Background
Colon cancer is the third most common cancer in men and the second most common cancer in women worldwide [
1]. Early diagnosis with colonoscopy and the removal of precancerous lesions has resulted in a recent decline in the incidence of colon cancer in the United States; however, its incidence is increasing in Asia and Eastern Europe [
2]. In Korea, despite the development of various treatment methods, colon cancer has now become the fourth leading cause of overall mortality, and its incidence is still increasing in both men and women.
Autophagy affects a wide range of processes, including homeostasis, developmental process, immune function, aging, and various cellular function [
3]. Autophagy is a catabolic process which involves the degradation of the large protein complexes and dysfunctional organelles. These components are sequestered and transported to lysosomes for degradation as a cytoprotective mechanism [
4]. Also, when cellular stress is extensive, autophagy acts degradation and recycling of process by the accumulation of acidic vesicular organelles (AVOs) through an alternative cell-death pathway as cytotoxic function [
5,
6]. Therefore, the dysfunction of autophagy can affect the incidence and treatment of diseases such as cancer [
7]. Recent reports have proposed autophagy as a novel strategy for cancer therapy [
8,
9]. However, the action of autophagy in cancer is highly complex and affected by genetic differences [
10,
11]. When apoptosis is excessive or deficient in the spontaneous destruction pattern of cells, this can contribute to the growth and recurrence of ischemia, neurodegenerative disease, autoimmune disease, viral infection, and tumors [
2,
12]. Recently, the complex interactions between autophagy and apoptosis have received attention, with studies showing that apoptosis can sometimes act as an inhibitor or inducer of autophagy, thus resulting in changed resistance to many anticancer drugs or to a clinical application [
13,
14]. Further studies are needed on the interaction between autophagy and apoptosis under various conditions [
15].
Chrysanthemum zawadskii Herbich (CZ) is a perennial herb from the family Asteraceae, which is grown in countries including China, Russia, Mongolia, and Japan [
16]. Extracts of CZ have been used in traditional medicine and as a tea in Korea and China. CZ has been shown to have effective therapeutic and medicinal properties, including antimicrobial, antioxidant, and antimycotic activity [
17‐
19]. Linarin, one of its physiologically active agents, has been reported to exhibit antiinflammatory, antipyretic, hepatoprotective, antibacterial, anticancer, and antioxidant activity [
20‐
22]. However, although the beneficial and pharmacological effects of CZ are established, the molecular mechanisms underlying its anticancer effects in colon cancer remain unknown. Therefore, the aim of the present study was to investigate the chemotherapeutic effects of an ethanol extract of CZ (ECZ) and to elucidate the interrelated mechanisms involving apoptosis and autophagy in mouse colon cancer CT-26 cells. The results showed that the production of reactive oxygen species (ROS) by ECZ may offer a therapeutic strategy to improve the treatment of colon cancer through the relationship between autophagy and apoptosis.
Materials and methods
Reagents
Chlorogenic acid and 3,5-di-caffeoylquinic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Luteolin was obtained from Faces Biochemical Co., Ltd. (Wuhan, China). HPLC-grade acetonitrile was purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA), and LC/MS-grade formic acid was purchased from Sigma-Aldrich. The ultrapure water used in the HPLC analysis was prepared using a Puris-Evo UP Water system with Evo-UP Dio VFT and Evo-ROP Dico20 (Mirae ST Co., Ltd., Anyang, Gyeonggi-do, Korea). Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin and fetal bovine serum (FBS) were obtained from Hyclone (Logan, UT, USA). Acridine orange (AO), dichlorodihydrofluorescein diacetate (DCF-DA), 3-methyladenine (3-MA), and N-Acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich. Cell counting kit (CCK)-8 assays and FITC Annexin V-Apoptosis Detection Kit were obtained from Dojindo Molecular Technologies, Inc. (Rockville, MD, USA) and BD Biosciences (San Jose, CA, USA), respectively. Primary antibodies against Bax, Bcl-2, caspase 3, poly (ADP-ribose) polymerase (PARP), microtubule-associated protein 1 light chain-3B (LC3B), p62/SQSTM1, and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA), and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. The antimouse IgG and goat antirabbit secondary antibodies were purchased from Enzo Life Science (Farmingdale, NY, USA).
