Article pubs.acs.org/JAFC
Gypenoside L, Isolated from Gynostemma pentaphyllum, Induces Cytoplasmic Vacuolation Death in Hepatocellular Carcinoma Cells through Reactive-Oxygen-Species-Mediated Unfolded Protein Response Kai Zheng,†,‡,§ Chenghui Liao,†,‡ Yan Li,†,∥ Xinmin Fan,‡ Long Fan,‡ Hong Xu,⊥ Qiangrong Kang,‡ Yong Zeng,∥ Xuli Wu,‡ Haiqiang Wu,‡ Lizhong Liu,‡ Xiaohua Xiao,# Jian Zhang,*,‡ Yifei Wang,*,§ and Zhendan He*,‡ ‡
Department of Pharmacy, School of Medicine, ⊥College of Life Sciences, and #First Affiliated Hospital of School of Medicine, Shenzhen University, Shenzhen, Guangdong 518060, People’s Republic of China § College of Life Science and Technology, Jinan University, Guangzhou, Guangdong 510632, People’s Republic of China ∥ First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, People’s Republic of China S Supporting Information *
ABSTRACT: Exploring novel anticancer agents that can trigger non-apoptotic or non-autophagic cell death is urgent for cancer treatment. In this study, we screened and identified an unexplored anticancer activity of gypenoside L (Gyp-L) isolated from Gynostemma pentaphyllum. We showed that treatment with Gyp-L induces non-apoptotic and non-autophagic cytoplasmic vacuolation death in human hepatocellular carcinoma (HCC) cells. Mechanically, Gyp-L initially increased the intracellular reactive oxygen species (ROS) levels, which, in turn, triggered protein ubiquitination and unfolded protein response (UPR), resulting in Ca2+ release from endoplasm reticulum (ER) inositol trisphosphate receptor (IP3R)-operated stores and finally cytoplasmic vacuolation and cell death. Interruption of the ROS−ER−Ca2+ signaling pathway by chemical inhibitors significantly prevented Gyp-L-induced vacuole formation and cell death. In addition, Gyp-L-induced ER stress and vacuolation death required new protein synthesis. Overall, our works provide strong evidence for the anti-HCC activity of Gyp-L and suggest a novel therapeutic option by Gyp-L through the induction of a unconventional ROS−ER−Ca2+-mediated cytoplasmic vacuolation death in human HCC. KEYWORDS: gypenoside L, vacuolation death, ROS, unfolded protein response, calcium release
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INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and is the third leading cause of cancerrelated mortality worldwide.1 The current therapeutic strategies for HCC include surgery, chemotherapy, ablation, liver transplantation, or combinations of them.2 Although there has been extensive research into HCC treatment, the overall survival rate in patients is still not optimistic as a result of the poor diagnosis, low curative resection ratio, and high recurrent metastasis ratio.3 Systematic chemotherapy is an important approach for HCC patients, and several compounds, such as sorafenib, 5-fluorouracil, and cisplatin, are clinically approved. However, increasing evidence exhibits their limited benefits as a result of toxic side effects and chemoresistance. Thus, it is urgent to develop novel effective therapeutic reagents without cytotoxicity. Apoptosis is a general way induced by most of the anticancer agents to trigger apoptotic networks and eliminate malignant cells. However, tumor cells frequently obtain their chemoresistance through deregulating apoptotic signaling and particularly activating an anti-apoptotic system and autophagy.4 Therefore, any compounds that can trigger non-apoptotic cell death may represent an alternative strategy for cancer treatment.5−8 There are several forms of non-apoptotic and © 2016 American Chemical Society
non-autophagic cell death with differential characteristics, including oncosis,9 necroptosis,10 entosis,11 paraptosis,12 and vacuolation death.13 Importantly, cell vacuolation death is a paraptosis-liked cell death and is characterized by extensive formation of cytoplasmic vacuoles, endoplasm reticulum (ER) swelling, insensitivity to caspase inhibitor, inhibition by cycloheximide, and increased expression of the autophagic marker LC3-II.14−17 To date, the list of cytoplasmic vacuolation inducers has been accumulating and the mechanisms underlying vacuolation death, particularly the signals responsible for triggering dilation of the ER and LC3-II accumulation, are still poorly defined. Natural products are key resources for anticancer drug discovery. Gynostemma pentaphyllum, also known as “cheap ginseng”, has been widely used as a traditional herb or tea in Asia, including China, northern Vietnam, southern Korea, and Japan. Gypenosides are the major extracts from G. pentaphyllum, and their anticancer activities have been welldocumented.18−22 However, the specific functional compoReceived: Revised: Accepted: Published: 1702
November 30, 2015 February 1, 2016 February 12, 2016 February 12, 2016 DOI: 10.1021/acs.jafc.5b05668 J. Agric. Food Chem. 2016, 64, 1702−1711
Article
Journal of Agricultural and Food Chemistry
Figure 1. Gyp-L induces cytoplasmic vacuolation and cell death of HCC cells. (A) Chemical structure of Gyp-L. (B) HepG2 cells were treated with different concentrations of Gyp-L (5, 10, 20, 40, 60, 80, 100, and 120 μg/mL) for 24 h, and cell viability was determined by the MTT assay. Data are the mean ± SD of at least three independent experiments. (C) Cell morphology change induced by Gyp-L. Cells were treated with Gyp-L for 24 h, and cell morphology was observed by microscopy. (D) (Left) Cytotoxicity of Gyp-L on SMMC-7721 and Huh7 cells. Different concentrations of Gyp-L (5, 10, 20, 40, 60, 80, 100, and 120 μg/mL) were used. (Right) Gyp-L induced cytoplasmic vacuolation. (E) Percentage of Gyp-L-induced vacuolation in three HCC cells. At least 100 cells from five representative fields were counted in each independent experiment. (F) Cytotoxicity of Gyp-L on normal cells (LO2). (C30), 106.19 (C1′), 82.91 (C2′), 78.89 (C3′), 72.35 (C4′), 78.66 (C5′), 63.40 (C6′), 104.99 (C1″), 77.21 (C2″), 79.04 (C3″), 71.39 (C4″), 78.81 (C5″), 62.82 (C6″). Electrospray ionization mass spectrometry (ESIMS): m/z 823.40 [M + Na]+ (calculated for C42H72O14, 800.49). Cell Culture. HepG2, SMMC-7721, and Huh7 cells were cultured in RPMI 1640 (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA) at 37 °C in a humid atmosphere with 5% CO2. LO2 cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco). Antibodies and Reagents. TUDCA (T0557), 2-APB (D9754), CHX (C7698), and NAC (A9165) were purchased from SigmaAldrich (St. Louis, MO). BAPTA-AM (B018) was purchased from Dojindo (Shanghai, China). Z-VAD-FMK (S7023), 3-MA (S2767), and Rapamycin (S1039) were purchased from Selleck (Houston, TX). Anti-LC3B rabbit polyclonal (L7543) and anti-p62/SQSTM1 antibody (P0067) were supplied by Sigma-Aldrich. Anti-calnexin (2679), anti-caspase 3 (9662), anti-caspase 9 (9504), anti-Ero1-Lα (3264), anti-IRE1α (3294), anti-PDI (3501), anti-PERK (5683), antiubiquitin (3936), anti-GAPDH (5174), anti-mouse IgG (7076), and anti-rabbit IgG (7074) horseradish peroxidase (HRP)-linked antibodies were purchased from Cell Signaling Technology (Danvers, MA). All inhibitors were used in a non-cytotoxic concentration. Cell Viability Assay. The cytotoxicity of Gyp-L and other chemical inhibitors was examined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich, M2128) assay. In brief, cells were cultured in 96-well plates with varying concentrations of drugs for 24 h. Following incubation with MTT (0.5 mg/mL) for 4 h, the supernatant of each well was discarded and the insoluble formazan product was dissolved in 100 μL of dimethyl sulfoxide (DMSO). The optical density was measured at 570
nents or the detailed mechanism of gypenoside-induced cell death remains to be clarified. In this study, we identified gypenoside L (Gyp-L), originally isolated from G. pentaphyllum, as a novel anticancer agent against several human HCC cells. We demonstrated that Gyp-L induces cytoplasmic vacuolation death through reactive oxygen species (ROS)-mediated ER−Ca2+ signaling. These finding suggest, for the first time, that Gyp-L may represent a novel therapeutic option for HCC treatment.
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MATERIALS AND METHODS
Isolation and Characterization of Gyp-L. The total saponins of G. pentaphyllum were kindly provided by William Chi-Shing Tai (Hong Kong Baptist University). A portion of total saponins (350 g) was first subjected to column chromatography over silica gel (6000 g, 300−400 mesh) and eluted with the isocratic gradient solvent system of CHCl3, methanol, and water (26:8:1) to yield 24 major fractions (fractions 1−24). Fraction 14 (4.7 g) was then subjected to column chromatography over octadecyl silica (ODS, 239.48 g, 100−200 mesh) and eluted with the isocratic gradient solvent system of 40% CH3CN in water to yield four major components A−D. B (265 mg) was identified as Gyp-L by 1H and 13C nuclear magnetic resonance (NMR) and liquid chromatography−mass spectrometry (LC−MS) (shown in Supplemental Figure 1 of the Supporting Information). 13C NMR (75 MHz, C5D5N) δ: 48.24 (C1), 67.15 (C2), 96.06 (C3), 41.47 (C4), 56.67 (C5), 18.96 (C6), 35.52 (C7), 40.43 (C8), 50.84 (C9), 38.30 (C10), 32.72 (C11), 71.74 (C12), 48.97 (C13), 52.18 (C14), 31.77 (C15), 27.52 (C16), 55.28 (C17), 16.26 (C18), 18.16 (C19), 73.35 (C20), 7.32 (C21), 36.37 (C22), 23.47 (C23), 126.82 (C24), 131.22 (C25), 26.30 (C26), 18.10 (C27), 28.77 (C28), 17.99 (C29), 17.43 1703
DOI: 10.1021/acs.jafc.5b05668 J. Agric. Food Chem. 2016, 64, 1702−1711
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Figure 2. Gyp-L induces non-apoptotic cell death. (A) HepG2 cells were treated with different concentrations of Gyp-L (40, 60, and 80 μg/mL) for 24 h, and the cells were then stained with Annexin V and PI and analyzed by flow cytometry. (B) Activation of caspase 3 or caspase 9. HepG2 cells were treated with different concentrations of Gyp-L for 24 h, and cell lysates were subjected to the western blot assay for caspase 3 and caspase 9. P.C. = SNX2112 (10 μM), a Hsp90 inhibitor, used as a positive control. Effect of Z-VAD on (C) Gyp-L-induced cytoplasmic vacuole formation and (D) cell death. HepG2 and SMMC-7721 cells were treated with Gyp-L (60 or 80 μg/mL) in the presence or absence of Z-VAD (50 μM) for 24 h, and cell morphology and viability were determined. Data are the mean ± SD of at least three independent experiments. n.s = no significance. nm, with a reference wavelength of 630 nm, on a multiscanner autoreader (M450, Bio-Rad, Hercules, CA). The optical density at a wavelength of 570 nm (OD570) in control cells was taken as 100% viability. Western Blotting. Cells treated with Gyp-L in the presence or absence of inhibitors for indicated times were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, P0013B, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor cocktail (Sigma-Aldrich, P8340). The cell lysates were normalized to equal amounts of protein and were separated by 8−12% gradient sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). After transfer to polyvinylidene fluoride (PVDF) membrane, the proteins were blocked with 5% bovine serum albumin (BSA) and probed with the specific primary antibodies. Detection was conducted by incubation with species-specific HRPconjugated secondary antibodies. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL) blotting detection reagents (Thermo, 34080, Waltham, MA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. ImageJ software was used to quantify the band intensity. For each point, the signal intensity for each protein was normalized to that measured for GAPDH and was expressed as the fold increase relative to the control. Acridine Orange (AO) or LysoTracker Red Staining. AO (Majorbio, A1398, China) produces red fluorescence (emission peak at about 650 nm) in lysosomal compartments and green fluorescence (emission between 530 and 550 nm) in the cytosolic and nuclear compartments. Cell staining with AO (Sigma-Aldrich, A6014) was performed according to published procedures, adding a final concentration of 5 μg/mL for a period of 30 min (37 °C and 5% CO2). For LysoTracker Red staining, cells were treated with chemicals (as indicated) for 12 h at 37 °C and then incubated with 50 nM
LysoTracker Red (Beyotime, C1043) for an additional 1 h. Images were captured and analyzed by fluorescence microscopy (Nikon Ti-u). Measurement of Intracellular ROS or Ca2+ by Flow Cytometry. Human HCC cells treated with indicated compounds for 8 h were stained with 10 μM 2′,7′-dichlorofluorescein diacetate (DCF-DA, Sigma-Aldrich, D6883) for 30 min. Then, ROS generation was determined at 525 nm by flow cytometry (BD FACS Calibur, Franklin Lakes, NJ) or fluorescence microscopy (Nikon Ti-u, Japan). Software ImageJ was used to measure the mean fluorescence intensity. The intracellular calcium concentration was measured by flow cytometry using specific probe Fluo-4/AM (Invitrogen, F-14201). The cells treated with different concentrations of Gyp-L were incubated with 5 μM Fluo-4/AM at 37 °C for 60 min and detected by flow cytometry. For apoptosis analysis, the cells were incubated with various concentrations of Gyp-L for 24 h, harvested, washed, and resuspended in 500 μL of 1× binding buffer containing Annexin V− fluorescein isothiocyanate (FITC) and propidium iodide (PI) (BD, 556547) for 10 min at room temperature. Statistical Analysis. Data shown in this study are representatives or statistics [mean value ± standard deviation (SD)] of the results from at least three independent experiments. Student’s two-tailed t test was used for all statistical analysis, with the level of significance set at (∗∗∗) p < 0.005, (∗∗) p < 0.01, and (∗) p < 0.05.
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RESULTS Gyp-L Induces Cytoplasmic Vacuolation and Nonapoptotic Cell Death in Human HCC Cell Lines. In previous pilot studies in our lab, several pure fragments isolated from G. pentaphyllum were tested for their cytotoxic activities in a series of human HCC cell lines. We characterized and identified Gyp-L (chemical structure shown in Figure 1A), 1704
DOI: 10.1021/acs.jafc.5b05668 J. Agric. Food Chem. 2016, 64, 1702−1711
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Figure 3. Gyp-L inhibits autophagy. (A) HepG2 cells were treated with Gyp-L (60 μg/mL) for 24 h and then stained with AO (5 μg/mL) or LysoTracker Red (50 nM) for 30 min at 37 °C. Fluorescence images were captured by fluorescence microscopy. (B) Cells were treated with different concentrations of Gyp-L for 12 h, and cell lysates were extracted and subjected to western blot for LC3-II and p62. GAPDH was used as a loading control. ImageJ densitometric analysis of the LC3-II/GAPDH or p62/GAPDH ratio from LC3 or p62 immunoblots (mean ± SD of three independent experiments). (∗∗∗) p < 0.005, (∗∗) p < 0.01, and (∗) p < 0.05 versus the control. (C and D) Gyp-L induced non-autophagic cell death. Cells were treated with Gyp-L in the presence or absence of 3-MA (10 μM) or rapamycin (1 μM) for 24 h. Data are the mean ± SD of at least three independent experiments. (E) Cells were pretreated with 3-MA (10 μM) for 4 h before incubation with Gyp-L for 24 h. Data are the mean ± SD of at least three independent experiments.
Gyp-L Inhibits Autophagy and Induces Non-autophagic Cell Death. Vacuoles formation is also a feature of autophagy; therefore, we tested the role of autophagy in Gyp-Linduced cell death. We first examined the effect of Gyp-L on autophagy. The fluorescence microcopy assay using AO, a versatile fluorescence dye used to stain autophagy, showed that Gyp-L significantly enhanced the accumulation of autophagic vacuoles (red fluorescence) when compared to control cells (Figure 3A). The lysosome staining assay using a specific probe (LysoTracker Red) also demonstrated that Gyp-L increased the number of autolysosomes (Figure 3A). These results suggested that Gyp-L increases autophagic vacuole formation. Treatment with Gyp-L for 12 h induced the accumulation of LC3-II in a dose-dependent manner in both HCC cell lines (Figure 3B). Because the increment of the LC3-II level may result from the increased autophagosome generation or the blockage of the autophagosome−lysosome fusion process, we performed an autophagic flux assay by measuring the total cellular amount of p62, an autophagic substrate that degrades during the autophagy process, to distinguish Gyp-L-medated LC3 accumulation. An immunoblot analysis showed that a remarkable increase of p62 was induced by Gyp-L in a dosedependent manner (Figure 3B), indicating that Gyp-L inhibits autophagic flux. Additionally, time-course experiments were performed to confirm the inhibitory effect of Gyp-L on
displaying the strongest cytotoxic activity on HCC cell lines. Gyp-L reduced cell survival of HepG2 cells in a dose-dependent manner (Figure 1B). Phase-contrast microcopy of Gyp-Ltreated HepG2 cells showed cytoplasmic vacuoles (Figure 1C). The vacuolated cells showed an intact nucleus, shrunk at later time points, and underwent cell death. Additionally, Gyp-L exhibited dose-dependent cytotoxicity on another two HCC cell lines (Huh7 and SMMC-7721) and induced extensive vacuolation (Figure 1D). Typically, the number and size of vacuoles appeared at a higher concentration (Figure 1E). The cytotoxicity of Gyp-L on normal cells has also been tested, and no obvious inhibitory effect was observed (Figure 1F). To find whether Gyp-L-induced vacuolation death was distinct from apoptosis, we first tested the effect of Gyp-L on apoptosis using Annexin V/PI double staining. As shown in Figure 2A, no apoptosis was observed under Gyp-L treatment. Besides, no cleaved caspase-3 or caspase-9 was detected by western blot (Figure 2B), further supporting the conclusion that Gyp-L induced a non-apoptotic cell death. Additionally, we examined the effect of apoptosis inhibitor, Z-VAD, on cytoplasmic vacuolation and cell death. Inclusion of Z-VAD prevented neither vacuolation (Figure 2C) nor cell death in HepG2 and SMMC-7721 cells (Figure 2D). These findings clearly suggested that Gyp-L induces non-apoptotic cytoplasmic vacuolation death in HCC cells. 1705
DOI: 10.1021/acs.jafc.5b05668 J. Agric. Food Chem. 2016, 64, 1702−1711
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Figure 4. Upregulation of protein ubiquitination and ER stress by Gyp-L. HepG2 and SMMC-7721 cells were treated with Gyp-L for 12 h, and the (A) total levels of protein ubiquitination or (B) activation of the UPR pathway were analyzed by western blot. (C) TUDCA inhibited Gyp-L-induced UPR activation. Cells were treated with Gyp-L (60 μg/mL) in the presence or absence of TUDCA (40 μM) for 12 h. The intensity ratios were calculated from three independent experiments. (∗∗∗) p < 0.005 versus the control. (D) TUDCA protected cells from Gyp-L-mediated cell death. Data are the mean ± SD of at least three independent experiments.
TUDCA remarkably attenuated UPR protein expression upregulated by Gyp-L (Figure 4C) as well as Gyp-L-induced cell death (Figure 4D). These results suggested that ER stress plays a crucial role in the Gyp-L-induced vacuolation-mediated death of human HCC cells. Inositol Trisphosphate Receptor (IP3R)-Mediated Ca2+ Release Enhances Gyp-L-Induced ER Stress. Because the ER is a major reservoir of Ca2+, we next investigated whether Gyp-L-induced ER stress perturbed the intracellular Ca2+ homeostasis. Flow cytometry using the specific Ca2+ indicator Fluo-4 demonstrated that treatment of Gyp-L dramatically increased the intracellular Ca2+ concentration in a dosedependent manner (Figure 5A). To explore the functional significance of this increase in the Ca2+ level, a cell-permeable acetoxymethyl ester of the Ca2+ scavenger BAPTA-AM was used. The MTT assay showed that BAPTA-AM significantly inhibited Gyp-L-induced cell death (Figure 5B). Inclusion with the ER stress inhibitor TUDCA reduced the Ca2+ level (Figure 5C), further confirmed the relation between ER stress and intracellular Ca2+ homeostasis, and gave a hint that Gyp-Linduced ER stress acts as the trigger of Ca2+ mobilization from ER to cytoplasm. IP3R is the major receptor for Ca2+ release from ER to cytosol; thus, we tested the function of IP3R in the intracellular Ca2+ increment using its specific inhibitor 2-APB. As shown in Figure 5D, 2-APB remarkably reduced the level of intracellular Ca2+. Surprisingly, 2-APB also reduced Gyp-Linduced ER stress (Figure 5E). These data suggested that IP3Rmediated Ca2+ release from ER is critical for ER stress. Furthermore, Gyp-L-induced ER stress was largely inhibited by BAPTA-AM (Figure 5F), indicating that intracellular calcium homeostasis potentiates Gyp-L-induced ER stress. Gyp-L-Induced Vacuolation and Cell Death Require Protein Synthesis. Considering that the ER stress stimulates expression of genes encoding protein synthesis, we next studied
autophagy, which demonstrated that p62 accumulated even at 24 h after Gyp-L treatment (Supplemental Figure 2A of the Supporting Information), supporting the conclusion that Gyp-L inhibits autophagic flux. We further tested the role of autophagy in Gyp-L-induced vacuolation death using autophagy chemical modulators. 3Methyladenine (3-MA) is a autophagy inhibitor, and its effect on Gyp-L-induced LC3-II accumulation was first confirmed by western blot (Supplemental Figure 2B of the Supporting Information). However, as shown in panels D and E of Figure 3, Gyp-L-induced cytoplasmic vacuolation and cell death were not altered in the presence of autophagy inhibitor 3-MA or autophagy activator rapamycin, respectively. Pretreatment of 3MA also did not affect Gyp-L-induced cell death (Figure 3E). Overall, these data indicated that the Gyp-L-induced vacuolation-mediated death in HCC cells does not involve the autophagic cell death pathway. Inhibition of autophagic flux by Gyp-L might be a side effect or have some unknown influences on cell death. Gyp-L Induces Protein Ubiquitination and ER Stress. Previous studies have demonstrated that ER stress and protein ubiquitination are characteristics of cytoplasmic vacuolationmediated cell death;16,17 thus, we examined whether changes in ER stress and protein ubiquitination are involved in Gyp-Linduced cytoplasmic vacuolation death. Indeed, Gyp-L increased polyubiquitinated protein levels in a concentrationdependent manner in HepG2 and SMMC-7721 cell lines (Figure 4A). Accumulation of misfolded or unfolded proteins after Gyp-L treatment would stimulate ER stress and the subsequent activation of the unfolded protein response (UPR) pathway to initiate gene expression and recapture the ER folding function. As expected, the western blot assay showed that Gyp-L significantly enhanced several UPR-related protein expressions (Figure 4B). In addition, the ER stress inhibitor 1706
DOI: 10.1021/acs.jafc.5b05668 J. Agric. Food Chem. 2016, 64, 1702−1711
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Figure 5. ER Ca2+ release is critical for Gyp-L-induced cell death. (A) HepG2 cells were treated various concentrations of Gyp-L for 12 h and then were incubated with 5 μM Fluo-4/AM at 37 °C for 60 min. The intracellular Ca2+ level was determined by flow cytometry. At least three experiments were performed, and a representative result was shown. (B) BAPTA-AM (10 μM) reduced the cytotoxicity of Gyp-L. Data are the mean ± SD of at least three independent experiments. (∗∗∗) p < 0.005, (∗∗) p < 0.01, and (∗) p < 0.05 versus the control. HepG2 cells were treated with Gyp-L (60 μg/mL) in the presence of (C) 40 μM TUDCA or (D) 20 μM 2-APB for 12 h, and flow cytometry was used to measure the total intracellular Ca2+ levels. (E and F) 2-APB or BAPTA-AM inhibited ER stress activated by Gyp-L. ImageJ densitometric analysis of targeting protein/ GAPDH ratios from three independent experiments with (∗∗∗) p < 0.005 versus the control.
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whether the reduction in cell viability and induction of cytoplasmic vacuole formation require active protein synthesis. As expected, blockage of the biosynthesis of new proteins using cycloheximide (CHX) significantly reduced the total ubiquitinated protein levels (Figure 6A) and Gyp-L-mediated UPR pathway activation (Figure 6B). In addition, CHX significantly prevented the vacuole formation (Figure 6C) and protected cells from Gyp-L-induced cell death (Figure 6D). ROS Generation Is Critical for Gyp-L-Induced ER Stress and Cell Death. On the basis of previous reports indicating that the generation of ROS plays an important role in cytoplasmic vacuolation death,17 we next evaluated the possible involvement of ROS release in Gyp-L-induced ER stress and cell death. Fluorescence microscopy using DCF-DA showed that Gyp-L increased the ROS level, whereas ROS inhibitor NAC antagonized the effect of Gyp-L (Figure 7A). Besides, treatment with NAC reduced Gyp-L-induced ER stress (Figure 7B) and the intracellular Ca2+ level (Figure 7C), implying that ER stress acted as a downstream effector of GypL-triggered ROS production. Finally, NAC also diminished cytoplasmic vacuoles (Figure 7D) and cell death induced by Gyp-L (Figure 7E). Taken together, these findings suggested that Gyp-L induced cytoplasmic vacuolation death of human HCC cells through the ROS−ER stress pathway.
DISCUSSION
Compounds from natural plants are important sources for cancer therapeutic drug discovery. In the present work, we identified the anti-liver cancer activity of Gyp-L and clarified the underlying mechanism. Previously Gyp-L has been demonstrated to act as a potential activator of AMP-activated protein kinase,23−25 and there is litter work on the anticancer activity of Gyp-L, except a recent work by Piao et al., who simply tested the inhibitory effect of Gyp-L on lung cancer A549 cells.23 Herein, for the first time, we showed that Gyp-L dosedependently induces non-apoptotic and non-autophagic cytoplasmic vacuolation death in three HCC cell lines. Mechanically, Gyp-L initially triggered the intracellular ROS production, which, in turn, activated the UPR pathway and Ca2+ release from the ER lumen. Additionally, Gyp-L showed strong induction and activation of protein ubiquitination and inhibited autophagic flux. Thus, in the case of cancer, there appears to be a defect in their ability to eliminate Gyp-Linduced misfolded proteins. These defects ultimately led to the accumulation of misfolded protein aggregates and cytoplasmic vacuolation death. We showed here that Gyp-L-induced vacuolation death is associated with ER stress and requires new protein synthesis. Accumulation of misfolded or unfolded proteins causes ER 1707
DOI: 10.1021/acs.jafc.5b05668 J. Agric. Food Chem. 2016, 64, 1702−1711
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Figure 6. Gyp-L-induced ER stress and cell death require protein synthesis. (A and B) Western blots showing the effect of CHX on the Gyp-Linduced UPR pathway activation and ubiquitinated proteins. HepG2 and SMMC-7721 cells were treated with Gyp-L (60 μg/mL) in the presence or absence of CHX (5 μg/mL) for 12 h, and cell lysates were analyzed by western blot. (C and D) CHX reduced vacuole formation and reversed the inhibitory effect of Gyp-L on HCC cell growth.
stress are currently unknown, and the role of the ER kinase PERK should be further investigated. Interestingly, we found that Gyp-L increases the intracellular Ca2+ level and treatment with Ca2+ scavenger BAPTA-AM effectively inhibits Gyp-L-induced cytoplasmic vacuolation and cell death (Figure 5). The loss of Ca2+ homeostasis or change of Ca2+ distribution can lead to cell death,33,34 and several recent works demonstrated the involvement of Ca2+ mobilization in cytoplasmic vacuolation and paraptosis-like cell death.35−38 In addition, instant Ca2+ influx and calmodulin are required for the initiation of vacuole formation.39,40 Lysosomal Ca2+-permeable channel P2X4 recruits and forms a complex with calmodulin at the membrane to promote vacuolation in a Ca2+-dependent fashion. Indeed, Gyp-L-induced vacuoles were partially colocalized with lysosomes, further suggesting that P2X4 might play a role in vacuole formation and enlargement. Furthermore, we also simply investigated the source of Ca2+ in response to Gyp-L treatment and showed that IP3R mediates the release of Ca2+ from the ER (Figure 5D). It is not surprising when considering that ER stress is accompanied by alterations in Ca2+ homeostasis, and depletion of ER Ca2+ levels can trigger the accumulation of misfolded proteins within the ER by impairing chaperone activity and protein processing.41−43 Further detailed studies are required to precisely clarify how
stress, which, in turn, triggered the UPR pathway through three stress sensors, including IRE1, PERK, and ATF6, to transduce information regarding ER protein folding status to the nucleus, to reduce global protein synthesis, and to reestablish ER protein-folding capacity.26 The UPR acts temporarily as a cytoprotective pathway to restore ER homeostasis, whereas continued ER stress results in the failure of ER-associated degradation and ultimately cell death. In our work, overwhelming cells by Gyp-L-induced protein ubiquitination might result from the failure of chaperones to repair unfolded proteins or failure of degradation by proteasome, a process that has also been observed to be associated with ER-derived cytoplasmic vacuole formation and enhanced antitumor activity.27 It is speculated that misfolded proteins trapped within the ER could exert an osmotic force to induce an water influx from the cytoplasm and to distend the ER luminal space into vacuoles,28 which is further supported by our finding that demolishing the protein synthesis by CHX significantly attenuated ER stress and protected HCC cells from Gyp-L-induced vacuole formation and cell death (Figure 6). Recently, ROS have been reported to act as a major source of ER stress via the activation of the PERK−ATF4-mediated UPR.29−32 In our study, we also found a ROS-mediated increase in ER stress by Gyp-L (Figure 7); however, the detailed molecular events linking ROS to ER 1708
DOI: 10.1021/acs.jafc.5b05668 J. Agric. Food Chem. 2016, 64, 1702−1711
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Figure 7. ROS generation mediates Gyp-L-induced ER stress and cell death. (A) HepG2 cells were treated with Gyp-L (60 μg/mL) in the presence or absence of NAC (5 mM) for 12 h before staining with 10 μM DCF-DA for 30 min. Then, ROS generation was determined by fluorescence microscopy, and fluorescence intensity was calculated using ImageJ software. (B) HepG2 cells were incubated with Gyp-L (60 μg/mL) and NAC (5 mM) for 12 h, and cell lysates were subjected to western blot for UPR-related proteins. (C) Flow cytometry experiment showing the inhibitory effect of NAC on Gyp-L-induced ROS generation. HCC cells were treated with Gyp-L and NAC for 24 h, and (D) cytoplasmic vacuolation or (E) cell death were examined. Data are the mean ± SD of at least three independent experiments. (∗∗∗) p < 0.005, (∗∗) p < 0.01, and (∗) p < 0.05 versus the control.
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ER stress-mediated Ca2+ influx leads to the vacuole formation that contributes to Gyp-L-induced cell death. In summary, we conclude that Gyp-L may induce cell death by a ROS−ER−Ca2+ pathway associated with cytoplasmic vacuolation. Our studies provide a new pattern to develop novel drugs for anticancer therapy, especially for apoptosisresistant cancer. Additionally, an agent that can induce cytoplasmic vacuolation death, such as Gyp-L, can be used as a combinatory strategy to overcome pro-survival autophagy.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05668. Spectroscopic data of Gyp-L (Supplementary Figure 1) and (A) HepG2 and SMMC-7721 cells treated with Gyp-L (60 μg/mL) for indicated times and cell lysates subjected to western blot assay for p62 and LC3-II and (B) cells treated with Gyp-L (60 μg/mL) in the presence 1709
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or absence of 3-MA (10 μM) for 24 h and total proteins extracted and analyzed using western blot (Supplemental Figure 2) (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Telephone/Fax: +86-755-86671909. E-mail: jzhanghappy@ szu.edu.cn. *Telephone/Fax: +86-20-85223426. E-mail: twang-yf@163. com. *Telephone/Fax: +86-755-86671909. E-mail: hezhendan@126. com. Author Contributions †
Kai Zheng, Chenghui Liao, and Yan Li contributed equally to this work. Funding
This work was supported by Grants from the Shenzhen Strategic Emerging Industry Development Project Fund (ZDSYS201506031617582, SFG2013-180, KQCX20140522111508785, CXZZ20150601110000604, JCYJ20140414170821276, JCYJ20140418091413497, and CXZZ20150529165110750), the Natural Micromolecule Drug Innovation Engineering Laboratory Fund [Shenfagai (2013) 180], the China Postdoctoral Science Foundation (Grant 2015M570726), the National Natural Science Foundation of China (31500285, 31540012, and 30570421), and the Natural Science Foundation of Guangdong Province (2015A030310529 and 2015A030313558). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED HCC, hepatocellular carcinoma; ER, endoplasm reticulum; Gyp-L, gypenoside L; ROS, reactive oxygen species; MTT, 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; AO, acridine orange; UPR, unfolded protein response; 3-MA, 3-methyladenine; IP3R, inositol trisphosphate receptor; CHX, cycloheximide
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