Abstract
Background/Aim: The aim of the present study was to investigate the efficacy of an ethanolic extract of gamboge (EEG), a traditional Chinese medicine (TCM), both in vitro on colon cancer cells and in vivo in an orthotopic mouse model of human colon cancer. Materials and Methods: The in vitro cytotoxicity of EEG on colon cancer cells was determined with the CCK8 proliferation assay and the Annexin V-PE/7-AAD apoptosis assay. Efficacy of EEG in vivo was evaluated in an orthotopic mouse model of human colon cancer implated with the green fluorescent protein-expressing human colon cancer cell line SW480-GFP. The tumor-bearing mice were treated with vehicle (0.2 ml/dose normal saline, po, daily), irinotecan (50 mg/kg/dose, ip, twice a week), 5-FU (15 mg/kg/dose, ip, every other day) as positive controls or EEG at doses of 12.5, 25 and 50 mg/kg/dose, po, daily. Real-time fluorescence imaging was performed to determine tumor inhibition in each treated group compared to the untreated controls. The protein expression of β-catenin, MMP-7, cyclin D1 and E-cadherin in the tumors was analyzed by immunohistochemistry. Results: EEG significantly induced proliferation inhibition and apoptosis of SW480 colon cancer cells in vitro in a dose-dependent manner. Tumor growth in the colon-cancer orthotopic model was significantly inhibited by irinotecan, 5-FU and all three doses of EEG. The efficacy of EEG was comparable to irinotecan and 5-FU. Irinotecan, 5-FU and 50 mg/kg EEG significantly decreased the protein expression of β-catenin and MMP-7. Cyclin D1 expression was decreased and E-cadherin expression was increased by irinotecan, 5-FU and all three doses of EEG. Conclusion: The present study demonstrates anti-tumor efficacy of EEG on colon cancer both in vitro and in vivo through inducing proliferation inhibition and apoptosis of SW480 colon cancer cells and inhibiting tumor growth, respectively. EEG exerts anti-tumor activity at least partly via down-regulation of the Wnt/β-catenin signaling pathway.
Colon cancer is a leading cause of cancer and cancer-death worldwide (1, 2). Standard chemotherapy has had limited efficacy in this disease (3, 4). Traditional Chinese medicine (TCM) offers an opportunity to improve outcomes in colon and other cancers (5, 6).
Garcinia hanburyi Hook.f., a plant belonging to the Guttiferae family, grows in southeastern Asia. Its resin is used as a dye and folk medicine for its potent purgative effects, and in the treatment of infected wounds (7, 8). Many extracts from plants of this family have been proven to possess anti-tumor activities (9-11). Gamboge, the yellow gum-resin secreted in latex-tubes (ducts) in the middle bark of Garcinia hanburyi, is used externally for infected wounds and systemically for pain and edema (12).
However, the anti-tumor efficacy of gamboges has not been investigated. Several solvents e.g. hexane, chloroform, ethyl acetate etc. were used for extracting gamboge. Ethyl acetate was found to be the best solvent to yield a relatively pure extract and also high crude yield.
The purpose of the present study was to evaluate the anti-tumor efficacy of an ethanolic extract of gamboge (EEG) from Garcinia hanburyi Hook.f. in an orthotopic mouse model of human colon cancer. Possible mechanisms of action were also examined.
Materials and Methods
Cell culture. The human colon cancer cell line SW480-GFP was obtained from AntiCancer, Inc., (San Diego, CA, USA). Cells were cultured in RPMI 1640 (GIBCO-BRL, Grand Island, New York, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT, USA) at 37°C in 5% CO2 saturated humidity. All media were supplemented with penicillin/streptomycin (GIBCO-BRL, Grand Island, New York, NY, USA).
Test agents. 5-fluorouracil (5-FU) and irinotecan (IRN) were used in this study. Ethanolic extract of gamboge (EEG) was supplied by Jiangsu Hospital of Traditional Chinese Medicine (Nanjing, P.R. China).
