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6-shogaol induces autophagic cell death then triggered apoptosis in colorectal adenocarcinoma HT-29 cells
Biomedicine & Pharmacotherapy 93 (2017) 208–217
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Original article
6-shogaol induces autophagic cell death then triggered apoptosis in colorectal adenocarcinoma HT-29 cells Ting-Yi Li, Been-Huang Chiang* Institute of Food Science and Technology, National Taiwan University, Taipei 10617, Taiwan
A R T I C L E I N F O
Article history: Received 31 March 2017 Received in revised form 23 May 2017 Accepted 12 June 2017 Keywords: Colorectal cancer HT 29 6-shogaol Autophagy Apoptosis
A B S T R A C T
6-shogaol is a phytochemical of dietary ginger, we found that 6-shogaol could induced both autophagic and apoptotic death in human colon adenocarcinoma (HT-29) cells. Results of this study showed that 6shogal induced cell cycle arrest, autophagy, and apoptosis in HT-29 cells in a time sequence. After 6 h, 6shogal induced apparent G2/M arrest, then the HT-29 cells formed numerous autophagosomes in each phase of the cell cycle. After 18 h, increases in acidic vesicles and LAMP-1 (Lysosome-associated membrane proteins 1) showed that 6-shogaol had caused autophagic cell death. After 24 h, cell shrinkage and Caspase-3/7 activities rising, suggesting that apoptotic cell death had increased. And after 48 h, the result of TUNEL assay indicated the highest occurrence of apoptosis upon 6-shogaol treatment. It appeared that apoptosis is triggered by autophagy in 6-shogaol treated HT-29 cells, the damage of autophagic cell death initiated apoptosis program. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Colorectal cancer (CRC) is the third most common cancer in the world. Its death toll in the last 50 y (1967–2016) has increased 10fold, perhaps due to changing diets. Some carcinogens from dietary factors, e.g., polycyclic aromatic hydrocarbons (PAHs), may increase the risk of CRC. However, several phytochemicals that are readily available in specific foods have been proven to reduce the risk of CRC [1]. Ginger (Zingiber officinale Roscoe), the most widespread seasoning used in Asia, is an important ingredient in both food preparation and folk medicine. The bioactive phytochemicals in ginger are polyphenols, including gingerols, paradols, gingerdiols, and shogaols, which have been reported to be potent chemopreventive agents [2]. For example, 6-gingerol has been thoroughly investigated for its role as an anticarcinogenic agent [3]. However,
Abbreviations: CRC, Colorectal cancer; PAHs, polycyclic aromatic hydrocarbons; PCD, program cell death; ER, endoplasmic reticulum; MOMP, mitochondrial outer membrane permeabilization; APAF1, apoptotic protease activating factor 1; DMSO, dimethyl sulfoxide; PI, propidium iodide; 3-MA, 3-methyladenine; LC3, microtubule associated protein 1 light chain 3; PBS, phosphate buffered saline; AO, acridine orange; AVOs, acidic vesicular organelles; LAMP-1, Lysosome-associated membrane proteins 1; DAPI, 4’6-diamidino-2-phenylindole. * Corresponding author at: Institute of Food Science and Technology, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan. E-mail address: [email protected] (B.-H. Chiang). http://dx.doi.org/10.1016/j.biopha.2017.06.038 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
the dehydrated form of 6-gingerol, 6-shogaol, was reported to have anti-inflammatory effects surpassing those of 6-gingerol [4,5]. Furthermore, 6-shogaol has also been reported to possess antitumor effects via multiple mechanisms. There are three types of programmed cell death (PCD): apoptosis, autophagy, and necroptosis. Apoptosis is a silent cell death elicited through extrinsic and intrinsic pathways. The extrinsic apoptotic pathway is initiated by ligation of death receptors. Then, caspase 8 activation can directly cleave and activate caspase 3 and caspase 7. In contrast, the intrinsic apoptotic pathway, typically stimulated by DNA damage or endoplasmic reticulum (ER) stress, can activate the BCL-2 family and mitochondrial outer membrane permeabilization (MOMP). Then, cytochrome c is released from mitochondria and binds apoptotic protease-activating factor 1 (APAF1), which recruits and also activates executioner caspases 3 and 7 [6]. Therefore, both intrinsic and extrinsic pathways ultimately activate caspase 3 and 7, leading to apoptosome body formation and DNA fragmentation. Autophagy, another type of PCD, occurs due to insufficient nutrition and other stress. A double-membrane autophagic vacuole which sequesters a portion of the cytoplasm (called an autophagosome) is formed as a maturation step of autophagy. In autophagosome formation, two ubiquitin-like conjugation systems, Atg12 and Atg8 (also called LC3), is important for the stability of double-membranes. The Atg12–Atg5 conjugate facilitates lipidation of Atg8. The Atg8–phosphatidylethanolamine will support membrane expansion and increase autophagosomes
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[7,8]. An autophagosome will fuse with a lysosome to form an autolysosome and then degrade the cytoplasmic material. Excessive self-digestion will threaten essential organelles and cause protein degradation, leading to autophagic cell death [9]. During necrotic cell death, several intracellular organelles dilate and the plasma membrane breaks down, causing a release of cell lysate and an inflammatory response [10]. Autophagy can be considered a protection mechanism through inhibition of cell necrosis [11]. In colon cancer development, autophagy negatively regulates Wnt signaling by targeting the dishevelled protein to inhibit proliferation [12]. Thus, utilizing autophagy to cause cell death in tumor cells is a potential therapeutic strategy [13]. 6shogaol has been reported that could induce microtubule damage, inhibiting breast cancer cell invasion, and apoptosis in human mahlavu, colorectal, and hepatocellular carcinoma cells [14–18]. Furthermore, 6-shogaol also initiates autophagy by inhibiting AKT/ mTOR in human non-small cell lung cancer A549 cells [19]. Recently, some reports have indicated that 6-shogaol might induce autophagic cell death in pancreatic, breast, and hepatoma cancer cell lines [20–22]. However, the evidence of 6-shogaol inducing autophagic cell “death” is insufficiently strong due to autolysosome formation and lysosomal digestion. Therefore, the objectives of this study were to elucidate the role of 6-shogaol in inducing both autophagic and apoptosis cell death in human colorectal cancer HT-29 cells in a time-dependent manner and to understand the relationship between them. 2. Materials and methods 2.1. Chemicals and reagents The 6-shogaol was obtained from Chromadex (Irvine, CA, USA). Rapamycin, digitonin, ethanol and 3-methyladenine (3-MA) were purchased from Merck (Darmstadt, Germany). Trypsin-EDTA, dimethyl sulfoxide (DMSO), propidium iodide (PI), ribonuclease A (RNase A), acridine orange (AO), Triton X-100, and Cell Counting Kit 8 were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2.2. Cell culture Human colorectal cancer cell line HT-29 (American Type Culture Collection [ATCC] HTB-38) was grown in RPMI-1640 medium (Gibco BRL, NY, USA) supplemented with 10% heatinactivated fetal bovine serum (FBS, Gibco BRL). It was then kept at 37 C in a humidified 5% CO2 incubator. When cell growth reached 80%, 3 ml trypsin was used for 3 min for cell harvesting. 2.3. Cell viability The HT-29 cells were seeded (density, 2 105 cells/ml) into 96well plates (100 ml per well). After 24 h (when cells were attached), an aliquot of 100 ml FBS-free medium was used to replace the original medium and incubated for another 24 h to stop cell growth [24]. Thereafter, 100 ml of 6-shogaol of various concentrations (20, 40, 60, 80, 100 mM dissolved in culture medium) was used to treat the cells for 6, 12, 18, 24, 48 h. A Cell Counting Kit 8 (CCK8; 10 ml) was added to each well, the plate was incubated for 1.5 h at 37 C, and absorbance was measured at 450 nm (ELISA reader) to detect an orange formazan product (proportional to the number of living cells). 2.4. Cell cycle assay Cells treated with 6-shogaol were analyzed by flow cytometric measurement of cellular DNA content. Briefly, 1 ml of HT-29 cells
