Ethanol extract of Chrysanthemum zawadskii Herbich induces autophagy and apoptosis in mouse colon cancer cells through the regulation of reactive oxygen species

Background Recent research has suggested that autophagy can provide a better mechanism for inducing cell death than current therapeutic strategies. This study investigated the effects of using an ethanol extract of Chrysanthemum zawadskii Herbich (ECZ) to induce apoptosis and autophagy associated with reliable signal pathways in mouse colon cancer CT-26 cells. Methods Using ECZ on mouse colon cancer CT-26 cells, cell viability, annexin V/propidium iodide staining, acridine orange staining, reactive oxygen species (ROS) and western blotting were assayed. Results ECZ exhibited cytotoxicity in CT-26 cells in a dose-dependent manner. ECZ induced apoptosis was confirmed by caspase-3 activation, poly (ADP-ribose) polymerase cleavage, and increased production of reactive oxygen species (ROS). Furthermore, it was shown that ECZ induced autophagy via the increased conversion of microtubule-associated protein 1 light chain 3II, the degradation of p62, and the formation of acidic vesicular organelles. The inhibition of ROS production by N-Acetyl-L-cysteine resulted in reduced ECZ-induced apoptosis and autophagy. Furthermore, the inhibition of autophagy by 3-methyladenine resulted in enhanced ECZ-induced apoptosis via increased ROS generation. Conclusion These findings confirmed that ECZ induced ROS-mediated autophagy and apoptosis in colon cancer cells. Therefore, ECZ may serve as a novel potential chemotherapeutic candidate for colon cancer.


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][18][19]. Linarin, one of its physiologically active agents, has been reported to exhibit antiinflammatory, antipyretic, hepatoprotective, antibacterial, anticancer, and antioxidant activity [20][21][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.

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.

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% CO 2 .

Cell viability
The cytotoxicity of ELT on CT-26 cells was calculated using CCK-8 assay. The cells were seeded at 1 × 10 4 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 × 10 4 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 × 10 4 cells/well in 6well 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 × 10 4 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).

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.

HPLC analysis of ECZ
The HPLC chromatograms for ECZ and a mix of the three standard components are shown in Fig. 1. The peaks for the three components in ECZ were identified by comparing their retention times, ultraviolet (UV) spectra, and chromatogram patterns with those of the standard mix. The target components were well separated and showed good selectivity, with no interference from other components within 40 min. The retention times of the compounds in the chromatogram of the standard mix were 9.45   Table 2.

ECZ inhibited proliferation and triggered apoptosis in mouse colon cancer CT-26 cells
We examined the impact on cell viability of mouse colon cancer CT-26 cells to ECZ. Exposure significantly reduced the viability of CT-26 cells in a dose-dependent manner (Fig. 2a). To examine the anticancer effects of ECZ in CT-26 cells, we quantitatively verified apoptosis by using Annexin V-FITC/propidium iodide (PI) staining. After treatment with 200 and 300 μg/ml of ECZ for 24 h, flow cytometry analysis showed significantly increased in early and late apoptotic CT-26 cells from 2.02% to 10.30 and 15.05%, respectively ( Fig. 2b and c). Addition, we confirmed that ECZ treatments also induced apoptosis in HT-29 cells (Additional file 1: Fig. S1A). To investigate the mechanism for ECZ-induced apoptosis in CT-26 cells, the expression levels of apoptosis-related proteins were evaluated by western blot analysis. ECZ exposure did not affect the expression of Bax, but reduced the expression of Bcl-2, and Bcl-2/Bax ratio was significantly reduced (Fig. 2d and e). Furthermore, ECZ reduced the pro-form of caspase-3 and increasingly induced the cleavage of capspase-3 and PARP in a dose-dependent manner (Fig. 2d, f and g). These results indicated that ECZ-induced apoptosis was functionally associated with caspase-3 dependent pathways.

ECZ induced autophagy in CT-26 cells
We confirmed the effects of ECZ regarding the induction of autophagy by using AO staining. After treatment with 200 and 300 μg/ml of ECZ for 24 h, there were significantly increases in the accumulation of acidic vesicular organelles (AVOs) from 9.04 to 16.08% and 18.72%, respectively ( Fig. 3a and b). Addition, we confirmed that ECZ treatments also induced autophagy in HT-29 cells (Additional file 1: Figure S1B). Furthermore, there were clear dose-dependent increased in the expression levels of LC3 conversion forms and p62/SQSTM1 (which are Apoptosis induction. c Representive graph of apoptotic cells. d Expression of representative apoptosis-related proteins. e Bax/Bcl-2 ratio. f Relative fold change of cleaved caspase-3. g Relative fold changes of cleaved PARP. The cells were treated with various concentrations (100-300 μg/ml) of ECZ for 24 h. Cell viability was determined using a standard CCK-8 assay. Cell viability is represented as the percent of relative absorbance relative to the controls. Apoptosis induction was evaluated using annexin V/propidium iodide (PI) staining and flow cytometry. Total cell lysates were analyzed by Western blot analysis. Data are presented as the mean ± standard deviation for at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 autophagy-specific markers) in ECZ-treated CT-26 cells (Fig. 3c, d and e). To reconfirm the ECZ-induced autophagy, we evaluated the formations of AVOs and the expression levels of LC3 conversion forms and p62/ SQSTM1 by using the autophagy inhibitor 3-MA. The levels of formation of AVOs in the presence or absence of 3-MA; were 9.97 or 21.13%, respectively (Fig. 3f). Furthermore, the expression of LC3 conversion forms and p62/SQSTM1 significantly reverted when ECZ was combined with 3-MA compared with ECZ alone (Fig. 3g, h  and i). These results indicated that ECZ induced autophagy by increasing autophagosome formations in CT-26 cells.

ECZ-induced apoptosis and autophagy were regulated by ROS production in CT-26 cells
Several recent studies have demonstrated that ROS plays an important role in the induction of apoptosis and autophagy [25,26]. Thus, we used flow cytometry with DCF-DA staining to examine whether there was a relationship between ROS production and ECZ-induced apoptosis and autophagy in CT-26 cells. ROS production was significantly increased in a dose-dependent manner (Fig. 4a); this was reversed by pretreatment with NAC, which is a ROS scavenger (Fig. 4b). We then examined the effects of NAC on the crosstalk of ROS production between ECZ-induced apoptosis and autophagy. Pretreatment with NAC reduced both apoptotic cells and autophagic cells in CT-26 cells (Fig. 4c and g). Addition, we confirmed that pretreatment with NAC reduced both apoptotic cells and autophagic cells in HT-29 cells (Additional file 1: Figure S1C and 1D). Consistent with the flow cytometry results, NAC reversed the increased expression of procaspase-3, PARP, LC3 conversion, and p62/SQSTM1 (Fig. 4d, e, f, h, i and j). These results indicated that ROS played a critical role not only in ECZ-triggered apoptosis via caspase-dependent pathways but also in autophagy via LC3-II expression and AVOs accumulation.

Inhibition of autophagy-enhanced ECZ-induced apoptosis via increased ROS production in CT-26 cells
To establish whether the inhibition of autophagy affected ECZ-induced apoptosis in CT-26 cells, we used flow cytometry with annexin-V-FITC staining after pretreatment with 3-MA to evaluate the induction of apoptosis. There was a greater increased in apoptotic cells among cells treated with a combination of ECZ and 3-MA than in cells treated with ECZ alone in CT-26 cells (Fig. 5a). Addition, we confirmed that pretreatment with 3-MA decreased ECZ-induced autophagy, but increased ECZ-induced apoptotic cells in HT-29 cells (Additional file 2: Figure S2A and S2B). Furthermore, there were greater increases in cleavage forms of caspase-3 and PARP proteins with the combination ECZ and 3-MA than with ECZ alone, thus indicating that the induction of apoptosis was further enhanced when autophagy was blocked with 3-MA (Fig. 5a-d). Thereafter, h Effects of NAC on LC3B and p62/SQSTM1 expression. i Relative fold change of LC3B-II. j Relative fold change of p62/SQSTM1. Cells were treated with 300 μg/ml of ECZ for 24 h prior to 1 h incubation with 1 mM NAC. ROS production, apoptotic cells, and autophagic cells were detected by flow cytometry. Total lysates were subjected to SDS-PAGE for Western blot analysis. Data are presented as the mean ± standard deviation for at least three independent experiments. ***p < 0.001 vs. the control group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. the ECZ treated group to evaluate whether the inhibition of autophagy affected the production of ROS, we analyzed ROS levels in CT-26 cells after treatment with ECZ and 3-MA. ROS production was significantly increased in cells treated with the combination of ECZ and 3-MA compared with those treated with ECZ alone (Fig. 5e), and this increase in ROS production was clearly attenuated by treatment with NAC (Fig. 5e). These results indicated that autophagy played a protective role against the induction of apoptosis in CT-26 cells by ECZ and that increasing ROS production by inhibiting autophagy enhanced the induction of apoptosis, consistent with the results in Fig. 5a.

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][28][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 ROSmediated 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 crosstalk survival and/or death signal pathway in many cancer cells [32][33][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, c Relative fold change of cleaved caspase-3. d Relative fold change of PARP. Cells were pretreated with 5 mM 3-MA prior to 1 h. Apoptotic cells were evaluated by flow cytometry, and the proteins were detected by Western blot analysis. e Effects of 3-MA on ROS production. Cells were treated with 300 μg/ml ECZ for 24 h prior to 1 h incubation with 5 mM 3-MA or/and 5 mM NAC. ROS production was evaluated by flow cytometry. Data are presented as the mean ± standard deviation for at least three independent experiments. ***p < 0.001 vs. the control group, #p < 0.05, ## p < 0.01 vs. the ECZ treated group 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 apoptosisand autophagy-related proteins procaspase-3 and LC3. Furthermore, the inhibition of autophagy enhanced ECZinduced 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.

Conclusion
The results of this study showed that ECZ induced apoptosis in mouse colon cancer CT-26 cells via caspasedependent pathways and that it triggered autophagy via an increase in the formation of autophagosomes. In particular, we showed that ROS production played a role in ECZinduced apoptosis and autophagy. The inhibition of autophagy enhanced ECZ-induced apoptosis resulting from increased ROS production, thus suggesting that autophagy may play a cytoprotective role via apoptosis resistance. Therefore, we believe that ECZ can be used for cancer chemoprevention or as a chemotherapeutic agent; however, further studies are needed to explore the molecular mechanisms and evaluate the anticancer activity of ECZ in vivo.
Additional file 1: Figure S1. Ethanol extracts of Chrysanthemum zawadskii Herbich (ECZ) induced reactive oxygen species (ROS)-mediated apoptosis and autophagy in human colon cancer HT-29 cells. (A) Apoptosis induction. (B) N-Acetyl-L-cysteine (NAC) attenuated apoptosis induced by ECZ. (C) Detection of acidic vesicular organelles (AVOs). (D) Effects of NAC on autophagy. Cells were treated with various concentrations (100-300 μg/ml) of ECZ for 24 h and stained with annexin V/propidium iodide and 1 μM acridine orange (AO) at 37°C in the dark for 20 min and then analyzed by flow cytometry. Cells were pretreated with 1 mM NAC prior to 1 h and annexin V/propidium iodide and AO-stained cells were evaluated by flow cytometry. Data are presented as the mean ± standard deviation for at least three independent experiments. ***p < 0.001 vs. the control group; ### p < 0.001 vs. the ECZ treated group.
Additional file 2: Figure S2. Inhibition of autophagy enhanced apoptosis induced by ethanol extracts of Chrysanthemum zawadskii Herbich (ECZ) in human colon cancer HT-29 cells. (A) Pretreatment with 3-methyladenine (3-MA) reversed the ECZ-induced increase in autophagy. (B) Treatment with 3-MA enhanced ECZ-induced apoptosis. Cells were pretreated with 5 mM 3-MA prior to 1 h. Apoptotic and autophagic cells were evaluated by flow cytometry. Data are presented as the mean ± standard deviation for at least three independent experiments. ***p < 0.001 vs. the control group; ### p < 0.001 vs. the ECZ treated group.