Cell proliferation assay. Cell proliferation was measured with the CCK8 assay. SW480-GFP cells (5×103 cells/well) were allowed to grow in 96-well overnight. Before treatment, all cells were switched to serum-free media with 1% BSA. Cells were then treated with EEG (0.2, 0.4, 0.8, 1.6, 3.2 μg/ml). After 24, 48 and 72 h treatment, 10 μl CCK8 reagent was added to each well for 4 h. The optical density (OD) at 450 nm was determined with a microplate reader (Bio-Rad, CA, USA). The results are presented as a percentage inhibition relative to the negative control. The median inhibitory concentration (IC50) was calculated from the dose-response curves. All experiments were repeated at least three times.
Detection of apoptotic cells. Apoptotic cells were identified with Annexin V-PE/7-AAD. Cells were seeded in 6-well plates (5×105 cells/well) and divided into four groups: untreated control, EEG 0.25, 0.5 and 1.0 μg/ml. After attachment to the dish, the cells in each group were treated with EEG and cultured for 24h. Cells were digested with 0.25% trypsin without EDTA (Thermo Fisher Scientific, CA, USA) and then were stained with the Annexin V-PE/7-AAD apoptosis kit (Nanjing KeyGen BioTech, Nanjing, P.R.China) according to the manufacturer's instructions. Stained cells were identified and analyzed using flow cytometry (Becton Dickinson, NJ, USA). Each experiment was performed in triplicate.
Animal care. BALB/C male nude mice (48), aged 4-6 weeks, 20-25 g, were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, P.R.China). All mice were maintained in a HEPA-filtered environment at 24-25°C. Humidity was maintained at 50-60%. All animals were fed with autoclaved laboratory rodent diet. Animal experiments were approved by the Animal Committee of Nanjing Origin Biosciences, P.R.China (OB1617).
Orthotopic mouse model of colon cancer. The surgical orthotopic implantation (SOI) method followed published procedures (13, 14). Stocks of SW480-GFP tumors were established by subcutaneously injecting SW480-GFP cells (5×106) in the flank of nude mice. Colon tumors, grown s.c. in nude mice, were harvested at the exponential growth phase and resected under aseptic conditions. Strong GFP expression of the SW480-GFP tumor tissue was confirmed by fluorescence microscopy. Necrotic tissues were removed and viable tissues were cut with scissors and minced into 1 mm3 pieces. Animals were anesthetized by injection of a 0.02 ml solution of 50% ketamine, 38% xylazine, and 12% acepromazine maleate. After proper exposure of the colon following a lower-midline abdominal incision, the serosa of the colon was removed and two 1 mm3 tumor fragments per mouse were implanted on the wall of the colon with an 8-0 surgical suture. The intestine was then returned to the abdominal cavity. The incision in the abdominal wall was closed with a 6-0 surgical suture in one layer. All procedures of the operation described above were performed under an 8 x magnification microscope (Shanghai Precision Instruments, YZ20P5, Shanghai, P.R. China).
Treatment. Treatments were initiated when mean tumor volume reached 100 mm3. The mice were randomly divided into six groups of eight. Group 1 (vehicle control) received saline by oral gavage daily at 0.2 ml/dose for four weeks. Group 2 (IRN) received intraperitoneal irinotecan twice a week at 50 mg/kg/dose for four weeks. Group 3 (5-fluorouracil [5-FU]) received intraperitoneal 5-FU every other day at 15 mg/kg/dose for two weeks. Group 4-6 received EEG by oral gavage daily for four weeks at 12.5, 25 and 50 mg/kg/dose, respectively. Tumor growth was measured by whole-body fluorescence imaging with a fluorescence stereo microscope (MZ650; Nanjing Optic Instrument Inc., Nanjing, P.R. China) equipped with band-pass HQ600/50 emission and HQ540/40 excitation filters (Chroma Technology, Brattleboro, VT, USA). Animal body weights and clinical signs were recorded over the course of the experiments. All animals were sacrificed 28 days after initial treatment. At autopsy open fluorescence imaging was performed and then the tumor was removed and weighed. Images were processed and analyzed with the use of IMAGE PRO Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).