Preparation of standard solutions and ECZ
Standard stock solutions in methanol (1 mg/ml) of chlorogenic acid, 3,5-di-caffeoylquinic acid, and luteolin were prepared. The standards were mixed from the stock solutions, and then freshly prepared by serial dilution in methanol to plot the calibration curves. The final concentrations of the calibration samples were in the range 25–400 μg/ml for chlorogenic acid and 3,5-di-caffeoylquinic acid, and 1.25–20.00 μg/ml for luteolin. CZ was purchased from Yeongcheon Oriental Herbal Market (Yeongcheon, Korea) and was authenticated by Professor Ki Hwan Bae, a medical botanist at the College of Pharmacy, Chungnam National University, Republic of Korea. A voucher (No. 203) was deposited at the Korean Medicine Application Center Korea Institute of Oriental Medicine in Daegu, Republic of Korea. Dried CZ (30 g) was ground to a fine powder, added to 300 ml of 70% ethanol, and then extracted by shaking it in an incubator at 100 rpm for 24 h at 40 °C. The extract was then filtered through a 150 μm testing sieve (Retsch, Haan, Germany), evaporated, concentrated through lyophilization, and then stored at − 20 °C (yield 13.83%). For the experiments, ECZ powder (10 mg) was dissolved in 1 ml of deionized distilled water (v/v) and filtered through a 0.22 μm disk filter.
The three components in ECZ were identified and quantified by HPLC via a previously reported method [
23,
24]. In this study, the HPLC analysis was performed using a Dionex UltiMate 3000 system (Dionex Corp., Sunnyvale, CA, USA) equipped with a binary pump, an auto-sampler, a column oven and a diode array UV/Vis detector (DAD). System control and data analysis were performed using Dionex Chromelon software. The three components were eluted in a gradient system based on 0.1% formic acid in deionized water (solvent A) and acetonitrile (solvent B). To improve the chromatographic separation capacity, the gradient elution system was programmed as follows: 5–12% B, 0–5 min; 12–15% B, 5–7 min; 15% B, 7–12 min; 15–20% B, 12–14 min; 20% B, 14–20 min; 20–60% B, 20–35 min; 60–90% B, 35–40 min; 90% B, 40–45 min; 90–95% B, 45–46 min; 5% B, 46–60 min at a flow rate of 1.0 ml/min. The components were separated on a Xbridge C18 column (250 × 4.60 mm, 5 μm; Waters, Milford, MA, USA), and the column oven temperature was kept at 40 °C. The injection volume was 5 μl. The detection wavelengths for the three components were set at 220, 254, 320 and 365 nm.
Cell culture
The mouse colon cancer CT-26 and human colon cancer HT-29 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured using DMEM containing 10% FBS, 100 units/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere with 5% CO2.
Cell viability
The cytotoxicity of ELT on CT-26 cells was calculated using CCK-8 assay. The cells were seeded at 1 × 104 cells/well in a 96-well plate. After incubation for 24 h, the cells in each well were treated with ELT at specific concentrations for 24 h. A CCK-8 assay was then performed in accordance with the manufacturer’s instructions. Absorbance was determined at 450 nm on a VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cell viability was calculated relative to untreated controls as follows: viability (% control) = 100 × absorbance of treated sample/absorbance of control.
Annexin V/propidium iodide (PI) assay
To verify the induction of apoptosis, CT-26 and HT-29 cells were cultured at a density of 1 × 104 cells and treated with ECZ for 24 h. The cells were then collected and double stained with Annexin-V-FITC and PI (BD Biosciences), following the manufacturer’s instructions. Apoptotic cells were determined by flow cytometry (Becton Dickinson Co.) and the percentages of apoptotic cells were calculated using Cell Quest software (Becton Dickinson Co.).
Detection of acidic vesicular organelles
To quantify the number of AVOs, CT-26 and HT-29 cells were cultured at a density of 1 × 104 cells/well in 6-well plates. After treatment with ECZ for 24 h, the cells were stained with AO (1 μg/ml) at 37 °C for 20 min in the dark. The cells were then washed with PBS, analyzed by a flow cytometry (Becton Dickinson Co.), and quantified using Cell Quest software (Becton Dickinson Co.).
Measurement of intracellular ROS generation
To determine intracellular ROS production, CT-26 cells were cultured in 6-well plates at a density of 1 × 104 cells/well. The cells were treated with ECZ and incubated with DCFH-DA (10 μM) at 37 °C for 30 min and then washed twice with PBS. For each experiment, the cells were analyzed by flow cytometry (Becton Dickinson Co.), and quantified using Cell Quest software (Becton Dickinson Co).
Western blotting
Cell extracts were prepared by incubating in lysis buffer [150 mM NaCl, 10 mM Tris (pH 7.4), 5 mM EDTA (pH 8.0), 1% Triton X-100, 1 mM PMSF, 20 μg/ml aprotinin, 50 μg/ml leupeptin, 1 mM benzidine, and 1 mg/ml pepstatin]. For separation using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, 50 μg of proteins were loaded onto 12–15% gel and transferred to a polyvinylidene fluoride membrane. After blocking with TBS-T buffer [20 mM Tris (pH 7.4), 150 mM NaCl, and 0.1% Tween 20] containing 5% skim milk, the membranes were incubated with primary and secondary antibodies, separately. The membranes were then washed with TBS-T buffer and visualized with ECL Western blotting detection reagents. The density of each band was determined with a fluorescence scanner (LAS 3000, Fuji Film) and analyzed with Multi Gauge V3.0 software.
Statistical analysis
Experiments were repeated to obtain three sets of consistent results. Unless otherwise stated, data are expressed as the mean ± standard deviation of the mean. ANOVA was used to compare experimental and control values. Comparisons between multiple groups were performed using Tukey’s multiple comparison tests, with the results considered statistically significant at *** p < 0.001.
Discussion
CZ has been reported to have diverse effects on several diseases, including pneumonia, bronchitis, coughs, colds, pharyngitis, bladder disease, hypertension, liver disease, and gastrointestinal disorders [
27‐
29]. However, the molecular mechanisms underlying ECZ’s anticancer effects are not yet understood. The aim of this study was to investigate the mechanism underlying the induction by ECZ of apoptosis and autophagy in mouse colon cancer CT-26 cells. The results showed that ECZ significantly inhibited cell viability in a dose-dependent manner in CT-26 cells. Furthermore, ECZ clearly increased both apoptosis and autophagy via caspase-dependent pathways and AVOs formation in CT-26 cells, respectively. Addition, ECZ induced ROS-mediated apoptosis and autophagy human colon cancer HT-29 cells. Recent findings have suggested that autophagy as a cytoprotective mechanism is closely associated with resistance to apoptosis [
8,
30]. In the present study, the inhibition of autophagy by 3-MA enhanced ECZ-induced apoptosis in CT-26 and HT-29 cells. These results showed that autophagy plays a protective role against cytotoxic effects in colon cancer cells via the inhibition of apoptosis.
Many reports have shown that apoptosis and autophagy induction share a common mechanism associated with ROS production [
12,
31]. Commonly, ROS play important roles in apoptosis and autophagy signaling and ROS production selectively induces cancer cell death [
32]. An autophagy has dual roles to regulate the cell fate in cancer, where autophagy and apoptosis share the cross-talk survival and/or death signal pathway in many cancer cells [
32‐
34]. Other study demonstrated that cross link between autophagy and apoptosis are associated with inhibit cancer cell death by autophagy inhibitors and antioxidants using potentiate or restore the cytotoxicity effect of the drug [
35,
36]. Consistent with these previous reports, the present study showed that ECZ-induced apoptosis and autophagy in CT-26 cells were significantly reversed by pretreatment with the ROS scavenger NAC. Treatment with NAC also restored the expression of the apoptosis- and autophagy-related proteins procaspase-3 and LC3. Furthermore, the inhibition of autophagy enhanced ECZ-induced apoptosis in CT-26 cells, and this was further enhanced by ROS production following treatment with 3-MA. These results demonstrated that intracellular ROS production plays a critical role during ECZ-induced apoptosis and autophagy and that increasing ROS generation by the inhibition of autophagy results in enhanced apoptosis.
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