Isoorientin Induces Apoptosis and Autophagy Simultaneously by

May 19, 2014 - autophagy and the interplay between apoptosis and autophagy in human ... activation of the apoptotic pathway in tumor cells is a major...
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Isoorientin Induces Apoptosis and Autophagy Simultaneously by Reactive Oxygen Species (ROS)-Related p53, PI3K/Akt, JNK, and p38 Signaling Pathways in HepG2 Cancer Cells Li Yuan, Shuping Wei, Jing Wang, and Xuebo Liu* Laboratory of Functional Chemistry and Nutrition of Food, College of Food Science and Engineering, Northwest Agriculture and Forestry (A&F) University, Yangling, Shaanxi 712100, People’s Republic of China ABSTRACT: Cell death is closely related to autophagy under some circumstances; however, the effect of isoorientin (ISO) on autophagy and the interplay between apoptosis and autophagy in human hepatoblastoma cancer (HepG2) cells remains poorly understood. The present study showed that ISO induced autophagy, which was correlated with the formation of autophagic vacuoles and the overexpression of Beclin-1 and LC3-II. The autophagy inhibitor 3-methyladenine (3-MA) markedly inhibited apoptosis, and the apoptosis inhibitor ZVAD-fmk also decreased ISO-induced autophagy. In addition, the PI3K/Akt inhibitor LY294002 enhanced Beclin-1, LC3-II, and poly(ADP-ribose) polymerase (PARP) cleavage levels. Also, the reactive oxygen species (ROS) inhibitor N-acetyl-L-cysteine (NAC), the JNK inhibitor SP600125, and the p38 inhibitor SB203580 efficiently downregulated the levels of these proteins. Moreover, the p53 inhibitor pifithrin-α and the nuclear factor (NF)-κB inhibitor pyrrolidinedithiocarbamic acid (PDTC) clearly suppressed Beclin-1 and LC3-II and increased cytochrome c release, caspase-3 activation, and PARP cleavage. These results demonstrated for the first time that ISO simultaneously induced apoptosis and autophagy by ROS-related p53, PI3K/Akt, JNK, and p38 signaling pathways. Furthermore, ISO-induced apoptosis by activating the Fas receptor-mediated apoptotic pathway and suppressing the p53 and PI3K/Akt-dependent NF-κB signaling pathway, with the subsequent increase in the release of cytochrome c, caspase-3 activation, and PARP cleavage. KEYWORDS: isoorientin, apoptosis, autophagy, ROS, interplay

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INTRODUCTION Apoptosis or type I programmed cell death involves the activation of catabolic enzymes, leading eventually to nuclear chromatin condensation, nuclear fragmentation, and formation of distinct apoptotic bodies.1 Apoptosis is driven by two major pathways: the cell death receptor-mediated (extrinsic) apoptotic pathway and the mitochondrial-mediated (intrinsic) apoptotic pathway, both of which are dependent upon the activation of caspases.2 Extensive evidence suggests that the activation of the apoptotic pathway in tumor cells is a major protective mechanism against the development and progression of cancer.3 Autophagy is known as type II programmed cell death; it involves membrane trafficking and causes the degradation of cytosolic proteins and organelles by lysosomes. In contrast to apoptosis, caspases are not activated during autophagic cell death; DNA degradation and nuclear fragmentation are also not apparent during autophagy. During autophagic cell death, organelles, such as mitochondria, the Golgi apparatus, polyribosomes, and the endoplasmic reticulum, are degraded before nuclear fragmentation occurs; in contrast, these organelles are preserved during apoptosis.4,5 Autophagy has pro-death or pro-survival functions depending upon the context. Under normal physiological conditions, autophagy is able to maintain cell homeostasis through eliminating superfluous, damaged, or aged cells or organelles and providing cells with building blocks and energy.6 On the other hand, beyond this homeostatic function, extensive autophagy results in cell death via the bulk elimination of cells. It has been reported that some agents exert their anticancer effects by regulating © 2014 American Chemical Society

autophagy; thus, there is potential for new drugs that modulate autophagy to be useful for anticancer treatment.7 Recently, the interplay between apoptosis and autophagy has received great attention. The molecular connections between apoptosis and autophagy are multifaceted, complex, and poorly understood currently. Apoptosis and autophagy may be triggered by common upstream signals that result in combined autophagy and apoptosis; in other instances, the cell switches between the two responses in a mutually exclusive manner.8 Isoorientin (3′,4′,5,7-tetrahydroxy-6-C-glucopyranosyl flavone, ISO) is a naturally occurring C-glycosyl flavone (Figure 1A) and is found in many plant species, such as Phyllostachys pubescens,9 Crataegus pentagyna,10 Patrinia,11 Fagopyrum esculentum,12 Drosophyllum lusitanicum,13 Arum palaestinum,14 Gentiana,15 Rumex, and Swertia.16 ISO is able to significantly reduce the proliferation of human hepatoblastoma cancer (HepG2) cells,17 and in our previous study, we found that ISO decreases HepG2 cell viability in a dose- and time-dependent manner. Also, ISO induces apoptosis in HepG2 cells by causing mitochondrial dysfunction, inhibiting the PI3K/Akt signaling pathway, and increasing the phosphorylation of JNK and p38 kinases.18,19 However, the effects of ISO on autophagy and the interplays between apoptosis and autophagy in HepG2 cells remain unknown. Received: Revised: Accepted: Published: 5390