Immunohistochemistry. At the end of the study, all mice were sacrificed and the tumors were removed and weighed. Tumor tissues were fixed in 10% buffered formalin and paraffin-embedded. For immunohistochemistry, sections were incubated with primary antibodies against β-catenin, MMP-7, cyclin D1 and E-cadherin (BD Biosciences, San Diego, CA, USA) overnight at 4°C after permeabilization with a solution of 0.1% sodium citrate and 0.1%. Triton X-100 and blocking with 10% rabbit serum. After washing with pH 7.4 phosphate-buffered saline (PBS), the sections were then incubated with secondary antibody (Dako REAL EnVision Detection System, Dako, UK) for 30 min at room temperature. Color development was performed with 3, 3’-diaminobenzidine (DAB). Nuclei were lightly counter stained with hematoxylin. The slides were viewed at ×400 magnification and positive cells were recognized by the appearance of brown staining. The expression level was quantified by the average optical density (AOD) of the positive cells in five fields per sample with ImagePro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) (15).
Statistical analysis. Data are expressed as means ± SD, and were analyzed by one-way analysis of variance (ANOVA), using SPSS software version 16.0, where p<0.05 is considered to be statistically significant.
Results and Discussion
EEG inhibits proliferation of colon cancer cells. EEG significantly inhibited SW480-GFP cell growth in a dose- and time-course-dependent manner (Figure 1). The IC50 values of EEG were 0.54 μg/ml at 24 h, 0.36 μg/ml at 48 h and 0.24 μg/ml at 72 h of treatment.
EEG induces apoptosis in colon cancer cells. EEG at doses of 0.25, 0.5 and 1 μg/ml significantly increased the frequency of early and late-stage apoptotic SW480-GFP cells compared to the untreated cells. Late-stage apoptosis was induced by EEG in a dose-dependent manner (Figure 2).
EEG inhibits orthotopic SW480 tumor growth. Tumor growth was significantly inhibited in the mice treated with all three doses of EGG as well as with IRN and 5-FU compared to the control group from day 7 after treatment initiation (p<0.05). Representative fluorescence images, tumor growth curves and final tumor weight in each group are shown in Figures 3A, B and C, respectively. The final tumor weight in the EEG-treated groups, as well as the IRN and 5-FU groups were also significantly reduced, compared to the control group (p<0.05).
EEG modulates the Wnt/beta-catenin signaling pathway. EGG (50 mg/kg), irinotecan and 5-FU significantly decreased the protein expression levels of β-catenin and MMP-7 compared to the control group (p<0.05) (Figure 4A, B). Cyclin D1 expression was significantly reduced in the groups treated with all three doses of EEG as well as in the IRN and 5-FU groups compared to the control group (p<0.01) (Figure 4C). All three doses of EEG as well as irinotecan and 5-FU increased protein expression of E-cadherin compared to control group (p<0.01) (Figure 4D).
Body weight and toxicity. No physical or behavioral signs indicated adverse effects due to EEG, IRN or 5-FU. No significant body weight loss was found in any treated groups (Figure 5).
The present study is the first to demonstrate anti-tumor activity of EEG on human colon cancer. EEG inhibited cell proliferation of SW480 human colon cancer cells and induced apoptosis, suggesting that EEG exerted its anti-tumor activity through apoptosis. EEG significantly inhibited tumor growth in an orthotopic mouse model of SW480 colon cancer comparable to 5-FU and IRN, first-line therapy for this disease.
Immunohistochemical analysis showed that EEG inhibited Wnt signaling by down-regulating β-catenin which resulted in decreased transcription of the downstream genes of β-catenin, including cyclin D1 and MMP-7 (16-19). EEG treatment increased the protein-expression level of E-cadherin, a marker of epithelial cells which is involved in the epithelial-mesenchymal transition (20, 21). Based on these findings, we infer that EEG exerts anti-tumor activity at least partly via down-regulation of the Wnt/β-catenin signaling pathway.
In conclusion, the present study demonstrates the efficacy of EEG on colon cancer both in vitro and in vivo by inducing apoptosis of SW480 cells and inhibiting tumor growth via down-regulation of the Wnt/β-catenin signaling pathway.
The orthotopic mouse tumor model, especially with the use of GFP for imaging, is revolutionizing the TCM field enabling discovery of potent anti-tumor and anti-metastatic agents (15, 22-30). The orthotopic model is the bridge to the clinic for TCM (13, 14, 31).
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 81473565) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province.
Footnotes
↵* These Authors contributed equally to this study.
Conflicts of Interest
None of the Authors have a conflict of interest in regard to this study.
- Received January 16, 2018.
- Revision received February 8, 2018.
- Accepted February 9, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved