Introduction

Colorectal cancer is one of the top three most common cancer and the third leading cause of cancer-related death in the United States1. In 2016, American Cancer Society (ACS) estimates that 134,490 persons will be diagnosed having colorectal cancer (CRC), and more than one-third will die from this cancer2. Although the incidence and mortality rate of CRC in developed countries declined during the last decade mainly due to the early screen in asymptomatic and average risk people2,3, incidence and mortality rate are still growing in develo** world with increasing westernized lifestyle and aging population3. Surgery is the primary treatment for most of the CRC patients4. For patients in higher level of metastatic stage, radiation and chemotherapy often accompany with surgery. Currently, fluorouracil (5-Fu) is often used alone or combined with folinic acid and oxaliplatin as FOLFOX to treat primary colon cancer. For advanced or metastatic CRC, FOLFOX and FOLFIRI (5-Fu, folinic acid and irinotecan) are the most commonly used chemotherapy combinations. Despite the effectiveness of the chemotherapy and radioactive therapy, the high incidence (up to 98%) of side effects, including hair loss, nausea, vomiting, neurotoxicity, increasing the chance of infection and immune system suppression often affect the quality of life5,6. Targeted therapy to vascular endothelial growth factor (e.g. bevacizumab) or epidermal growth factor receptor (e.g. cetuximab) are common adjuvant/alternative treatments for CRC7. Although they are reported to increase survival rates for cancer patients, the costs for these treatments are high8. Hence, the searching of new compounds from natural source with high efficacy, low toxicity and low cost for CRC remains highly desirable.

Traditional Chinese medicines (TCM) have been used for thousands of years for treating various diseases, however, the active components and mechanisms are still often unanswered. In the past, natural compounds have been proven as rich sources of anticancer drugs, such as paclitaxel, camptothecin9,10. Bigelovin, a sesquiterpene lactone isolated from Inula helianthus-aquatica, was identified as a selective retinoid X receptor α agonist11, possessed anti-emetic12 activities, anti-angiogenic activities13, and down-regulated the gene expressions of inflammatory-related cell adhesion molecules and monocyte adhesion14. Previous studies demonstrated that bigelovin could induce apoptosis on a panel of cancer cell lines including leukemia, lung, liver, glioma, kidney, gastric, cervix and breast in vitro15,16. However, the anti-CRC effect and the underlying mechanisms of bigelovin have not been investigated.

Death receptor 5 (DR5) is a TNF-related apoptosis inducing ligand (TRAIL) receptor, which has been identified as a novel target with better selectivity for cancer therapy as shown to induce apoptosis in a diversity of cell types17. Engagement of DR 5 results in the activation of caspase 8, which in turn activates downstream effector caspases in the extrinsic apoptosis pathway. While reactive oxygen species (ROS) are known to be regulator of TRAIL receptor induction18. Furthermore, emerging evidences illustrated that other terpenoids such as eriocalyxin B19, celastrol20, tagalsins21 and zerumbone22 can cause ROS-mediated apoptosis due to their structure of α, β-unsaturated ketone moieties19. Bigelovin also has two α, β-unsaturated ketone moieties (Fig. 1a), thus we hypothesized that bigelovin-induced apoptosis may be mediated by ROS.

Figure 1: Bigelovin inhibited cell viability and proliferation in human colon cancer cells.
figure 1

(a) Chemical structure of bigelovin with two α, β-unsaturated ketone moieties. (b) Bigelovin was selectively toxic to colorectal cancer cells comparing to primary normal colon cells. IC50 values from 48 h incubation in HT-29 and HCT 116 cell lines and primary normal colon cells by MTT assay. (Mean ± SD; ***p < 0.001, vs primary normal colon cells; n = 4). (c) Cell proliferation assay of two cell lines treated with bigelovin for indicated dose and time points (**p < 0.01, ***,###,&&&p < 0.001 vs. medium control at the corresponding time point; n = 3–4). HT-29 (d) and HCT 116 (e) were seeded in 100 mm dish, and after 48 h bigelovin treatment, they were reseeded and maintained for 8 or 11 days to form colonies (**p < 0.01, ***p < 0.001 vs. medium control; n = 4–5).

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The present study aimed to investigate the inhibitory effect of bigelovin on CRC through evaluating its anti-tumor effect in vivo and elucidating the underlying mechanisms of actions in vitro.