February 25, 2014 May 19, 2014 May 19, 2014 May 19, 2014 dx.doi.org/10.1021/jf500903g | J. Agric. Food Chem. 2014, 62, 5390−5400

Journal of Agricultural and Food Chemistry

Article

Figure 1. ISO induces apoptosis in HepG2 cells. (A) Chemical structure of ISO. (B) HepG2 cells were treated with ISO at the indicated concentrations for 24 h. The levels of TNF-α in the supernatant was measured using an ELISA assay kit, the results are shown as the mean ± standard deviation (SD) of nine separate experiments. Representative immunoblots for (C) Fas, FasL, caspase-8, and Bid and (D) p53, IκB, p-IκB, and NF-κB are shown. α-Tubulin and lamin B were used as internal controls. Measurement of Cell Viability. Cell viability was determined using the MTT assay. Cells were seeded at a density of 1 × 106 cells/ mL in 96-well polystyrene culture plates at 37 °C with 5% (v/v) CO2 for 1 day. After 24 h of incubation, 100 μL of medium was removed from each well and 100 μL aliquots of different concentrations of ISO were added to the cells and incubated for 24 h. Subsequently, 100 μL of 0.5% (w/v) MTT dissolved in phosphate-buffered saline was added to each well and incubated for 4 h. The formazan crystals formed by live cells were solubilized with 100 μL of DMSO, and absorbance at 490 nm was measured with a microplate reader (Bio-Rad Laboratories, Ltd., China). Cell viability was expressed as a percentage of the control (untreated cells). Measurement of TNF-α. Cells were treated with ISO for 24 h. The levels of TNF-α in the culture medium were measured using an ELISA kit according to the instructions of the manufacturer. Detection of Autophagic Vacuoles by AO. Autophagy is the process of sequestering cytoplasmic proteins into the lytic component and is characterized by the formation and promotion of acidic vesicular organelles, as described previously. AO was always used to detect the acidic cellular compartment, which emits bright red fluorescence in acidic autophagic vacuole vesicles, but green fluoresces in the cytoplasm and nucleus. After treatment, cells were incubated with 1 μg/mL AO for 15 min. Then, the AO was removed and observed using a fluorescence microscopy (Olympus Optical Co., Ltd., Tokyo, Japan). Observation and Quantification of Monodansylcadaverine (MDC) Staining. MDC staining was introduced to analyze autophagy induction in cells. After treatment, cells were rinsed with phosphatebuffered saline (PBS) and stained with 50 m/L MDC at 37 °C for 1 h. Finally, cells were washed with PBS, and the cellular fluorescence changes were observed using fluorescence microscopy (Olympus Optical Co., Ltd., Tokyo, Japan), and the MDC fluorescent intensity changes were determined by the microplate reader with excitation light at 488 nm and emission light at 530 and 650 nm (Molecular Devices Co., Sunnyvale, CA). Transmission Electron Microscopy (TEM). HepG2 cells were treated with ISO, and then the cell pellets were fixed with a 0.1 M PBS solution containing 2.5% glutaraldehyde for 12 h. They were then washed with 0.1 M PBS, embedded in 2% agarose gel, and postfixed in 4% osmium tetroxide solution for 1 h. Next, the examples were

In the present study, we investigated the mechanisms by which ISO affects autophagy in HepG2 cells and demonstrated the interplays between apoptosis and autophagy induced by ISO in HepG2 cells.

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MATERIALS AND METHODS

Reagents and Antibodies. ISO (purity of ≥98%) was purchased from Forever Biotechnology, Ltd. (Shanghai, China). RPMI-1640 cell cultures, fetal bovine serum (FBS), and the BCA protein kit were purchased from Thermo Fisher (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliunbromide (MTT) (purity of ≥93%) was obtained from Wolsen Biotechnology, Ltd. (Xian, China). NAcetyl-L-cysteine (NAC) (purity of ≥99%), pyrrolidinedithiocarbamic acid (purity of ≥99%), 3-methyladenine, rapamycin (purity of ≥95%), and pifithrin-α (purity of ≥95%) were purchased from Sigma. Acridine orange (AO), LY294002 (purity of ≥98%), and ZVAD-fmk were provided by Beyotime Institute of Biotechnology (Jiangsu, China). Antibodies against Bcl-2 (SC-492), Bax (SC-493), cytochrome c (SC-7159), caspase-8 (SC-56070), FasL (SC-6237), α-tubulin (SC5286), lamin B (SC-6216), and horseradish-peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Beclin-1 (1353R) and cleaved caspase-3 (p17) (BS7004) were obtained from Abcam (Hong Kong) and Bioworld Technology, Inc. (Louis Park, MN). Nuclear factor (NF)-κB p65 (8242), IκB (9242), p-IκB (Ser32/36) (9246), poly(ADP-ribose) polymerase (PARP) (9542), LC3A/B (4108), p53 (9282), Fas (8023), p-Akt (Ser473) (9271), and Akt (9272) polyclonal antibodies were purchased from Cell Signaling Technology Company (Shanghai, China). An enzyme-linked immunosorbent assay (ELISA) kit for TNF-α was obtained from the Xinle Biology Technology (Shanghai, China). All other chemicals made in China were of analytical grade. Cell Culture and Treatment. Human hepatoblastoma HepG2 cells were obtained from the Fourth Military Medical University (Xian, China) and cultured in RPMI-1640 medium with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified incubator (5% CO2 and 95% air). For the ISO treatment, ISO was dissolved in dimethyl sulfoxide (DMSO) and further diluted to final concentrations with serum-free culture medium. 5391

dx.doi.org/10.1021/jf500903g | J. Agric. Food Chem. 2014, 62, 5390−5400

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Figure 2. ISO induces autophagy in HepG2 cells. (A) Cells were seeded at a density of 1 × 105 cells/mL in 96-well polystyrene culture plates at 37 °C with 5% (v/v) CO2 for 1 day. Then, cells were incubated with variable concentrations of ISO for 24 h, then exposed to AO and MDC, and observed by fluorescent microscopy. Arrows indicated the cells containing autophagic vacuoles. (B) Cells were incubated with variable concentrations of ISO for 12, 24, and 48 h, respectively, and were determined by the microplate reader of MDC staining at the emission wavelength of 525 nm. The results are shown as the mean ± SD of nine separate experiments. (∗∗) p < 0.01 versus the corresponding control. (C) TEM shows the ultrastructure of cells treated with 80 μM ISO for 24 h. Arrows indicate autophagosomes, including residual digested material and empty vacuoles. (D) Cells were incubated with variable concentrations of ISO for 24 h, and (E) cells were treated with ISO at 80 μM for 0, 6, 12, 24, and 48 h before collection and lysis, respectively. Beclin-1 and LC3 were assessed by western blot analysis, and α-tubulin was used as an internal control. seeding density of 1 × 104 cells/mL and cultured with 2 mL of cell culture medium overnight. After treatment, cells were washed with PBS twice, and then cells were incubated with 5 μL of 100 μg/mL AO−EB at room temperature in the dark, followed by observation under a fluorescence microscope. Cells were stained with 1 mg/mL Hoechst 33258 for 30 min at 37 °C and observed using a fluorescence microscope (Olympus Optical Co., Ltd., Tokyo, Japan). Sodium Dodecyl Sulfate (SDS)−Polyacrylamide Gel Electrophoresis (PAGE) and Western Blot Analysis. After the cells were subjected to the indicated treatments, they were harvested and lysed with cell lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) and nuclear extraction reagent (Xianfeng Biotechnology, Xian, China) as cytosolic extract (cytosol) and nuclear extract (nucleus), respectively. The total protein concentration was determined using the BCA protein kit (Thermo Fisher, Shanghai, China). The homogenates were treated with SDS sample buffer and then immediately heated at 95 °C for 10 min. The proteins were separated by SDS−PAGE and electrotransferred onto a polyvinylidene fluoride (PVDF) membrane (0.45 μm, Millipore) using a semi-dry transfer apparatus (Bio-Rad, Shanghai, China). Blocking was carried out for 2 h in 5% nonfat dry milk in TBST (20 mM Tris, 166 mM NaCl, and 0.05% Tween 20 at pH 7.5). Then, the membrane was washed with TBST for 15 min. The primary antibodies were added at the manufacturer-recommended dilutions in TBST buffer overnight at 4 °C. Secondary antibodies were added and incubated at 25 °C for 2 h. The blots were subjected to another three washes with TBST, developed with a chemiluminescent substrate (Thermo Fisher, China), and exposed using a Molecular Imager ChemiDoc XRS system (Bio-Rad, Shanghai, China). Statistical Analysis. All experiments were performed 3 times, and the data are presented as the mean ± standard error (SE). Significant differences between measurements for the control and treated samples were analyzed using one-way factorial analysis of variance (ANOVA), followed by Duncan’s post-hoc test (SPSS 16.0).

desiccated with a range of ethanol concentrations and embedded in epoxy resin. The resin was polymerized at 30 °C for 24 h and 60 °C for 48 h, respectively. Ultrathin sections obtained with an ultramicrotome were stained with uranyl acetate and lead citrate and examined under TEM (HT7700, Hitachi, Japan). Measurement of Mitochondrial Membrane Potential (MMP) by JC-1. The MMP was measured using the mitochondria-specific lipophilic cationic fluorescence dye JC-1. As a monomer, JC-1 is capable of selectively entering the mitochondria. Under normal conditions, JC-1 aggregates within the mitochondria and emits red fluorescence, but when the MMP collapses during apoptosis, JC-1 emits green fluorescence. The ratio of red/green JC-1 fluorescence reflects the change in MMP. Cells were seeded into 96-well polystyrene culture plates. After treatment, medium was removed from each well and 100 μL of 5 μg/mL JC-1 was added to the cells for 1 h. Finally, cells were washed twice with PBS and then quantitatively analyzed by a microplate reader (Molecular Devices Co., Sunnyvale, CA). Detection of Intracellular Reactive Oxygen Species (ROS) Production. Cellular ROS was measured with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Sigma). This dye is a stable nonpolar compound that diffuses readily into cells and yields DCFH. Intracellular ROS in the presence of peroxidase changes DCFH to the highly fluorescent compound DCF. Thus, the fluorescent intensity is proportional to the amount of ROS produced by the cells. After treatment, 10 μM H2DCFDA was added to the wells for 30 min at 37 °C. Then, cells were washed twice with PBS, lysed in cell lysates, and centrifuged at 15000g for 10 min at 4 °C. The DCF fluorescence intensity of the supernatant was measured via a fluorescence microplate reader at 485 nm excitation and 535 nm emission. Cellular ROS levels were expressed as relative DCF fluorescence per microgram of protein.20 AO−Ethidium Bromide (EB) and Hoechst 33258 Staining Assay. Cells were seeded into 35 mm polystyrene culture dishes at a 5392

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Figure 3. ISO induces autophagic cell death in HepG2 cells. (A) Cells were incubated with ISO at the indicated concentrations for 24 h and then processed for MTT assay. (B) Representative immunoblots for Beclin-1 and LC3 are shown. (C) Cells were incubated with variable concentrations of ISO for 24 h in the presence of 3-MA or RAP, respectively, and then processed for the MTT assay. The results are shown as the mean ± SD of nine separate experiments. (∗∗) p < 0.01 and (∗) p < 0.05 versus the corresponding control. (D) Cells were pretreated with 3-MA or RAP prior to exposure to ISO, respectively. Beclin-1 and LC3 were assessed by western blot analysis. α-Tubulin was used as an internal control.

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untreated cells. Furthermore, the fluorescence intensity of MDC was significantly (p < 0.01) enhanced by ISO in a doseand time-dependent manner and reached its maximum intensity at an ISO dose of 80 μM for 24 h (Figure 2B). A TEM assay confirmed that numerous autophagic vacuoles and empty vacuoles were present in the HepG2 cells treated with 80 μM ISO (Figure 2C). Additionally, the expression level of Beclin-1 and the conversion of LC3-I to LC3-II, both of which indicate autophagy, were increased after ISO treatment in a dose- and time-dependent manner and reached their maximum levels at an ISO dose of 80 μM for 24 h (panels C and D of Figure 2). Taken together, the results suggest that ISO induces autophagy in HepG2 cells. Effect of Autophagy on ISO-Induced Cell Death in HepG2 Cells. A significant inhibition in cell viability was observed in the ISO high-concentration groups (10, 20, and 40 μM), while no significant effects were observed in the lower concentration groups (0.1 and 1 μM) (Figure 3A), and the IC50 of ISO was 71.77 μM. There were also no significant changes in the protein expression of Beclin-1 and LC3-II in cells treated with ISO at 0.1 and 1 μM (Figure 3B). More importantly, the effects of the autophagy inhibitor 3-methyladenine (3-MA) and the autophagy activator rapamycin (RAP) on cell viability were also examined (Figure 3C); the results showed that 3-MA significantly (p < 0.05) increased cell viability and weakened the inhibitory effect of ISO on cell viability. However, RAP notably (p < 0.01) decreased cell viability and strengthened the inhibitory effect of ISO on cell viability. Additionally, 3-MA diminished the expression levels of Beclin-1 and LC3-II, and RAP increased their expression in HepG2 cells (Figure 3D). These results indicated that ISO induced autophagic cell death to decrease cell viability of HepG2 cells.

RESULTS Effect of ISO on Apoptosis in HepG2 Cells. Apoptosis includes two major pathways: the cell-death-receptor-mediated (extrinsic) apoptotic pathway and the mitochondrial-mediated (intrinsic) apoptotic pathway. We found that ISO induces apoptosis in HepG2 cells through the mitochondrial-mediated (intrinsic) apoptotic pathway. In the present study, we examined the effect of ISO on the cell-death-receptor-mediated (extrinsic) apoptotic pathway. As shown in Figure 1B, ISO increased the levels of TNF-α in HepG2 cells in a dosedependent manner, but there was no significant difference in the TNF-α levels between the ISO-treated group and control group. ISO also notably increased the protein expression of Fas and FasL, decreased caspase-8 levels, and eventually caused the cleavage of Bid, increasing the protein levels of t-Bid (Figure 1C). In addition, we also found that ISO markedly increased p53 levels and the protein expression of NF-κB in the cytosol and decreased the phosphorylation of IκB and the level of NFκB in the nucleus (Figure 1D). These results indicated that ISO induced apoptosis in HepG2 cells by activating the Fas receptor-mediated apoptotic pathway, increasing p53 levels and blocking the nuclear translocation of NF-κB. Effect of ISO on Autophagy in HepG2 Cells. To investigate whether ISO induces autophagy in HepG2 cells, we examined the formation of autophagic vacuoles using the specific fluorescent dyes AO and MDC. Figure 2A shows predominantly green fluorescence in the control cells and considerable red fluorescence in a dose-dependent manner in the cells treated with ISO. ISO treatment also resulted in an increase in the accumulation of MDC, indicating an increased formation of the MDC-labeled vacuoles compared to the 5393

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Figure 4. Effect of autophagy inhibitor and activator on ISO-induced apoptosis. (A) Cells were incubated with variable concentrations of ISO for 24 h in the presence of 3-MA or RAP, respectively. Then, cells were stained with JC-1 at 37 °C for 1 h. The fluorescence intensity of JC-1 was detected by a multifunctional microplate reader. The results are expressed as the mean ± SD of nine separate experiments. (∗∗) p < 0.01 and (∗) p < 0.05 versus the corresponding control. Representative image of immunoblots for (B) autophagic marker proteins and (C) Akt and p-Akt. α-Tubulin was used as an internal control.

Figure 5. Effect of the apoptosis inhibitor on ISO-induced autophagy in HepG2 cells. (A) Cells were incubated with ISO for 48 h in the presence of ZVAD-fmk and then processed for the MTT assay. The results are shown as the mean ± SD of nine separate experiments. (∗∗) p < 0.01 versus other treatments and (##) p < 0.01 versus the ISO treatment group. (B) Cells were incubated with ISO for 24 h in the presence of ZVAD-fmk, and then cells were exposed to AO−EB or Hoechst 33258. Green cells were counted as live cells; yellow cells were counted as early apoptotic cells; and orange cells were counted as late apoptotic cells. Chromatin condensation and nuclear fragmentation were considered to indicate apoptotic cells. Scale bar = 50 μm. (C) Caspase-8, cleaved caspase-3, and PARP and (D) Beclin-1 and LC3 were assessed by western blot analysis. NADPH was used as an internal control.

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Figure 6. Effect of ROS on ISO-induced autophagy and NF-κB signaling pathway. (A) After treatment, cells were stained with H2DCFDA for 30 min prior to detection using a fluorescence microplate reader. The results are expressed as the mean ± SD of nine separate experiments. (∗∗) p < 0.01 versus the control group. (B) Beclin-1 and LC3 were assessed by western blot analysis. (C) Protein expressions of p53, caspase-8, and PARP were analyzed by western blotting. (D) Protein expressions of the NF-κB signaling pathway were analyzed by western blotting. α-Tubulin and lamin B were used as an internal control.

A complicated interplay exists between autophagy and apoptosis. To demonstrate whether ISO-triggered autophagy promotes or inhibits apoptosis, we examined apoptotic markers after treatment with the autophagy inhibitor 3-MA and the autophagy activator RAP. As shown in Figure 4A, 3-MA significantly (p < 0.01) attenuated the MMP collapse induced by ISO, while RAP increased MMP levels. In addition, in comparison to ISO alone, 3-MA markedly inhibited the expression of Bax, cytochrome c (in the cytoplasm), cleaved caspase-3, and PARP and increased the levels of Bcl-2 and caspase-8 in the presence of ISO (Figure 4B). In contrast, RAP notably enhanced the effects of ISO on the expression of these proteins. Furthermore, 3-MA significantly increased the phosphorylation of Akt. RAP strongly suppressed the phosphorylation of Akt, not only by itself but also in the presence of ISO (Figure 4C). These results suggest that the suppression of autophagy diminished the ISO-induced apoptosis in HepG2 cells. To confirm these results, we further investigated the effect of the apoptosis inhibitor ZVAD-fmk on HepG2 cells. As shown in Figure 5A, the cell viability inhibition caused by ISO was strongly (p < 0.01) decreased in the presence of ZVAD-fmk and ZVAD-fmk alone did not have any cytotoxic effects. The fluorescence microscopy showed that the typical characteristics of apoptosis, such as cell shrinkage, chromatin condensation, and EB signals, were obviously attenuated in the cells treated with ZVAD-fmk and that ZVAD-fmk strongly reversed the apoptosis-inducing effect of ISO (Figure 5B). Other apoptotic hallmarks, including caspase-8, cleaved caspase-3, and PARP cleavage, were also suppressed by ZVAD-fmk in ISO-treated cells (Figure 5C). Additionally, ZVAD-fmk markedly decreased Beclin-1 and LC3-II levels, which had been increased by ISO, indicating that the inhibition of apoptosis blocks autophagy (Figure 5D). Taken together, these results suggest that ISO

decreased cell viability simultaneously by inducing apoptosis and autophagy in HepG2 cells. Effect of ISO-Stimulated ROS on Apoptosis and Autophagy. The molecular crosstalk between apoptosis and autophagy processes is complex, and they both may be triggered by common upstream signals; therefore, we next determined which interactions are demonstrated in the apoptosis and autophagy in HepG2 cells. Accumulating evidence indicates that intracellular ROS can trigger apoptosis and autophagy. We examined whether ROS stimulated by ISO induced apoptosis and autophagy in HepG2 cells. The ROS inhibitor NAC markedly (p < 0.01) decreased ROS generation, not only by itself but also in the presence of ISO (Figure 6A). NAC also significantly suppressed the protein expression of Beclin-1 and LC3-II and strongly reduced the effects of ISO on the expression of those proteins (Figure 6B). This suggests that a higher level of ROS may directly initiate autophagy. Similarly, a significant decrease in p53 and PARP cleavage and a increase in caspase-8, p-IκB, and NF-κB p65 (in the nucleus) were observed in NAC-treated cells (panels C and D of Figure 6). Taken together, these results showed that ROS is involved in the p53 signaling pathway, the phosphorylation of IκB, and the nuclear translocation of NF-κB as an upstream signal and that intracellular ROS controls apoptosis and autophagy induced by ISO. Effect of the PI3K/Akt Signaling Pathway on Apoptosis and Autophagy. The PI3K/Akt signaling pathway is involved in the regulation of the cell cycle, growth, survival, metabolism, and tumorigenesis. In a previous study, we found that ISO induced apoptosis by inhibiting the PI3K/Akt pathway in HepG2 cells. To investigate the effect of the PI3K/ Akt pathway on the crosstalk between the processes of apoptosis and autophagy, cells were pretreated with the PI3K/Akt inhibitor LY294002 prior to being exposed to ISO. 5395

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Figure 7. Effect of PI3K/Akt on ISO-induced autophagy and NF-κB. (A) Cells were incubated with 20 μM PI3K/Akt inhibitor LY294002 in the presence or absence of ISO. Beclin-1 and LC3 were assessed by western blot analysis. (B) Representative image of immunoblots for caspase-8 and PARP. (C) Protein expressions of NF-κB signaling pathway were analyzed by western blotting. α-Tubulin and lamin B were used as an internal control. The graph shows the levels of NF-κB in cytoplasm and nucleus. The densitometric analysis shows the mean ± SD of four independent experiments. (∗∗) p < 0.05 and (##) p < 0.05 versus the control of cytoplasm and nucleus, repectively.

Figure 8. Effect of the MAPK signaling pathway on ISO-induced autophagy and apoptosis. Cells were treated with 5 μM U0126 (an ERK1/2 inhibitor), 10 μM SP600125 (a JNK inhibitor), or 10 μM SB203580 (a p38 inhibitor) for 30 min prior to exposure to ISO (80 μM) for 24 h. Representative images of immunoblots for (A) Beclin-1 and LC3 and (B) caspase-8 and PARP. α-Tubulin was used as an internal control.

and SB203580 decreased the levels of Beclin-1 and LC3-II individually and in the presence of ISO and weakened the induced effect of ISO on the protein expression of Beclin-1 and LC3-II. It was interesting that the significant increase in Beclin1 and LC3-II levels was observed in cells that were treated with U0126 alone, while U0126 inhibited the effect of ISO on Beclin-1 and LC3-II levels. In addition, U0126 markedly increased cleaved caspase-8 levels and PARP cleavage in the absence of ISO but did not change the activation effects of ISO on those protein levels. In contrast, SP600125 and SB203580 notably inhibited the levels of PARP cleavage, increased the protein expression of caspase-8, and reversed the activation effects of ISO on those protein levels (Figure 8B). Taken together, these results indicate that ISO induced apoptosis and autophagy by activating JNK and p38.

In comparison to the control group, LY294004 markedly increased the protein expression of Beclin-1 and LC3-II, not only by itself but also in the presence of ISO (Figure 7A). Additionally, LY294004 significantly decreased caspase-8 levels and increased PARP cleavage levels in both the presence and absence of ISO (Figure 7B). Also, LY294004 obviously inhibited the phosphorylation of IκB and the nuclear translocation of NF-κB (Figure 7C). These data indicate that ISO induced apoptosis and autophagy simultaneously by inhibiting the PI3K/Akt signaling pathway. Effect of the MAPK Signaling Pathway on ISOInduced Apoptosis and Autophagy. To investigate the effect of the MAPK pathway on apoptosis and autophagy, cells were pretreated with U0126 (an ERK1/2 inhibitor), SP600125 (a JNK inhibitor), and SB203580 (a p38 inhibitor) prior to being exposed to ISO. As shown in Figure 8A, both SP600125 5396

dx.doi.org/10.1021/jf500903g | J. Agric. Food Chem. 2014, 62, 5390−5400

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Figure 9. Effect of p53 on ISO-induced autophagy, apoptosis, and NF-κB. Cells were treated with 20 μM pifithrin-α (p53 inhibitor) for 30 min prior to exposure to ISO (80 μM) for 24 h. (A) Representative images of immunoblots for Beclin-1 and LC3 and (B) protein expressions of the NF-κB signaling pathway. (C) Apoptotic marker proteins were analyzed by western blotting. α-Tubulin was used as an internal control. The graph shows the ratio of Bax/Bcl-2 values. The densitometric analysis shows the mean ± SD of four independent experiments. (∗∗) p < 0.05 versus the control.

Figure 10. Effect of NF-κB on ISO-induced autophagy and apoptosis. Cells were treated with 50 μM PDTC (NF-κB inhibitor) for 30 min prior to exposure to ISO (80 μM) for 24 h. (A) Protein expressions of NF-κB signaling pathway were analyzed by Western blotting. (B) Representative images of immunoblots for Beclin-1 and LC3 and (C) apoptotic marker proteins. α-Tubulin was used as an internal control. The graph shows the relative expression of cleaved caspase-3. The densitometric analysis shows the mean ± SD of four independent experiments. (∗∗) p < 0.05 versus the control.

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Effect of p53 on ISO-Induced Apoptosis and Autophagy. The effect of p53 on autophagy depends upon the cellular context. To determine the effects of p53 on autophagy and apoptosis, cells were exposed to the p53 inhibitor pifithrin-α prior to treatment with ISO. As shown in Figure 9A, pifithrin-α significantly inhibited the protein expression of p53, Beclin-1, and LC3-II and reversed the effects of ISO on p53, Beclin-1, and LC3-II (Figure 9A). This suggests that p53 promotes ISO-induced autophagy in HepG2 cells. In contrast, pifithrin-α upregulated the phosphorylation of IκB and the protein expression of NF-κB in the nuclear fraction of the cells (Figure 9B), indicating that p53 is involved in the activation of NF-κB as an upstream signaling molecule. Additionally, the upregulation of the Bax/Bcl-2 ratio, the release of cytochrome c into the cytosol, and an increase in the levels of cleaved caspase-3 and PARP cleavage were observed in pifithrin-α-treated cells in both the absence and presence of ISO (Figure 9C). Taken together, these data indicated that the activation of p53 induced apoptosis and autophagy in HepG2 cells. Effect of NF-κB Activation on ISO-Induced Apoptosis and Autophagy. Accumulating evidence indicates that the activation of NF-κB inhibits apoptosis, activates autophagy, and regulates cell survival pathways. To confirm this evidence, cells were treated with the NF-κB inhibitor pyrrolidinedithiocarbamic acid (PDTC) prior to being exposed to ISO. PDTC notably decreased the phosphorylation of IκB and the protein levels of NF-κB in the nuclear fraction of the cells (Figure 10A). A significant decrease in the expression of Beclin-1 and LC3-II was also observed in cells treated with PDTC alone and in cells treated with PDTC and ISO (Figure 10B). In contrast, PDTC alone and with ISO caused a strong upregulation of cytochrome c (in the cytosolic fraction), cleaved caspase-3, and PARP cleavage levels, and PDTC enhanced the effects of ISO on these protein levels (Figure 10C). These results suggest that the inhibition of NF-κB suppresses autophagy and promotes apoptosis.

inflammation, processes that are induced by oncogenes; thus, autophagy eventually limits tumor growth.22 A low level of constitutive autophagy has an important housekeeping role in cellular metabolism and is crucial for maintaining healthy cells. However, excessive autophagy or the activation of autophagy in a specific context has been implicated in autophagic cell death and may be harmful.23 Numerous studies have revealed that autophagy is linked to cell death under certain circumstances. For example, the upregulation of autophagy contributes to the progress of cadmium-induced nephrotoxicity.24 Also, our results showed that ISO decreased cell viability by inducing autophagic cell death in HepG2 cells (Figure 3). Both apoptosis and autophagy are essential cellular degradation pathways that are often induced by similar stimuli and regulated by similar pathways. However, the relationship between apoptosis and autophagy is multifaceted, complex, and controversial. In some cellular settings, autophagy is necessary for apoptosis to occur. In other cellular settings, autophagy suppresses or delays apoptosis as a stress adaptation, and in some cell systems, the two processes occur independently.8,25,26 In summary, autophagy and apoptosis can cooperate, antagonize, or assist each other to differentially influence the fate of the cell. It has been repotred that vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cell,4 and the survivin suppressant YM155 inhibits growth in both PC3 and LNCaP prostate cancer cells by inducing the autophagydependent apoptosis.27 In the present study, the autophagy inhibitor 3-MA markedly inhibited ISO-induced apoptosis, and the apoptosis inhibitor ZVAD-fmk significantly decreased ISOinduced autophagy (Figures 4 and 5), suggesting that apoptosis and autophagy could exist simultaneously and work cooperatively in ISO-treated HepG2 cells. ROS regulates a number of cellular pathways and, thus, plays critical roles in determining the fate of cells.28 Accumulating evidence has revealed that autophagy can be stimulated in response to ROS injury in cancer cells, and ROS may have synergetic effects in apoptosis and autophagy.29 Consistent with previous studies, we also found that both the autophagic markers (Beclin-1 and LC3-II) and apoptotic markers (caspase8 and PARP) were suppressed by the ROS inhibitor NAC in ISO-treated cells (panels B and C of Figure 6). Our previous research showed that ROS is an upstream signal involved in the effects of ISO on the PI3K/Akt and MAPK signaling pathways, which impact apoptosis.18,19 Numerous studies have demonstrated that autophagic cell death is triggered by the MAPK and PI3K/Akt pathways.30−32 We further investigated the roles of the PI3K/Akt and MAPK pathways on the interactions of the apoptosis and autophagy processes. The results showed that the PI3K/Akt inhibitor LY294004 markedly increased autophagy; similarly, LY294004 notably promoted ISO-induced apoptosis (Figure 7), indicating that ISO simultaneously induced apoptosis and autophagy by inhibiting the PI3K/Akt signaling pathway. The mechanism behind this is the inhibition of the phosphorylation of mTOR by LY294002, resulting in the phosphorylation of autophagy protein-13 and the suppression of autophagy.1 LY294002 also inhibits the phosphorylation of pro-apoptotic proteins, such as Bad, Bax, and caspase-9.33,34 In addition, SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) obviously decreased autophagy that was induced by ISO (Figure 8A), and both SP600125 and SB203580 significantly inhibited ISO-induced apoptosis (Figure 8B). The effects of JNK and p38 on apoptosis and autophagy were consistent, because JNK and p38 kinases modulate the

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DISCUSSION ISO is a naturally occurring C-glycosyl flavone and has been reported to reduce the proliferation of HepG2 cells, with no significant cytotoxicity on human liver cells (HL-7702) and buffalo rat liver cells (BRL-3A).18 The present study showed that ISO induced cell death by activating both apoptosis and autophagy, which coexist and promote each other in HepG2 cells. ISO was able to induce apoptosis by activating the Fas receptor-mediated apoptotic pathway and suppressing the p53 and PI3K/Akt-dependent NF-κB signaling pathway. Also, we found that ROS-mediated p53, PI3K/Akt, JNK, and p38 were the common upstream signals that simultaneously triggered apoptosis and autophagy. Apoptosis is a programmed cell death process, and a multitude of studies have suggested that the activation of apoptosis in tumor cells is an effective form of chemotherapy designed to hinder the development and progression of cancer.3 Autophagy is another programmed cell death process, and it can have two effects on tumors. First, by removing damaged organelles, regenerating metabolic precursors, and reducing chromosomal instability, it aids the survival of cancer cells in a low-nutrient environment and increases the resistance of cancer cells to anticancer treatments preventing tumor suppression.21 On the other hand, autophagy prevents tumor transformation and facilitates senescence, necrosis, and 5398

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expression and translocation of Bcl-2 and Bax in apoptotic cells.35,36 JNK is able to trigger autophagy by targeting Bcl2/ BclXL proteins and abrogating their binding to Beclin-1,37 and p38 catalyzes the phosphorylation of mTOR.38 Furthermore, we found that the ROS inhibitor NAC also reversed the inhibitory effect of ISO on the protein expression of p53 and the nuclear translocation of NF-κB (panels C and D of Figure 6). The tumor suppressor p53 plays a major role in cell cycle arrest and/or apoptosis during the response of mammalian cells to stress, and p53 mutations and functional inactivation are linked to the pathology of more than half of all cancers.39,40 Furthermore, p53 can both promote and inhibit autophagy, depending upon the cellular conditions.8 In the current study, a p53 inhibitor promoted the induction of apoptosis by ISO and NF-κB nuclear translocation and inhibited autophagy (Figure 9). The transcription factor NFκB is involved in the regulation of survival, death, differentiation, and migration.41 The present study has shown that the NF-κB inhibitor PDTC efficiently suppressed ISO-induced autophagy and enhanced ISO-induced apoptosis (Figure 10). The results from several other studies also indicated that p53 and NF-κB promote apoptosis over autophagy.42,43 In summary, this study demonstrated that ISO simultaneously induced apoptosis and autophagy by inhibiting the ROS-related PI3K/Akt signaling pathway and activating the ROS-related JNK and p38 pathways. Moreover, ISO induced apoptosis by activating the Fas receptor-mediated apoptotic pathway and suppressing the p53 and PI3K/Akt-dependent NF-κB signaling pathway, with the subsequent increase in the release of cytochrome c, caspase-3 activation, and PARP cleavage. A schematic diagram of these observed effects of ISO is shown in Figure 11. These findings indicate that ISO inhibited cell viability by both apoptosis and autophagy and that autophagy induction by ISO may be a novel therapeutic method for the treatment of cancer.

Article

AUTHOR INFORMATION

Corresponding Author

*Telephone: 86-029-87092325. Fax: 86-029-87092817. E-mail: [email protected]. Funding

This work was financially supported by the National Natural Science Foundation of China (Grant 31271810) and the National “Twelfth Five-Year” Plan for Science and Technology Support (2012BAH30F03). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED HepG2 cells, human hepatoblastoma cancer cell line; ISO, isoorientin; PARP, poly(ADP-ribose) polymerase; ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine; PDTC, pyrrolidinedithiocarbamic acid; 3-MA, 3-methyladenine; RAP, rapamycin

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Figure 11. Possible molecular interplays between apoptosis and autophagy induced by ISO in HepG2 cells. → indicates activation or induction; ⊥ indicates inhibition or blockade; and ↔ indicates mutual promotion. 5399

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