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were seeded (2 105 per well) in 6-well plates and allowed to adhere and grow for 24 h, followed by 6-shogaol treatment for various durations. Culture medium containing detached cells was collected and the attached cells were trypsinized. Attached and floating cells were pooled, pelleted, and washed with PBS. They were then re-suspended in 70% ethanol overnight. Hypotonic buffer containing 20 mg/ml propidium iodide, 0.01% Triton X-100, and 10 mg/ml RNase A was added and the mixture was incubated for 30 min in the dark. Then cells were analyzed on a Beckman Coulter flow cytometer (FC500, Fullerton, CA, USA). Data analysis was performed using Flow Jo software (Treestar, Inc., San Carlos, CA, USA). 2.5. Immunocytochemistry for LC3 punctate and lysosome membrane The HT-29 cells (4 105 cells/ml, 0.5 ml) were cultured on fourwell chamber slides (Lab-Tek II, Nunc, Rochester, NY, USA) for 24 h, treated with 6-shogaol, and incubated for various durations. Cells were fixed on slides by immersing them in 4% formaldehyde for 30 min and then washed three times with PBS. Bovine serum albumin (BSA, 5%, with 0.1% Triton X-100. Sigma) was added and incubated for 60 min. For autophagosome detection, primary antibody LC3-B (1:200, Genetex, Taiwan, diluted with 1% BSA and 0.1% Triton X-100 PBS solution) was added to cells and incubated for 2 h. Secondary antibody Dylight-488 anti-rabbit IgG (1:100 dilution, Thermo, Waltham, MA, USA) was then added and incubated for 1 h. For autolysosome detection, after LC3 staining, PE Mouse anti-human CD107a (BD, San Jose, CA, USA) diluted to 200X was used to bind LAMP-1 on lysosome membrane. Finally, nuclei were labeled with 300 nM of 40 , 6-diamidino-2-phenylindole (DAPI, Lonza, Walkersville, MD, USA) for 15 min. Then the chamber was removed and mounting medium was added onto the slide with a cover slip on the top. Confocal microscopy (Leica TCS SP5 II, Solms, Germany) was used for assessment. 2.6. Monitoring autophagosomes in each cell cycle The HT-29 cells (2 105 cells/ml, 2 ml) lwere seeded on 6-well plate and treated with 6-shogaol for varying durations. Then the media were collected and cells were harvested by trypsinization, suspended in a 1.8 ml Eppendorf tube, and immediately centrifuged. After being washed with PBS, the cell pellets were treated with digitonin (150 mg/ml) for 7 min. Cells were then centrifuged (1000 g for 5 min) to remove LC3-I and washed twice, primary antibody LC3 (1:200, PM036, MBL, Tokyo, Japan) and FITC IgG antirabbit (1:200, GTX27079, Genetex, Irvine, CA, USA) were added, and then the mixture was incubated in dark for 30 min [25]. After incubation, PBS buffer containing propidium iodide (5 mg/ml) and RNaseA (10 mg/ml) was used to stain cells (which were vortexed before analysis). 2.7. Acidic vesicular organelles assay The HT-29 cells (2 105 cells/ml, 2 ml) were cultured on a 6well plate and treated with 6-shogaol for varying durations. Then the treated cells were stained with Acridine Orange (1.5 mg/ml) for 15 min at 37 C. After the cells were harvested, red fluorescence on FL3 was analyzed by flow cytometry using Win Midi 2.9 software (Joseph Trotter, Scripps Institute, La Jolla, CA, USA). 2.8. Lysosome expression assay The HT-29 cells (2 105 cells/ml, 2 ml) were seeded on a 6-well plate and treated with 6-shogaol for various durations. PharmingenTM Transcription Factor Buffer Set (BD, San Jose, CA, USA) was used for intracellular staining. After washing, fixing, and
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Fig. 1. Effects of 6-shogaol on cell cytotoxicity in HT-29 cells. (A) Cell viability of HT-29 cells following treatment with 6-shogaol for 6, 12, 18, 24, or 48 h. (B) Effects of 6-shogaol on morphology of HT-29 cells after 12 or 24 h (light microscopy, 400X). Data are expressed as mean SD (n = 3).
permeabilization, PE Mouse anti-human CD107a (LAMP-1, BD, San Jose, CA, USA) was added and the cells were incubated for 40 min in the dark. After the cells were harvested, red fluorescence of FL3 was analyzed by flow cytometry.
Diego, CA, USA; * for P < 0.05, ** for P < 0.01 and *** for P < 0.001). All results are reported as mean SD
2.9. Apoptotic cells analysis
3.1. Effect of 6-shogaol on cell proliferation inhibition
An annexin V-FITC/PI double staining apoptosis assay (BD, San Jose, CA, USA) was used to quantify 6-shogaol induced apoptosis in HT-29 cells. Cells were seeded at a concentration of 2 105 cells/ well in a 6-well plate, treated with 6-shogaol for 18, 24, or 48 h, and then washed twice with PBS buffer. Cells were re-suspended in 1X binding buffer (1 106 cells/ml) and transferred to 500 ml of 1X binding buffer in a 1.8 ml Eppendorf tube. Then 5 ml of annexin VFITC and 5 ml PI were added into the tubes and allowed to react for 15 min in the dark. Finally, after addition of 500 ml of 1X binding buffer, samples were analyzed by flow cytometry (within 1 h).
To evaluate the anticancer effects of 6-shogaol, we investigated the proliferation inhibition of 6-shogaol on the human colorectal cancer HT-29 cell line. Cell viabilities of HT-29 cells treated with 6shogaol (various doses and times) are shown in Fig. 1A. The cell death rate was dose- and time-dependent after 18 h (the 24-h IC50 of 6-shogaol-treated HT-29 cells was 40 mM). However, cell death phenomena after treatment for 6 and 12 h were unexpected, particularly at high doses. This motivated us to study the time
3. Results and discussion
2.10. Caspase-3, 7 activity detection Active caspase was detected with a CaspaTagTM Caspase-3/7 In Situ assay kit (Chemicon, Temecula, CA, USA), based on fluorochrome inhibitors of caspases (FLICA). The 6-shogaol-treated cell suspension (290 ml, 106 cells) and 10 ml of FLICA were mixed in 1.8 ml Eppendorf tubes. The tubes were gently flicked and then incubated for 1 h in a cell culture incubator, after which the contents were washed twice by centrifugation (500 x g for 5 min). The cell pellet was re-suspended and immediately analyzed by flow cytometry.
2.11. Assessment of DNA damage The TUNEL method (BD, San Jose, CA, USA) was used for analyzing DNA fragmentation. Briefly, 6-shogaol-treated HT-29 cells were suspended in 1% (w/v) paraformaldehyde and fixed in 70% (v/v) ethanol, and DNA Labeling Solution (TdT Enzyme with FITC dUTP) was added. After 60 min, the cell suspension was washed twice with rinsing buffer, centrifuged (300 x g for 5 min), and then analyzed by flow cytometry. Both positive and negative cells were also used as controls.
2.12. Statistical analyses Differences between the 6-shogaol-treated and control (0 mM) groups were analyzed by one way analysis of variance (ANOVA) and Duncan's multiple comparison tests using GraphPad (San
Fig. 2. Autophagy inhibitor 3-MA promoted apoptosis in 6-shogaol-treated HT-29 cells. (A) Annexin-V results in HT-29 cells at 48 h of exposure to 60 mM 6-shogaol and co-treatment with 5 mM 3-MA.
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effect of 6-shogaol treatment on cell death. With light microscopy, it was observed that cell shrinkage, the defining morphological characteristic of apoptosis, was not obvious at 12 h, but it became more apparent after 24 h (Fig. 1B). Therefore, we suspected that there was another cell death mechanism affecting 6-shogaoltreated HT-29 cells at 12 h. 3.2. An autophagy inhibitor increased apoptosis in 6-shogaol-treated HT-29 cells In the present study, cell death was dose-dependent in 6shogaol-treated HT-29 cells when the treatment exceeded 18 h (Fig. 1A). To check how the cell death mechanism autophagy affects apoptosis, 3-methyladenine (3-MA; an autophagic sequestration blocker via class III PI3 K inhibition) was used for annexin-V analysis of 6-shogaol-treated HT-29 cells. As shown by the data in Fig. 2, 60 mM of 6-shogaol induced only approximately 15.3% apoptosis in 48 h. Conversely, co-treatment of 6-shogaol with 3methyladenine for 48 h increased apoptotic cells to 80.9%. Therefore, we suspected that autophagy could happen in 6shogaol-treated HT-29 cells. 3.3. 6-shogaol induced G2/M phase arrest in HT-29 cells starting from 6h To investigate the potential anticancer properties and apoptotic cells of 6-shogaol-treated HT-29 cells, the cell cycle distributions following 6-shogaol treatment for 24 and 48 h are shown (Fig. 3A). The rate of apoptotic cells (Sub-G1) increased in a dose-dependent
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manner. However, the counts of apoptotic cells (Fig. 3A) comprised less than half of total cell death (Fig. 1A). Furthermore, from 6 to 48 h, we could see that 6-shogaol significantly induced cell cycle arrest at the G2/M phase (Fig. 3B). Treatment for 12 h induced the highest percentage of G2/M arrest; the G2/M phase comprised 43%, as compared to the 17% of control cells (cells not treated with 6shogaol) (Fig. 3C). Cell cycle arrest is a very important phenomenon in cancer pathology. It has been reported that treatment with 6-shogaol induces G2/M arrest and aberrant mitotic cell death at proapoptotic concentrations by causing microtubule damage through a specific reaction with sulfhydryl groups in tubulin [5,16]. Moreover, 6-shogaol inhibited cell proliferation by suppressing phosphorylation of mitogen-activated protein kinase (MAPK) and PI3 K/Akt signaling [17,23]. As a G2/M phase initiator, the suppression of Akt in turn inhibits binding of CDK1 and cyclin B, causing cells to arrest at the G2/M phase. Moreover, under autophagy progression, the suppression of Akt together with its downstream target mTOR contributes to initiation of autophagy [26]. In human non-small cell lung cancer A549 cells, 6-shogaol induces autophagy by inhibiting the Akt/mTOR pathway [19]. Based on the current results and the literature, we inferred that 6shogaol induced autophagy in colorectal cancer cells. 3.4. 6-shogaol induced autophagosome formation in HT-29 cells in 6 h Autophagy in tumorigenesis is gaining attention in the field of cancer therapy [27]. There is increasing evidence that the death of
Fig. 3. 6-shogaol induced cell cycle arrest in HT-29 cells. (A) Distribution of cell cycles in 6-shogaol-treated cell at 24 and 48 h. (B) G2/M phase exhibition of HT-29 cells after various intervals of treatment with 6-shogaol. (C) Cells were treated with 6-shogaol for 12 h and cell cycle distribution assessed. Data are expressed as mean SD (n = 3).*, P < 0.05, **, P < 0.01, ***, P < 0.001, control versus 6-shogaol-treated cells.
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cancer cells (unable to undergo apoptosis) is caused by autophagy, in a caspase-independent fashion, corresponding to type II programmed cell death. Since deficiencies of autophagy may enhance tumor progression, many autophagy-related genes (Atg) have been identified as tumor suppressor genes. Beclin1, UVRAG,
and Bif-1s, for example, promote carcinogenesis in knockout and xenograft mouse models [28–30]. In addition, PTEN, a tumor suppressor, provides signals to activate autophagy via Akt inhibition. Under this signaling transduction, many oncogenes, such as Bcl-2 and mTOR, are decreased, resulting in inhibition of
Fig. 4. The formation of autophagosomes in 6-shogaol treated HT-29 cells. LC3 puncta (yellow color) were labeled for autophagosome and stained with DAPI (blue color). (A) Based on confocal microscopy, HT-29 cells treated with 6-shogaol for 9 or 12 h had autophagosomes (white arrow). Rapamysin (positive control) had LC3 puncta, similar to the treatment group. (B) Individual HT-29 cells exposed to 60 mM 6-shogaol for 12 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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cancer proliferation [27]. For clinical induction of autophagy, direct mTOR inhibition with rapamycin has been widely used with numerous types of tumors [31]. In the initiation stage of autophagy, decline of mTOR causes double membrane formation. The membrane encapsulates some vital organelles in cancer cells to form autophagosomes. Thus, we investigated whether 6-shogaol could induce autophagy by
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staining the marker of autophagosome, LC3, and examining the formation of LC3 puncta using confocal microscopy. Cytoplasmic LC3 puncta (white arrow) were observed after treatment for 6 h and became more obvious after 12 h, indicating conversion of LC3-I into LC3-II (yellow dot) due to 6-shogaol and rapamycin (positive control) treatments, as compared to controls, which did not show any sign of autophagosome formation (Fig. 4A). There were LC3
Fig. 5. Effects of 6-shogaol treated HT-29 cells on LC3-II accumulation in each cell cycle phase. (A) Percentages of LC3-II-positive cells are shown under various concentrations of 6-shogaol for 12 h. Double-stained LC3 with PI demonstrated the distribution of LC3-II-positive cells in each cell cycle. (B) Percentages of performance LC3-II-positive cells in each phase of the cell cycle for control/treatment are shown. Data are expressed as mean SD (n = 3).
Fig. 6. Acidic vesicular organelles performance in 6-shogaol treated HT-29 cells. (A) Overlay of the control/treatment for acridine orange staining by flow cytometry. (B) Fold increase of fluorescence intensity as compared to control when cells were treated with various concentrations of 6-shogaol. Data are expressed as mean SD (n = 3). *, P < 0.05, **, P < 0.01, ***, P < 0.001, control versus 6-shogaol-treated cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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puncta in individual cells at 12 h (Fig. 4B). DAPI was used for nuclear staining (blue), there was no chromatin condensation during autophagosome formation (a, b, c) for either DNA content N (a, b) or 2N(c). Fluorescence intensity increased from 3 to 12 h, but then became obscure at 18 h (Fig. 4A). The decrease in LC3 puncta after 18 h treatment was attributed to fusion of the autophagosome with lysosomes (autolysosome formation). When this occurs, Atg4 recycles LC3-I and phosphatidylethanolamine from LC3-II on the cytosolic face of the autolysosome membrane [32]. Thus, the LC3 puncta would appear blurred. 3.5. 6-shogaol induced G2/M arrest and formed LC3-II puncta in every phase of the cell cycle in HT-29 cells Based on confocal images, we suspected that autophagy might occur in every phase of the cell cycle. Therefore, the autophagosome contents in every phase of the cell cycle of 6-shogaol-treated HT-29 cells were quantitated by flow cytometry [25]. In this assay, digitonin was used to increase cell membrane permeability to remove LC3-I in the cytoplasm, thereby allowing measurement of the amount of LC3-II. The percentages of LC3-II-positive cells and the distribution of the cell cycle in 6-shogaol-treated HT-29 cells are shown in Fig. 5A. Treatment with 6-shogaol significantly induced LC3-II-positive cells at 12 h to increase, in a dosedependent manner, from 1.62% (control) to 66.25% (60 mM). Based on DNA content, each cell cycle phase was gated (as shown in the dot plot of the control). The amounts of LC3-II-positive cells in each
phase of the cell cycle were approximately equal in 6-shogaoltreated HT-29 cells (Fig. 5B). Also, it should be noted that sub-G1 apoptotic cell phase was not apparent under the autophagic flux condition. Taken together, the above findings led us to presume that cell cycle arrest would promote the formation of autophagosomes due to 6-shogaol treatment. Furthermore, a large amount of autophagosome production in cells might result in cell cycle progression blockage, which in turn would induce G2/M phase arrest. 3.6. Acidic vesicular organelles increase in 6-shogaol-treated HT-29 cells in 18 h From Fig. 4A, we inferred that after 18 h, the autophagosome might fuse with lysosome to form autolysosome and then be degraded by acidic lysosomal hydrolases. Acridine orange (AO) is an acidotropic dye generally used for labeling autolysosomes during the maturation stage. In that regard, AO stains the acidic vesicular organelles (AVOs), facilitating their measurement with flow cytometry. Treatment with 6-shogaol for 18 h increased acidic vesicular organelles in a dose-dependent manner (Fig. 6A). For the dose of 60 mM, more than one-third of the cells had AVOs, whereas a dose of 80 mM caused 64.97% of cells to form AVOs. Furthermore, the fold increase in fluorescence intensity was also dose dependent (Fig. 6B). Based on LC3 staining, there was an obvious amount of autophagosome at 12 h. However, at 18 h, AVOs increased, so fewer
Fig. 7. Autolysosome performance in 6-shogaol treated HT-29 cells. (A) LC3 puncta (green) were labeled for autophagosomes and DAPI (blue) stained for nuclear, LAMP-1 (red) were marked for lysosome to colocalize autolysosomes. (B) A single cell treated with 60 mM 6-shogaol is shown. (C) Display of the control/treatment for LAMP-1 PE staining by flow cytometry. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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LC3 puncta were visible under confocal microscopy, as the LC3-II in the autolysosomal lumen had degraded. 3.7. 6-shogaol induced autolysosome formation and autophagic cell death Since an autolysosome is merely a kind of AVO, we needed to seek further evidence of the fusion of autophagosomes and lysosomes. Therefore, immunocytochemical staining for Lysosomal-associated membrane protein 1 (LAMP-1) and LC3 was performed to colocalize lysosomes and autophagosomes. The colors used were LAMP-1 (red), LC3 (green), and DAPI (blue); the combination of LAMP-1 and LC3 was yellow. Pictures of control cells and those treated with 60 mM of 6-shogaol for 15 h are shown in Fig. 7A. Unlike controls, some of the 60 mM-treated cells had a merged yellow color, demonstrating that 6-shogaol induced fusion of autophagosome with lysosome to form an autolysosome. A single cell treated with 60 mM 6-shogaol is shown in Fig. 7B; the green LC3 and red LAMP-1 puncta are readily apparent (c, d and g, h). Furthermore, note the bright merged puncta, which further confirmed autolysosome formation (Fig. 7B, pictures a and e). Comparing the LAMP-1 histograms of the flow cytometry analyses of the cells treated with 6-shogaol for 18 and 24 h, the fluorescent intensity of cells treated for 24 h was significantly higher than that of cells treated for 18 h (Fig. 7C), indicating a significant increase in lysosome formation induced by 6-shogaol from 18 h to 24 h. Furthermore, the increase in lysosome formation by 6-shogaol
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treatment was also dose-dependent. These results substantiated the observation that 6-shogaol treatment induced autophagic cell death in colorectal cancer cells. Furthermore, such evidence supported our hypothesis that the major cell-death type of 6-shogaol treated HT-29 cells was autophagy at 24 h. When cells were treated with 6-shogaol for 6 to 12 h, the cell death pattern may have been irregular due to induced autophagy and G2/M arrest. However, from 18 h to 24 h, the mortality of 6-shogaol treated-HT-29 cells was dose-dependent due to autophagic cell death (Fig. 1A). In this study, we found that 6-shogaol induced autophagic cell death via formation of acidic vesicular organelles and autolysosomes in human colon adenocarcinoma HT-29 cells (Figs. 6 and 7). This finding has considerable importance. Other phytochemicals, such as resveratrol, also induce autophagy in breast and ovarian cancer [33]. The B-group triterpenoid saponins in soybean can induce autophagy in colon cancer [34]. However, only a limited number of studies have provided sufficient evidence to confirm that some phytochemicals, such as 6-shogaol, can induce autophagic cell death. This form of cell death can occur independently of other forms of cell death, such as apoptosis and necrosis [35]. Autophagic cell death is an important strategy in cancer therapy [36]. In breast carcinoma cell line MCF-7, for example, tamoxifen, an estrogen antagonist, causes autophagic cell death and thereby inhibits metastasis. In colon cancer, radiation treatment also induces autophagic cell death. Moreover, there is growing evidence that autophagic cell death may inhibit angiogenesis [13]. Consequently, the autophagic response of angiogenesis
Fig. 8. Apoptotic cell death induced by 6-shogaol in HT-29 cells. (A) Annexin V staining showed early and late apoptosis cell percentages of 6-shogaol-treated HT-29 at 18, 24, or 48 h. (B) Caspase 3/7 activity of 6-shogaol-treated HT-29 at 18, 24, or 48 h. (C) DNA fragmentation of 6-shogaol-treated HT-29 at 18, 24, or 48 h, assessed by TUNEL. Data are expressed as mean SD (n = 3). *, P < 0.05, **, P < 0.01, ***, P < 0.001, cells treated with 6-shogaol for 24 or 48 h versus those treated for 18 h.
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Fig. 9. Effect of 6-shogaol on HT-29 cells from 6 to 48 h. 6-shogaol induced both autophagic and apoptotic cell death.
inhibitors has become a new target for enhancing therapeutic efficacy [37]. 3.8. Apoptosis happens after autophagy After autophagic cell death, we found that apoptotic began to increase. Results of the annexin-V assay after 18, 24, and 48 h treatment are shown in Fig. 8A. At 18 h, apoptotic cells accounted for only less than 8% of the total cells, even when treated with 80 mM of 6-shogaol. However, by 24 and 48 h, they comprised 19% and 29%, respectively, at a dose of 60 mM. Since the annexin-V assay reflects the early stage of apoptosis, it could be concluded that 6-shogaol triggered apoptosis profoundly after autophagy. In addition, through lysosomal degradation, autophagic cell death might also trigger apoptotic cell death [38,39]. For the apoptosis process, extrinsic and intrinsic pathway stimulation would both activate caspase 3 and 7. These effector caspases are responsible for proteolytic cleavage of a broad spectrum of cellular targets and lead to programmed cell death. When cells were treated with 60 mM of 6-shogaol, the activity of caspase 3/7 increased to 1.9 fold after 24 h, relative to the untreated control. But it increased to 5.6 fold after 48 h (Fig. 8B). When the dosage was increased to 80 mM, the caspase activity increased to 6.7 fold in 24 h, but it then decreased to 2.8 fold after 48 h (Fig. 8 B). Since the cell viability was less than 10% after 80 mM of 6-shogaol treatment for 48 h (Fig. 1A), which consequently caused decreases of caspase activity. It appeared that the apoptotic cell death caused by 6-shogaol was not apparent at 18 h regardless of the dose applied. For the dose of 60 mM, the apoptotic activity reached its maximum at approximately 48 h. When the dose was increased to 80 mM, the maximal activity occurred at 24 h after 6-shogaol treatment. DNA fragmentation of cells was analyzed by TUNEL assay. After 48 h, the HT-29 cells treated with 60 mM 6-shogaol had approximately 2-fold increases in FITC fluorescence intensity as compared to untreated counterparts, and the fluorescence intensity increased to 2.5 fold when the cells were treated with 80 mM of 6-shogaol. This observation confirmed that the cells had progressed to late-stage apoptosis. Treatment of 6-shogaol activated effector caspases, followed by DNA fragmentation at 48 h.
4. Conclusion In this study, we utilized different analytical methods that might reflect different stages of PCDs to investigate the effect of 6shogaol on HT-29 cells. We found that the cells reached the highest G2/M arrest and autophagosome accumulation in 12 h. 6-shogaol induced numerous acidic vesicular organelles in 18 h, and autolysosome formation caused autophagic cell death in 24 h. After 24 h, 6-shogaol treatment resulted in characteristic apoptotic cell death via effector caspase activation, and DNA fragmentation reached its highest point in 48 h (Fig. 9). This study demonstrates that autophagy and apoptosis may be closely interlinked and occur in a time series. Widespread autophagosome and autolysosome might cause essential organelles degradation and trigger apoptosis. In conclusion, 6-shogaol induces autophagic and apoptostic cell death in colorectal adenocarcinoma HT-29 cells, and therefore it has potential as a chemo-preventive agent for colorectal cancer. Acknowledgements We thank the staff of the Joint Center for Instruments and Research, College of Bioresources and Agriculture, National Taiwan University. References [1] C.S. Yang, J.M. Landau, M.-T. Huang, H.L. Newmark, Inhibition of carcinogenesis by dietary polyphenolic compounds, Annu. Rev. Nutr. 21 (1) (2001) 381–406. [2] Q.-Y. Wei, J.-P. Ma, Y.-J. Cai, L. Yang, Z.-L. Liu, Cytotoxic and apoptotic activities of diarylheptanoids and gingerol-related compounds from the rhizome of Chinese ginger, J. Ethnopharmacol. 102 (2) (2005) 177–184. [3] M.-H. Pan, C.-T. Ho, Chemopreventive effects of natural dietary compounds on cancer development, Chem. Soc. Rev. 37 (11) (2008) 2558–2574. [4] S. Dugasani, M.R. Pichika, V.D. Nadarajah, M.K. Balijepalli, S. Tandra, J.N. Korlakunta, Comparative antioxidant and anti-inflammatory effects of [6]gingerol,[8]-gingerol,[10]-gingerol and [6]-shogaol, J. Ethnopharmacol. 127 (2) (2010) 515–520. [5] F.-F. Gan, A.A. Nagle, X. Ang, O.H. Ho, S.-H. Tan, H. Yang, W.-K. Chui, E.-H. Chew, Shogaols at proapoptotic concentrations induce G2/M arrest and aberrant mitotic cell death associated with tubulin aggregation, Apoptosis 16 (8) (2011) 856–867. [6] S.W. Tait, D.R. Green, Mitochondria and cell death: outer membrane permeabilization and beyond, Nat. Rev. Mol. Cell Biol. 11 (9) (2010) 621–632. [7] J. Geng, D.J. Klionsky, The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy, EMBO Rep. 9 (9) (2008) 859–864.
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Original article
6-shogaol induces autophagic cell death then triggered apoptosis in colorectal adenocarcinoma HT-29 cells Ting-Yi Li, Been-Huang Chiang* Institute of Food Science and Technology, National Taiwan University, Taipei 10617, Taiwan
A R T I C L E I N F O
Article history: Received 31 March 2017 Received in revised form 23 May 2017 Accepted 12 June 2017 Keywords: Colorectal cancer HT 29 6-shogaol Autophagy Apoptosis
A B S T R A C T
6-shogaol is a phytochemical of dietary ginger, we found that 6-shogaol could induced both autophagic and apoptotic death in human colon adenocarcinoma (HT-29) cells. Results of this study showed that 6shogal induced cell cycle arrest, autophagy, and apoptosis in HT-29 cells in a time sequence. After 6 h, 6shogal induced apparent G2/M arrest, then the HT-29 cells formed numerous autophagosomes in each phase of the cell cycle. After 18 h, increases in acidic vesicles and LAMP-1 (Lysosome-associated membrane proteins 1) showed that 6-shogaol had caused autophagic cell death. After 24 h, cell shrinkage and Caspase-3/7 activities rising, suggesting that apoptotic cell death had increased. And after 48 h, the result of TUNEL assay indicated the highest occurrence of apoptosis upon 6-shogaol treatment. It appeared that apoptosis is triggered by autophagy in 6-shogaol treated HT-29 cells, the damage of autophagic cell death initiated apoptosis program. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Colorectal cancer (CRC) is the third most common cancer in the world. Its death toll in the last 50 y (1967–2016) has increased 10fold, perhaps due to changing diets. Some carcinogens from dietary factors, e.g., polycyclic aromatic hydrocarbons (PAHs), may increase the risk of CRC. However, several phytochemicals that are readily available in specific foods have been proven to reduce the risk of CRC [1]. Ginger (Zingiber officinale Roscoe), the most widespread seasoning used in Asia, is an important ingredient in both food preparation and folk medicine. The bioactive phytochemicals in ginger are polyphenols, including gingerols, paradols, gingerdiols, and shogaols, which have been reported to be potent chemopreventive agents [2]. For example, 6-gingerol has been thoroughly investigated for its role as an anticarcinogenic agent [3]. However,
Abbreviations: CRC, Colorectal cancer; PAHs, polycyclic aromatic hydrocarbons; PCD, program cell death; ER, endoplasmic reticulum; MOMP, mitochondrial outer membrane permeabilization; APAF1, apoptotic protease activating factor 1; DMSO, dimethyl sulfoxide; PI, propidium iodide; 3-MA, 3-methyladenine; LC3, microtubule associated protein 1 light chain 3; PBS, phosphate buffered saline; AO, acridine orange; AVOs, acidic vesicular organelles; LAMP-1, Lysosome-associated membrane proteins 1; DAPI, 4’6-diamidino-2-phenylindole. * Corresponding author at: Institute of Food Science and Technology, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan. E-mail address: [email protected] (B.-H. Chiang). http://dx.doi.org/10.1016/j.biopha.2017.06.038 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
the dehydrated form of 6-gingerol, 6-shogaol, was reported to have anti-inflammatory effects surpassing those of 6-gingerol [4,5]. Furthermore, 6-shogaol has also been reported to possess antitumor effects via multiple mechanisms. There are three types of programmed cell death (PCD): apoptosis, autophagy, and necroptosis. Apoptosis is a silent cell death elicited through extrinsic and intrinsic pathways. The extrinsic apoptotic pathway is initiated by ligation of death receptors. Then, caspase 8 activation can directly cleave and activate caspase 3 and caspase 7. In contrast, the intrinsic apoptotic pathway, typically stimulated by DNA damage or endoplasmic reticulum (ER) stress, can activate the BCL-2 family and mitochondrial outer membrane permeabilization (MOMP). Then, cytochrome c is released from mitochondria and binds apoptotic protease-activating factor 1 (APAF1), which recruits and also activates executioner caspases 3 and 7 [6]. Therefore, both intrinsic and extrinsic pathways ultimately activate caspase 3 and 7, leading to apoptosome body formation and DNA fragmentation. Autophagy, another type of PCD, occurs due to insufficient nutrition and other stress. A double-membrane autophagic vacuole which sequesters a portion of the cytoplasm (called an autophagosome) is formed as a maturation step of autophagy. In autophagosome formation, two ubiquitin-like conjugation systems, Atg12 and Atg8 (also called LC3), is important for the stability of double-membranes. The Atg12–Atg5 conjugate facilitates lipidation of Atg8. The Atg8–phosphatidylethanolamine will support membrane expansion and increase autophagosomes
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[7,8]. An autophagosome will fuse with a lysosome to form an autolysosome and then degrade the cytoplasmic material. Excessive self-digestion will threaten essential organelles and cause protein degradation, leading to autophagic cell death [9]. During necrotic cell death, several intracellular organelles dilate and the plasma membrane breaks down, causing a release of cell lysate and an inflammatory response [10]. Autophagy can be considered a protection mechanism through inhibition of cell necrosis [11]. In colon cancer development, autophagy negatively regulates Wnt signaling by targeting the dishevelled protein to inhibit proliferation [12]. Thus, utilizing autophagy to cause cell death in tumor cells is a potential therapeutic strategy [13]. 6shogaol has been reported that could induce microtubule damage, inhibiting breast cancer cell invasion, and apoptosis in human mahlavu, colorectal, and hepatocellular carcinoma cells [14–18]. Furthermore, 6-shogaol also initiates autophagy by inhibiting AKT/ mTOR in human non-small cell lung cancer A549 cells [19]. Recently, some reports have indicated that 6-shogaol might induce autophagic cell death in pancreatic, breast, and hepatoma cancer cell lines [20–22]. However, the evidence of 6-shogaol inducing autophagic cell “death” is insufficiently strong due to autolysosome formation and lysosomal digestion. Therefore, the objectives of this study were to elucidate the role of 6-shogaol in inducing both autophagic and apoptosis cell death in human colorectal cancer HT-29 cells in a time-dependent manner and to understand the relationship between them. 2. Materials and methods 2.1. Chemicals and reagents The 6-shogaol was obtained from Chromadex (Irvine, CA, USA). Rapamycin, digitonin, ethanol and 3-methyladenine (3-MA) were purchased from Merck (Darmstadt, Germany). Trypsin-EDTA, dimethyl sulfoxide (DMSO), propidium iodide (PI), ribonuclease A (RNase A), acridine orange (AO), Triton X-100, and Cell Counting Kit 8 were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2.2. Cell culture Human colorectal cancer cell line HT-29 (American Type Culture Collection [ATCC] HTB-38) was grown in RPMI-1640 medium (Gibco BRL, NY, USA) supplemented with 10% heatinactivated fetal bovine serum (FBS, Gibco BRL). It was then kept at 37 C in a humidified 5% CO2 incubator. When cell growth reached 80%, 3 ml trypsin was used for 3 min for cell harvesting. 2.3. Cell viability The HT-29 cells were seeded (density, 2 105 cells/ml) into 96well plates (100 ml per well). After 24 h (when cells were attached), an aliquot of 100 ml FBS-free medium was used to replace the original medium and incubated for another 24 h to stop cell growth [24]. Thereafter, 100 ml of 6-shogaol of various concentrations (20, 40, 60, 80, 100 mM dissolved in culture medium) was used to treat the cells for 6, 12, 18, 24, 48 h. A Cell Counting Kit 8 (CCK8; 10 ml) was added to each well, the plate was incubated for 1.5 h at 37 C, and absorbance was measured at 450 nm (ELISA reader) to detect an orange formazan product (proportional to the number of living cells). 2.4. Cell cycle assay Cells treated with 6-shogaol were analyzed by flow cytometric measurement of cellular DNA content. Briefly, 1 ml of HT-29 cells
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were seeded (2 105 per well) in 6-well plates and allowed to adhere and grow for 24 h, followed by 6-shogaol treatment for various durations. Culture medium containing detached cells was collected and the attached cells were trypsinized. Attached and floating cells were pooled, pelleted, and washed with PBS. They were then re-suspended in 70% ethanol overnight. Hypotonic buffer containing 20 mg/ml propidium iodide, 0.01% Triton X-100, and 10 mg/ml RNase A was added and the mixture was incubated for 30 min in the dark. Then cells were analyzed on a Beckman Coulter flow cytometer (FC500, Fullerton, CA, USA). Data analysis was performed using Flow Jo software (Treestar, Inc., San Carlos, CA, USA). 2.5. Immunocytochemistry for LC3 punctate and lysosome membrane The HT-29 cells (4 105 cells/ml, 0.5 ml) were cultured on fourwell chamber slides (Lab-Tek II, Nunc, Rochester, NY, USA) for 24 h, treated with 6-shogaol, and incubated for various durations. Cells were fixed on slides by immersing them in 4% formaldehyde for 30 min and then washed three times with PBS. Bovine serum albumin (BSA, 5%, with 0.1% Triton X-100. Sigma) was added and incubated for 60 min. For autophagosome detection, primary antibody LC3-B (1:200, Genetex, Taiwan, diluted with 1% BSA and 0.1% Triton X-100 PBS solution) was added to cells and incubated for 2 h. Secondary antibody Dylight-488 anti-rabbit IgG (1:100 dilution, Thermo, Waltham, MA, USA) was then added and incubated for 1 h. For autolysosome detection, after LC3 staining, PE Mouse anti-human CD107a (BD, San Jose, CA, USA) diluted to 200X was used to bind LAMP-1 on lysosome membrane. Finally, nuclei were labeled with 300 nM of 40 , 6-diamidino-2-phenylindole (DAPI, Lonza, Walkersville, MD, USA) for 15 min. Then the chamber was removed and mounting medium was added onto the slide with a cover slip on the top. Confocal microscopy (Leica TCS SP5 II, Solms, Germany) was used for assessment. 2.6. Monitoring autophagosomes in each cell cycle The HT-29 cells (2 105 cells/ml, 2 ml) lwere seeded on 6-well plate and treated with 6-shogaol for varying durations. Then the media were collected and cells were harvested by trypsinization, suspended in a 1.8 ml Eppendorf tube, and immediately centrifuged. After being washed with PBS, the cell pellets were treated with digitonin (150 mg/ml) for 7 min. Cells were then centrifuged (1000 g for 5 min) to remove LC3-I and washed twice, primary antibody LC3 (1:200, PM036, MBL, Tokyo, Japan) and FITC IgG antirabbit (1:200, GTX27079, Genetex, Irvine, CA, USA) were added, and then the mixture was incubated in dark for 30 min [25]. After incubation, PBS buffer containing propidium iodide (5 mg/ml) and RNaseA (10 mg/ml) was used to stain cells (which were vortexed before analysis). 2.7. Acidic vesicular organelles assay The HT-29 cells (2 105 cells/ml, 2 ml) were cultured on a 6well plate and treated with 6-shogaol for varying durations. Then the treated cells were stained with Acridine Orange (1.5 mg/ml) for 15 min at 37 C. After the cells were harvested, red fluorescence on FL3 was analyzed by flow cytometry using Win Midi 2.9 software (Joseph Trotter, Scripps Institute, La Jolla, CA, USA). 2.8. Lysosome expression assay The HT-29 cells (2 105 cells/ml, 2 ml) were seeded on a 6-well plate and treated with 6-shogaol for various durations. PharmingenTM Transcription Factor Buffer Set (BD, San Jose, CA, USA) was used for intracellular staining. After washing, fixing, and
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Fig. 1. Effects of 6-shogaol on cell cytotoxicity in HT-29 cells. (A) Cell viability of HT-29 cells following treatment with 6-shogaol for 6, 12, 18, 24, or 48 h. (B) Effects of 6-shogaol on morphology of HT-29 cells after 12 or 24 h (light microscopy, 400X). Data are expressed as mean SD (n = 3).
permeabilization, PE Mouse anti-human CD107a (LAMP-1, BD, San Jose, CA, USA) was added and the cells were incubated for 40 min in the dark. After the cells were harvested, red fluorescence of FL3 was analyzed by flow cytometry.
Diego, CA, USA; * for P < 0.05, ** for P < 0.01 and *** for P < 0.001). All results are reported as mean SD
2.9. Apoptotic cells analysis
3.1. Effect of 6-shogaol on cell proliferation inhibition
An annexin V-FITC/PI double staining apoptosis assay (BD, San Jose, CA, USA) was used to quantify 6-shogaol induced apoptosis in HT-29 cells. Cells were seeded at a concentration of 2 105 cells/ well in a 6-well plate, treated with 6-shogaol for 18, 24, or 48 h, and then washed twice with PBS buffer. Cells were re-suspended in 1X binding buffer (1 106 cells/ml) and transferred to 500 ml of 1X binding buffer in a 1.8 ml Eppendorf tube. Then 5 ml of annexin VFITC and 5 ml PI were added into the tubes and allowed to react for 15 min in the dark. Finally, after addition of 500 ml of 1X binding buffer, samples were analyzed by flow cytometry (within 1 h).
To evaluate the anticancer effects of 6-shogaol, we investigated the proliferation inhibition of 6-shogaol on the human colorectal cancer HT-29 cell line. Cell viabilities of HT-29 cells treated with 6shogaol (various doses and times) are shown in Fig. 1A. The cell death rate was dose- and time-dependent after 18 h (the 24-h IC50 of 6-shogaol-treated HT-29 cells was 40 mM). However, cell death phenomena after treatment for 6 and 12 h were unexpected, particularly at high doses. This motivated us to study the time
3. Results and discussion
2.10. Caspase-3, 7 activity detection Active caspase was detected with a CaspaTagTM Caspase-3/7 In Situ assay kit (Chemicon, Temecula, CA, USA), based on fluorochrome inhibitors of caspases (FLICA). The 6-shogaol-treated cell suspension (290 ml, 106 cells) and 10 ml of FLICA were mixed in 1.8 ml Eppendorf tubes. The tubes were gently flicked and then incubated for 1 h in a cell culture incubator, after which the contents were washed twice by centrifugation (500 x g for 5 min). The cell pellet was re-suspended and immediately analyzed by flow cytometry.
2.11. Assessment of DNA damage The TUNEL method (BD, San Jose, CA, USA) was used for analyzing DNA fragmentation. Briefly, 6-shogaol-treated HT-29 cells were suspended in 1% (w/v) paraformaldehyde and fixed in 70% (v/v) ethanol, and DNA Labeling Solution (TdT Enzyme with FITC dUTP) was added. After 60 min, the cell suspension was washed twice with rinsing buffer, centrifuged (300 x g for 5 min), and then analyzed by flow cytometry. Both positive and negative cells were also used as controls.
2.12. Statistical analyses Differences between the 6-shogaol-treated and control (0 mM) groups were analyzed by one way analysis of variance (ANOVA) and Duncan's multiple comparison tests using GraphPad (San
Fig. 2. Autophagy inhibitor 3-MA promoted apoptosis in 6-shogaol-treated HT-29 cells. (A) Annexin-V results in HT-29 cells at 48 h of exposure to 60 mM 6-shogaol and co-treatment with 5 mM 3-MA.
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effect of 6-shogaol treatment on cell death. With light microscopy, it was observed that cell shrinkage, the defining morphological characteristic of apoptosis, was not obvious at 12 h, but it became more apparent after 24 h (Fig. 1B). Therefore, we suspected that there was another cell death mechanism affecting 6-shogaoltreated HT-29 cells at 12 h. 3.2. An autophagy inhibitor increased apoptosis in 6-shogaol-treated HT-29 cells In the present study, cell death was dose-dependent in 6shogaol-treated HT-29 cells when the treatment exceeded 18 h (Fig. 1A). To check how the cell death mechanism autophagy affects apoptosis, 3-methyladenine (3-MA; an autophagic sequestration blocker via class III PI3 K inhibition) was used for annexin-V analysis of 6-shogaol-treated HT-29 cells. As shown by the data in Fig. 2, 60 mM of 6-shogaol induced only approximately 15.3% apoptosis in 48 h. Conversely, co-treatment of 6-shogaol with 3methyladenine for 48 h increased apoptotic cells to 80.9%. Therefore, we suspected that autophagy could happen in 6shogaol-treated HT-29 cells. 3.3. 6-shogaol induced G2/M phase arrest in HT-29 cells starting from 6h To investigate the potential anticancer properties and apoptotic cells of 6-shogaol-treated HT-29 cells, the cell cycle distributions following 6-shogaol treatment for 24 and 48 h are shown (Fig. 3A). The rate of apoptotic cells (Sub-G1) increased in a dose-dependent
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manner. However, the counts of apoptotic cells (Fig. 3A) comprised less than half of total cell death (Fig. 1A). Furthermore, from 6 to 48 h, we could see that 6-shogaol significantly induced cell cycle arrest at the G2/M phase (Fig. 3B). Treatment for 12 h induced the highest percentage of G2/M arrest; the G2/M phase comprised 43%, as compared to the 17% of control cells (cells not treated with 6shogaol) (Fig. 3C). Cell cycle arrest is a very important phenomenon in cancer pathology. It has been reported that treatment with 6-shogaol induces G2/M arrest and aberrant mitotic cell death at proapoptotic concentrations by causing microtubule damage through a specific reaction with sulfhydryl groups in tubulin [5,16]. Moreover, 6-shogaol inhibited cell proliferation by suppressing phosphorylation of mitogen-activated protein kinase (MAPK) and PI3 K/Akt signaling [17,23]. As a G2/M phase initiator, the suppression of Akt in turn inhibits binding of CDK1 and cyclin B, causing cells to arrest at the G2/M phase. Moreover, under autophagy progression, the suppression of Akt together with its downstream target mTOR contributes to initiation of autophagy [26]. In human non-small cell lung cancer A549 cells, 6-shogaol induces autophagy by inhibiting the Akt/mTOR pathway [19]. Based on the current results and the literature, we inferred that 6shogaol induced autophagy in colorectal cancer cells. 3.4. 6-shogaol induced autophagosome formation in HT-29 cells in 6 h Autophagy in tumorigenesis is gaining attention in the field of cancer therapy [27]. There is increasing evidence that the death of
Fig. 3. 6-shogaol induced cell cycle arrest in HT-29 cells. (A) Distribution of cell cycles in 6-shogaol-treated cell at 24 and 48 h. (B) G2/M phase exhibition of HT-29 cells after various intervals of treatment with 6-shogaol. (C) Cells were treated with 6-shogaol for 12 h and cell cycle distribution assessed. Data are expressed as mean SD (n = 3).*, P < 0.05, **, P < 0.01, ***, P < 0.001, control versus 6-shogaol-treated cells.
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cancer cells (unable to undergo apoptosis) is caused by autophagy, in a caspase-independent fashion, corresponding to type II programmed cell death. Since deficiencies of autophagy may enhance tumor progression, many autophagy-related genes (Atg) have been identified as tumor suppressor genes. Beclin1, UVRAG,
and Bif-1s, for example, promote carcinogenesis in knockout and xenograft mouse models [28–30]. In addition, PTEN, a tumor suppressor, provides signals to activate autophagy via Akt inhibition. Under this signaling transduction, many oncogenes, such as Bcl-2 and mTOR, are decreased, resulting in inhibition of
Fig. 4. The formation of autophagosomes in 6-shogaol treated HT-29 cells. LC3 puncta (yellow color) were labeled for autophagosome and stained with DAPI (blue color). (A) Based on confocal microscopy, HT-29 cells treated with 6-shogaol for 9 or 12 h had autophagosomes (white arrow). Rapamysin (positive control) had LC3 puncta, similar to the treatment group. (B) Individual HT-29 cells exposed to 60 mM 6-shogaol for 12 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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cancer proliferation [27]. For clinical induction of autophagy, direct mTOR inhibition with rapamycin has been widely used with numerous types of tumors [31]. In the initiation stage of autophagy, decline of mTOR causes double membrane formation. The membrane encapsulates some vital organelles in cancer cells to form autophagosomes. Thus, we investigated whether 6-shogaol could induce autophagy by
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staining the marker of autophagosome, LC3, and examining the formation of LC3 puncta using confocal microscopy. Cytoplasmic LC3 puncta (white arrow) were observed after treatment for 6 h and became more obvious after 12 h, indicating conversion of LC3-I into LC3-II (yellow dot) due to 6-shogaol and rapamycin (positive control) treatments, as compared to controls, which did not show any sign of autophagosome formation (Fig. 4A). There were LC3
Fig. 5. Effects of 6-shogaol treated HT-29 cells on LC3-II accumulation in each cell cycle phase. (A) Percentages of LC3-II-positive cells are shown under various concentrations of 6-shogaol for 12 h. Double-stained LC3 with PI demonstrated the distribution of LC3-II-positive cells in each cell cycle. (B) Percentages of performance LC3-II-positive cells in each phase of the cell cycle for control/treatment are shown. Data are expressed as mean SD (n = 3).
Fig. 6. Acidic vesicular organelles performance in 6-shogaol treated HT-29 cells. (A) Overlay of the control/treatment for acridine orange staining by flow cytometry. (B) Fold increase of fluorescence intensity as compared to control when cells were treated with various concentrations of 6-shogaol. Data are expressed as mean SD (n = 3). *, P < 0.05, **, P < 0.01, ***, P < 0.001, control versus 6-shogaol-treated cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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puncta in individual cells at 12 h (Fig. 4B). DAPI was used for nuclear staining (blue), there was no chromatin condensation during autophagosome formation (a, b, c) for either DNA content N (a, b) or 2N(c). Fluorescence intensity increased from 3 to 12 h, but then became obscure at 18 h (Fig. 4A). The decrease in LC3 puncta after 18 h treatment was attributed to fusion of the autophagosome with lysosomes (autolysosome formation). When this occurs, Atg4 recycles LC3-I and phosphatidylethanolamine from LC3-II on the cytosolic face of the autolysosome membrane [32]. Thus, the LC3 puncta would appear blurred. 3.5. 6-shogaol induced G2/M arrest and formed LC3-II puncta in every phase of the cell cycle in HT-29 cells Based on confocal images, we suspected that autophagy might occur in every phase of the cell cycle. Therefore, the autophagosome contents in every phase of the cell cycle of 6-shogaol-treated HT-29 cells were quantitated by flow cytometry [25]. In this assay, digitonin was used to increase cell membrane permeability to remove LC3-I in the cytoplasm, thereby allowing measurement of the amount of LC3-II. The percentages of LC3-II-positive cells and the distribution of the cell cycle in 6-shogaol-treated HT-29 cells are shown in Fig. 5A. Treatment with 6-shogaol significantly induced LC3-II-positive cells at 12 h to increase, in a dosedependent manner, from 1.62% (control) to 66.25% (60 mM). Based on DNA content, each cell cycle phase was gated (as shown in the dot plot of the control). The amounts of LC3-II-positive cells in each
phase of the cell cycle were approximately equal in 6-shogaoltreated HT-29 cells (Fig. 5B). Also, it should be noted that sub-G1 apoptotic cell phase was not apparent under the autophagic flux condition. Taken together, the above findings led us to presume that cell cycle arrest would promote the formation of autophagosomes due to 6-shogaol treatment. Furthermore, a large amount of autophagosome production in cells might result in cell cycle progression blockage, which in turn would induce G2/M phase arrest. 3.6. Acidic vesicular organelles increase in 6-shogaol-treated HT-29 cells in 18 h From Fig. 4A, we inferred that after 18 h, the autophagosome might fuse with lysosome to form autolysosome and then be degraded by acidic lysosomal hydrolases. Acridine orange (AO) is an acidotropic dye generally used for labeling autolysosomes during the maturation stage. In that regard, AO stains the acidic vesicular organelles (AVOs), facilitating their measurement with flow cytometry. Treatment with 6-shogaol for 18 h increased acidic vesicular organelles in a dose-dependent manner (Fig. 6A). For the dose of 60 mM, more than one-third of the cells had AVOs, whereas a dose of 80 mM caused 64.97% of cells to form AVOs. Furthermore, the fold increase in fluorescence intensity was also dose dependent (Fig. 6B). Based on LC3 staining, there was an obvious amount of autophagosome at 12 h. However, at 18 h, AVOs increased, so fewer
Fig. 7. Autolysosome performance in 6-shogaol treated HT-29 cells. (A) LC3 puncta (green) were labeled for autophagosomes and DAPI (blue) stained for nuclear, LAMP-1 (red) were marked for lysosome to colocalize autolysosomes. (B) A single cell treated with 60 mM 6-shogaol is shown. (C) Display of the control/treatment for LAMP-1 PE staining by flow cytometry. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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LC3 puncta were visible under confocal microscopy, as the LC3-II in the autolysosomal lumen had degraded. 3.7. 6-shogaol induced autolysosome formation and autophagic cell death Since an autolysosome is merely a kind of AVO, we needed to seek further evidence of the fusion of autophagosomes and lysosomes. Therefore, immunocytochemical staining for Lysosomal-associated membrane protein 1 (LAMP-1) and LC3 was performed to colocalize lysosomes and autophagosomes. The colors used were LAMP-1 (red), LC3 (green), and DAPI (blue); the combination of LAMP-1 and LC3 was yellow. Pictures of control cells and those treated with 60 mM of 6-shogaol for 15 h are shown in Fig. 7A. Unlike controls, some of the 60 mM-treated cells had a merged yellow color, demonstrating that 6-shogaol induced fusion of autophagosome with lysosome to form an autolysosome. A single cell treated with 60 mM 6-shogaol is shown in Fig. 7B; the green LC3 and red LAMP-1 puncta are readily apparent (c, d and g, h). Furthermore, note the bright merged puncta, which further confirmed autolysosome formation (Fig. 7B, pictures a and e). Comparing the LAMP-1 histograms of the flow cytometry analyses of the cells treated with 6-shogaol for 18 and 24 h, the fluorescent intensity of cells treated for 24 h was significantly higher than that of cells treated for 18 h (Fig. 7C), indicating a significant increase in lysosome formation induced by 6-shogaol from 18 h to 24 h. Furthermore, the increase in lysosome formation by 6-shogaol
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treatment was also dose-dependent. These results substantiated the observation that 6-shogaol treatment induced autophagic cell death in colorectal cancer cells. Furthermore, such evidence supported our hypothesis that the major cell-death type of 6-shogaol treated HT-29 cells was autophagy at 24 h. When cells were treated with 6-shogaol for 6 to 12 h, the cell death pattern may have been irregular due to induced autophagy and G2/M arrest. However, from 18 h to 24 h, the mortality of 6-shogaol treated-HT-29 cells was dose-dependent due to autophagic cell death (Fig. 1A). In this study, we found that 6-shogaol induced autophagic cell death via formation of acidic vesicular organelles and autolysosomes in human colon adenocarcinoma HT-29 cells (Figs. 6 and 7). This finding has considerable importance. Other phytochemicals, such as resveratrol, also induce autophagy in breast and ovarian cancer [33]. The B-group triterpenoid saponins in soybean can induce autophagy in colon cancer [34]. However, only a limited number of studies have provided sufficient evidence to confirm that some phytochemicals, such as 6-shogaol, can induce autophagic cell death. This form of cell death can occur independently of other forms of cell death, such as apoptosis and necrosis [35]. Autophagic cell death is an important strategy in cancer therapy [36]. In breast carcinoma cell line MCF-7, for example, tamoxifen, an estrogen antagonist, causes autophagic cell death and thereby inhibits metastasis. In colon cancer, radiation treatment also induces autophagic cell death. Moreover, there is growing evidence that autophagic cell death may inhibit angiogenesis [13]. Consequently, the autophagic response of angiogenesis
Fig. 8. Apoptotic cell death induced by 6-shogaol in HT-29 cells. (A) Annexin V staining showed early and late apoptosis cell percentages of 6-shogaol-treated HT-29 at 18, 24, or 48 h. (B) Caspase 3/7 activity of 6-shogaol-treated HT-29 at 18, 24, or 48 h. (C) DNA fragmentation of 6-shogaol-treated HT-29 at 18, 24, or 48 h, assessed by TUNEL. Data are expressed as mean SD (n = 3). *, P < 0.05, **, P < 0.01, ***, P < 0.001, cells treated with 6-shogaol for 24 or 48 h versus those treated for 18 h.
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Fig. 9. Effect of 6-shogaol on HT-29 cells from 6 to 48 h. 6-shogaol induced both autophagic and apoptotic cell death.
inhibitors has become a new target for enhancing therapeutic efficacy [37]. 3.8. Apoptosis happens after autophagy After autophagic cell death, we found that apoptotic began to increase. Results of the annexin-V assay after 18, 24, and 48 h treatment are shown in Fig. 8A. At 18 h, apoptotic cells accounted for only less than 8% of the total cells, even when treated with 80 mM of 6-shogaol. However, by 24 and 48 h, they comprised 19% and 29%, respectively, at a dose of 60 mM. Since the annexin-V assay reflects the early stage of apoptosis, it could be concluded that 6-shogaol triggered apoptosis profoundly after autophagy. In addition, through lysosomal degradation, autophagic cell death might also trigger apoptotic cell death [38,39]. For the apoptosis process, extrinsic and intrinsic pathway stimulation would both activate caspase 3 and 7. These effector caspases are responsible for proteolytic cleavage of a broad spectrum of cellular targets and lead to programmed cell death. When cells were treated with 60 mM of 6-shogaol, the activity of caspase 3/7 increased to 1.9 fold after 24 h, relative to the untreated control. But it increased to 5.6 fold after 48 h (Fig. 8B). When the dosage was increased to 80 mM, the caspase activity increased to 6.7 fold in 24 h, but it then decreased to 2.8 fold after 48 h (Fig. 8 B). Since the cell viability was less than 10% after 80 mM of 6-shogaol treatment for 48 h (Fig. 1A), which consequently caused decreases of caspase activity. It appeared that the apoptotic cell death caused by 6-shogaol was not apparent at 18 h regardless of the dose applied. For the dose of 60 mM, the apoptotic activity reached its maximum at approximately 48 h. When the dose was increased to 80 mM, the maximal activity occurred at 24 h after 6-shogaol treatment. DNA fragmentation of cells was analyzed by TUNEL assay. After 48 h, the HT-29 cells treated with 60 mM 6-shogaol had approximately 2-fold increases in FITC fluorescence intensity as compared to untreated counterparts, and the fluorescence intensity increased to 2.5 fold when the cells were treated with 80 mM of 6-shogaol. This observation confirmed that the cells had progressed to late-stage apoptosis. Treatment of 6-shogaol activated effector caspases, followed by DNA fragmentation at 48 h.
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