Results

Bigelovin inhibited growth and colony formation of human colon cells

Cell viability was assessed by MTT assay on HT-29 and HCT 116 cells. Cells were treated with bigelovin (0.037 to 9 μM) or 5-Fu/cisplatin (0.11 to 27 μM) for 24, 48 and 72 h. As shown in the Supplementary Table S1, colon cancer cell lines were more sensitive to bigelovin treatment rather than 5-Fu or cisplatin. Bigelovin induced cytotoxicity in these two cancer cell lines in time-dependent and dose-dependent manners. To test the selectivity of bigelovin, primary human colon cells were used (mixture of fibroblast and epithelial cells, data not shown). From IC50 values, primary colon cells were less sensitive to bigelovin treatment (8.55 μM for 48 h treatment) comparing to colon cancer cell lines (0.8 and 1.2 μM for 48 h treatment, Fig. 1b and Table S1). To test the effects of bigelovin on cell proliferation, HT-29 and HCT 116 cells were treated with bigelovin at 1.4–5.4 μM (1 to 3 folds of 24 h IC50 values for each cell line) for 24, 48 and 72 h. As shown in Fig. 1c, bigelovin significantly reduced cell proliferation of both cell lines in a time- and dose- dependent manners. Further more, to determine cell lethal- and sub-lethal damage repair after bigelovin treatment, HT-29 and HCT 116 cells were reseeded and maintained for 8 or 11 days to allow colony formation. Cells which were treated by bigelovin showed significantly decreased colony formation ability as compared with vehicle control (Fig. 1d,e). The decreasing of colony formation ability indicated that bigelovin could decrease damage repair ability of colon cancer cell lines. Taken together, our results showed that bigelovin suppressed the growth of colorectal cancer cells.

Bigelovin induced apoptosis through caspases activation

Suppression of cancer growth arises from inducing of apoptosis, inhibition of cell proliferation, or bothSiRNA transfection

For siRNA transfection, HT-29 and HCT 116 cells were plated in 6-well plates/60 mm dish and cultured for 16 h. Thirty pmol siRNAs (s225038 and s16756) /negative control, 9 μL Lipofectamine™ RNAiMAX were mixed with 150 μL Opti-MEM medium separately for 5 min. Lipofectamine was added to siRNAs for 20 min of incubation. Culture medium of HT-29 and HCT 116 cells was changed to Opti-MEM medium and then a mixture of siRNAs were added to cells. After 24 h of incubation, culture medium was changed to fresh, full-culture medium plus bigelovin/ vehicle DMSO at the indicated dose for 24 h.

In vivo xenograft studies

All animal experiments were carried out under institutional guidelines, and experimental procedures were approved by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong (Ref. No. 14/170/MIS). Male BALB/c nude mice (6–8 weeks of age) were supplied by the Laboratory Animal Services Centre of CUHK. Mice were bred and maintained in pathogen-free conditions (sterile water and food) in specifically designed air-controlled rooms with a 12-h light/dark cycle. HCT 116 cells (5 × 106) were suspended in 100 μL PBS and injected subcutaneously into the back of nude mice. After tumor reaching 50 mm3 in volume, mice were randomized into groups of 5 animals and treated with either vehicle, bigelovin (5 mg/kg, 10 mg/kg, and 20 mg/kg) or FOLFOX (15 mg/kg fluorouracil daily; 5 mg/kg folinic acid daily and 5 mg/kg oxaliplatin once a week) as positive control. Bigelovin or vehicle was administered intraperitoneally every two days for 10 times while FOLFOX was administered intraperitoneally daily for 7 days. Tumors were measured twice per week using calipers and tumor volumes were calculated using the formula length × width × depth/2 (mm3), and at the same time, each mouse was weighed. Bigelovin was dissolved in PBS (with 2% DMSO). At the end of experiment, plasma, tumors, livers, hearts, kidneys and lungs were collected. The plasma enzymes, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were used to assess tissue damage; creatine kinase (CK) was used to assess muscle damage. They were evaluated according to manufacturer’s instructions (Stanbio Laboratory, USA).

Immunohistochemistry

Tumor tissue specimens were collected from mice and separated into two halves, one half was fixed in 10% formalin and embedded in paraffin whereas the other half was embedded in Optimal Cutting Temperature compound (OCT) and frozen at −80 °C. Other organs (liver, lung, heart, kidney) were fixed in 10% formalin, embedded in paraffin and sectioned at 5 μm, collected on coated slides for staining. Tumor sections on slides were dewaxed, rehydrated, incubated with Cova Decloater and incubated with primary antibodies (Ki 67, DR5, Factor VIII) overnight at 4 °C as described in other study51. Formalin-fixed paraffin embedded sections were also used in TUNEL assay as per kit’s instruction. Frozen sections of tumors were sectioned at 10 μm using a cryostat (CM 1100, Leica, Germany), and stained with dihydroethidium (DHE, 2.5 μM in Krebs-HEPES buffer) dye for 15 min at 37 °C in the dark50. Fluorescence captured by fluorescence microscope (Olympus IX71, Japan) and intensity of fluorescence was calculated by Image J software (Image J 1.50b).

Statistical analysis

All in vitro experiments were performed at least three times. Quantitative results were analyzed by Student’s t test or one-way ANOVA. Statistical significance was considered when p < 0.05. All statistical analysis was assessed by SPSS 20 software.

Additional Information

How to cite this article: Li, M. et al. Bigelovin triggered apoptosis in colorectal cancer in vitro and in vivo via upregulating death receptor 5 and reactive oxidative species. Sci. Rep. 7, 42176; doi: 10.1038/srep42176 (2017).

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