cancer, bladder cancer, pancreatic cancer, gastric cancer, ovarian cancer, melanoma, and lymphoma (13-19). Growth inhibition, cell cycle regulation and apoptosis induction are exhibited in honokiol-mediated tumor suppression in various human cancers (13, 16, 19). Moreover, multiple pathways including NF-κB activation, reactive oxygen species (ROS) production, endoplasmic reticulum (ER) stress, extracellular signal-regulated kinases (ERKs), JNK, p38 pathways, RhoA/ROCK (Rho-associated protein kinase), c-src/JAK/STAT3, and protein kinase B (AKT)/mTOR signaling pathways have been demonstrated to influence honokiol-mediated cellular death or tumor migration (16).
In addition, 5-fluorouracil (5-FU) is a pyrimidine analog that is widely used in the treatment of various solid cancers, including human urothelial carcinoma (20, 21). When it enters cancer cells, it is converted into three active metabolites including fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine triphosphate (FUTP), which disrupt the action of thymidylate synthase and exert cytotoxic effects by incorporating into RNA and DNA (21). Ultimately, these activities lead to the induction of apoptosis.
Furthermore, it has been reported that honokiol exerts anti-UCC activities through the induction of mitochondria-mediated apoptosis (22). However, it is still unclear whether honokiol can influence the anti-UCCs activity of 5-FU. In the present study, we verified the anti-tumor effects of honokiol alone or in combination with 5-FU in human UCC cells by determining its effect on cell growth, tumorigenesis, cell cycle regulation and apoptosis. Our data demonstrated that honokiol is an effective therapeutic agent for treating human UCC, and most importantly it exerts a synergistic anti-UCC activity in combination with 5-FU.
Materials and Methods
Urothelial cell carcinoma cell lines and cell culture. The two human UCC cell lines, 5637 and HT1197, and SV-HUC-1 cells (a SV40 immortalized human uroepithelial cell line) were obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) (23, 24). 5637, HT1197 and SV-HUC-1 cells were maintained in minimum essential medium (MEM) (Gibco BRL, Grand Island, NY, USA), RPMI1640 medium (Gibco) and Ham's F12 medium (Gibco) with 10% FBS (Gibco), respectively, and were cultured at 37°C in a 5% CO2 atmosphere according to BCRC's recommendations. 5637 cells were derived from a patient with grade II UCC, who was PIK3CA, FGFR3 wildtype and TERT and TP53 mutant. H1197 cells were generated from a patient with grade IV UCC, who was TP53 wildtype, and carried mutations in PIK3CA, FGFR3, and TERT.
Cell proliferation assay. Human UCC cells (5×103/well) were seeded in 96-well culture plates and incubated with medium only (containing 0.01% DMSO as the negative control) or with medium containing various concentrations of honokiol (Sigma, St. Louis, MO, USA) or 5-FU (Sigma). Then, cell viability was examined by the Cell Counting Kit-8 (CCK-8) (Sigma) (25). Moreover, Z-VAD-fmk (BioVision, Mountain View, CA, USA), a pan-caspase inhibitor, was used to block caspase activation, and evaluate the effect of honokiol or honokiol in combination with 5-FU on apoptosis of UCC cells using CCK-8 analysis (19, 26-29).
Colony formation assay. To determine the effect of honokiol on in vitro tumorigenesis, UCC cells (600 cells/well) were plated in a 6-well culture dish and pre-incubated with or without honokiol for 72 h. Then, cells were grown for 14 days to form colonies, and the colonies were stained with crystal violet for further counting (24, 26).
Cell cycle analysis. Cells (1×105 cells/plate) were seeded and then starved in serum-free medium for synchronizing cell cycle progression. After starvation, cells were incubated with DMSO or various concentrations of honokiol and/or 5-FU at different time intervals. Cells were then harvested and fixed with 70% methanol at 4°Covernight. Fixed cells were further incubated with RNase (10 mg/ml; Sigma) and propidium iodide (PI; 1 mg/ml; Sigma) at room temperature (RT) in the dark for 30 min. DNA content was determined by FACScan (Becton– Dickinson, San Diego, CA, USA) with the ModFit LT 3.3 software (19, 30).
Apoptosis analysis. Cells (1×106) were treated without or with honokiol and/or 5-FU. After incubation, cells were incubated with FITC-labelled annexin-V and PI (Sigma) for 15 min at RT, and the intensity of annexin-V or PI fluorescence was analyzed by FACScan (Becton–Dickinson). In addition, the mechanism underlying honokiol-mediated apoptosis was examined by western blotting with specific antibodies against the activation/cleavage of caspase-9 and -3 (Cell Signaling, Danvers, MA, USA), and PARP (Cell Signaling) (13, 19, 27-29, 31, 32).
Statistical analysis. Data are presented as mean±standard deviation. Significance of differences was analyzed by one-way ANOVA and Fisher's least significant difference test. Statistical significance was defined as p<0.05.
Results
Honokiol inhibited cellular proliferation and colony formation capacity of human UCC cells. To determine the anti-tumor activity of honokiol on UCC cells, SV-HUC-1, 5637 and HT1197 cells were incubated with DMSO (as a negative control) or various concentrations of honokiol. CCK-8 assay was performed to evaluate the effects of honokiol on cell viability. Our data showed that honokiol suppressed the proliferation of all cells in a time- and dose-dependent manner (Figure 1). The half maximal inhibitory concentration (IC50) of honokiol in SV-HUC-1 cells was 54.5, 42.3, and 32.5 μM at 24, 48 and 72 h, respectively. In addition, the IC50 in 5637 cells was 37.8, 28.2, and 22.6 μM at 24, 48 and 72 h, respectively. Furthermore, the IC50 in HT1197 ells was 17.3, 13.1, and 12.5 μM at 24, 48 and 72 h, respectively. These data demonstrated that honokiol can effectively inhibit the growth of UCC cell lines, and that HT1197 cells were more sensitive to the treatment of honokiol (Figure 1). Importantly, SV-HUC-1 cells were more resistant to honokiol treatment (Figure 1A). Because SV-HUC-1 cells are SV40 immortalized human uroepithelial cells, it can be considered as a benign cell line. Therefore, these data suggested that honokiol may be a safe chemotherapeutic agent in human UCCs. In addition, to determine the anti-tumorigenesis property of honokiol in 5637 and HT1197 cells, the colony formation assay was performed. Figures 2A and 2B demonstrate that honokiol suppresses tumorigenesis in a dose-dependent manner. However, 5637 cells were more sensitive to honokiol than HT1197 cells. Altogether, our results indicated that honokiol may be a useful and safe chemotherapeutic agent for treating human UCC.
Honokiol induced cell cycle arrest at G0/G1 phase. Because honokiol suppressed cellular proliferation in 5637 and HT1197 cells, we further examined its effect on cell cycle regulation. Figures 3A and 3B illustrate that honokiol causes cell cycle arrest at G0/G1 phase in 5637 and HT1197 cells in a dose-dependent manner at 24 h of treatment. Moreover, a significant increase in the percentage of cells in the sub-G1 phase was observed in both 5637 and HT1197 cells after honokiol treatment (Figure 3), suggesting that honokiol induces cell death after 48 and 72 h of treatment.
Honokiol regulated apoptosis in human UCC cells. To further investigate the effect of honokiol on apoptosis, 5637 and HT1197 cells were stained with annexin V and PI. The results showed that honokiol induced apoptosis in 5637 and HT1197 cells after 48 and 72 h of treatment (Figure 4A and B). Furthermore, the underlying molecular mechanism of honokiol-mediated apoptosis in UCC cells was explored by western blotting, by examining the expression of caspases. Figures 4A and 4B show that honokiol increased cleavage of caspases-9, and -3 and PARP. In contrast, caspase-8 was not activated in both 5637 and HT1197 cells under honokiol treatment (data not shown). These results suggest that the intrinsic pathway is involved in the honokiol-mediated apoptosis in human UCC cells.
To confirm the role of caspases in honokiol-mediated anti-UCC effect, Z-VAD-FMK, a pan-caspase-inhibitor, was used. The levels of uncleaved caspase-3 and PARP were increased after treatment with Z-VAD-FMK in both 5637 and HT1197 cells following honokiol treatment (Figure 5A). Moreover, expression of the cleaved forms of caspase-3 and PARP was reduced in cells incubated with Z-VAK-FMK and honokiol, compared to cells incubated with honokiol only (Figure 5A and 5B). Most importantly, the effect on cell viability was significantly reversed following treatment of 5637 and HT1197 cells with the combination of honokiol and Z-VAD-FMK (Figures 5A and 5B), suggesting that honokiol induced caspase-dependent apoptosis. Because the combination treatment of honokiol with Z-VAD-FMK was not able to cause a complete reversal of honokiol-mediated apoptosis, the existence of caspase-independent apoptosis cannot be rule out.
Honokiol exhibited synergistic anti-human UCCs activity with 5-fluorouracil. Previous studies have demonstrated that honokiol has an anti-cancer activity in various human cancer and UCC cells. Here, we further evaluated the therapeutic potency of honokiol and that of the clinical chemotherapeutic agent 5-Fluorouracil (5-FU), a common chemotherapeutic agent used to treat several human cancers. We therefore compared the anti-tumor activity of honokiol and that of 5-FU in 5637 cells after treatment for 24 h. The EC50 of honokiol in 5637 cells was 37.8 μM after 24 h treatment, and 128.38 μM in the 5-FU treated group (Data not shown). These data suggested that 5637 cells were more sensitive to treatment with honokiol than 5-FU. To address whether honokiol can modulate 5-FU-mediated anti-human UCC activity, human UCC cells were treated for 24 or 48 h with honokiol and/or 5-FU. Our results indicated that 40 μM honokiol had a similar effect with 100 μM 5-FU regarding cell growth suppression (Figure 6A). Importantly, a synergistic effect was determined following treatment of 5637 cells with both honokiol and 5-FU (Figure 6A). Further study illustrated that the 5-FU mediated anti-tumorigenesis activity could be enhanced at honokiol dose that does not inhibit colony formation (Figure 6B), suggesting their synergistic effect on anti-tumorigenesis in vitro in 5637 cells. In addition, apoptosis of 5637 cells was also observed following treatment with honokiol and 5-FU indicating their synergistic effect against human UCCs (Figure 6C and D). These findings demonstrate that honokiol in combination with 5-FU, can synergistically inhibit UCC proliferation, tumorigenesis and apoptosis and that honokiol is an alternative therapeutic agent for UCC.
Discussion
In the present study, we demonstrated an anti-tumor activity of honokiol in human UCC cells. Honokiol was able to inhibit proliferation, cause cell cycle arrest, induce apoptosis and suppress tumorigenesis. Most importantly, we also illustrated that honokiol enhanced the cytotoxicity of 5-FU, a clinical chemotherapeutic agent used for treating various cancer. The anti-cancer activity of honokiol has also been demonstrated in various cancers, including neuroblastoma, glioblastoma, thyroid cancer, lung cancer, breast cancer, hepatoma, colon cancer, prostate cancer, bladder cancer, pancreatic cancer, gastric cancer, ovarian cancer, melanoma, and lymphoma, in accordance with the findings of this study (13-19). It has also been reported that honokiol suppresses human bladder tumor growth and tumor stemness in vivo, accompanied by dysregulation of EZH2 and miR-143 expression (33). Moreover, honokiol has been shown to inhibit bladder cancer cell invasion by repressing SRC-3 expression and epithelial-mesenchymal transition (34). In addition, honokiol has been reported to induce apoptotic cell death through reactive oxygen species accumulation and mitochondrial hyperpolarization in bladder cancer cells (22). In addition, honokiol has exerted a synergistic effect with 5-FU on apoptosis induction in oral squamous cell carcinoma cells (35). Here, it was also demonstrated that honokiol exerts a synergistic anti-tumor activity with 5-FU in human UCC cells. Our findings and those of others strongly suggest that honokiol whether used alone or in combination with conventional chemotherapeutics such as 5-FU can be a potential therapeutic agent for treating human UCC.
In addition, evaluation of novel therapeutic agents that have less toxicity in normal cells is an important and challenging task. Honokiol has been reported to be safe for in vitro and in vivo use (15). Moreover, a preparation containing honokiol has been used in preclinical models of asthma, and clinical trials are currently undergoing to test this in Japan (36, 37). This study suggests that honokiol is safe to use in humans.
Additionally, honokiol has been reported to act as an anti-cancer agent in human T-cell leukemia, hepatoma, glioblastoma, pleural mesothelioma, epidermoid squamous carcinoma, prostate cancer, thyroid cancer, bladder cancer, pancreatic cancer, oral cancer and breast cancer (13). We and others have demonstrated that honokiol induces apoptosis in human UCC and other cancers. Honokiol induces ROS-mediated apoptosis through the intrinsic apoptosis pathway (BFTC-909 cells) (15). Here, induction of the extrinsic-apoptotic pathway was observed in UCC cells after honokiol treatment (Figures 4 and 5). However, by blocking the activity of caspases with Z-VAD-FMK in 5637 and HT1197 cells, the effect of honokiol on the viability of 5637 cells was slightly reversed (Figure 5). These data suggest that in addition to caspase-dependent apoptosis, other death pathways (38) may play a key role in the honokiol-mediated anticancer effect in human UCC cells. Therefore, a comprehensive investigation of the anti-tumor activity of honokiol in all human UCC cell lines is recommended.
In addition, NF-κB, STAT3, EGFR, mTOR and MAPK have been reported to be therapeutic targets of honokiol (18). MAPK signaling through ERK, P38, and JNK as well as the Akt/mTOR is known to be involved in cell differentiation, proliferation, and apoptosis. Moreover, activation of NF-κB as well as EGFR signaling is required to protect cancer cells from apoptotic cell death in certain advanced human bladder cancer cells and to increase the malignant potential of the disease (18, 39). Other studies have demonstrated that STAT3 promotes the progression from carcinoma in situ to invasive bladder cancer by increasing the expression of HIF1α and VEGF (40). Also, the role of NF-κB, STAT3, EGFR, mTOR or MAPK pathways in honokiol-induced apoptosis should be further evaluated in UCC.
Honokiol and 5-FU have been shown to exert a synergistic effect both in vitro and in vivo on oral squamous cell carcinoma cells, and the intrinsic apoptotic pathway has been shown to be involved in this effect (35). 5-FU is known to exert cytotoxic effects via its incorporation into RNA and DNA, and has been shown to induce apoptosis that critically depended on the presence of p53 and the cleavage of BID (35, 41). Here, we demonstrated that honokiol could synergistically enhance the anti-proliferative and anti-tumorigenic actions of 5-FU (Figure 6). However, whether p53 and Bid are involved in the synergistic effect of honokiol and 5-FU in UCC needs further investigation.
In the present study, we demonstrated that treatment with honokiol inhibited cellular proliferation, tumorigenesis, cell cycle progression, and induced apoptosis in human UCC cells through the intrinsic pathway. The synergistic activity of honokiol with 5-FU was also illustrated in the present study. Therefore, administration of honokiol alone or in combination with 5-FU is a possible chemotherapeutic strategy for the treatment of UCC and warrants further investigation.
Acknowledgements
This study was supported by grants from the Ministry of Science and Technology of the Republic of China, Taiwan MOST 108-2314-B-705-001, Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chiayi city, Taiwan (R106-39), and Chang Gung Memorial Hospital, Chaiyi, Taiwan (CMRPG6D0091~2). The Authors thank Mr. Myron for the critical reading of the manuscript.
Footnotes
Authors' Contributions
MYL, CSS YCH, KJH, YCH and YRL conceived and designed the experiments; YCH, KJH, SHC, PWZ, HCC and YRL analysed the data; MYL, CSS, YCH and YRL wrote the first draft of the manuscript; MYL, CSS, YCH, KJH, SHC, PWZ, HCC, YCH, and YRL read and confirm the criteria for authorship.
Conflicts of Interest
The Authors confirm that there are no conflicts of interest regarding this study.
- Received October 15, 2019.
- Revision received October 30, 2019.
- Accepted October 31, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved