Dig Dis Sci (2015) 60:2985–2995 DOI 10.1007/s10620-015-3696-7
ORIGINAL ARTICLE
Hesperetin Induces the Apoptosis of Gastric Cancer Cells via Activating Mitochondrial Pathway by Increasing Reactive Oxygen Species Jixiang Zhang1 • Dandan Wu1 • Vikash1 • Jia Song1 • Jing Wang1 Jiasheng Yi1 • Weiguo Dong1
•
Received: 5 January 2015 / Accepted: 29 April 2015 / Published online: 14 May 2015 Ó Springer Science+Business Media New York 2015
Abstract Background Hesperetin, has been shown to exert biological activities on various types of human cancers. However, few related studies on gastric cancer are available. Aim In this study, we sought to investigate the effect of hesperetin on gastric cancer and clarify its specific mechanism. Materials and Methods Cell Counting Kit-8, 20 ,70 dichlorofluorescin diacetate, JC-1, Hoechst 33258 staining, and western bolt were used to detect cell viability, levels of intracellular reactive oxygen species (ROS), changes in mitochondrial membrane potential (4wm), cell apoptosis, and expressions of mitochondrial pathway proteins, respectively. Meanwhile, xenograft tumor models in nude mice were made to evaluate the effect of hesperetin on gastric cancer in vivo. Results Compared with the control group, the proliferation of gastric cancer cells in hesperetin groups was significantly inhibited (P \ 0.05), and dose- and time-dependent effects were observed. Pretreatment with H2O2 (1 mM) or N-acetyl-L-cysteine (5 mM) enhanced or attenuated the hesperetin-induced inhibition of cell viability (P \ 0.05). Percentages of apoptotic cells, levels of intracellular ROS, and 4wm varied with the dose and treatment time of hesperetin (P \ 0.05), and hesperetin caused an
& Weiguo Dong [email protected] Jixiang Zhang [email protected] 1
Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
increase in the levels of AIF, Apaf-1, Cyt C, caspase-3, caspase-9, and Bax and a decrease in Bcl-2 levels (P \ 0.05). Meanwhile, hesperetin significantly inhibited the growth of xenograft tumors (P \ 0.05). Conclusion Our study suggests that hesperetin could inhibit the proliferation and induce the apoptosis of gastric cancer cells via activating the mitochondrial pathway by increasing the ROS. Keywords Hesperetin Apoptosis Gastric cancer Mitochondria Reactive oxygen species
Introduction Gastric cancer is the fourth most prevalent malignant disease and the second leading cause of cancer-related mortality worldwide [1]. Based on the data of GLOBOCAN 2012, about 951 thousand gastric cancer cases and 723 thousand gastric cancer deaths are estimated to have occurred in 2012 [2]. Currently, main therapeutic modalities involve surgery, radiation, and chemotherapy. However, about 70 % gastric cancer patients have a locally advanced or metastatic disease at the time of initial diagnosis which compromises the effects of surgery and radiation greatly [3, 4]. Chemotherapeutic drugs, including 5-fluorouracil, cisplatin, and adriamycin, are used commonly. But chemotherapeutic options have become restricted due to drug resistance and cell toxicity [5, 6]. Therefore, it is of great value to discover the natural anticancer compounds that have high efficacy and low toxicity in the treatment of gastric cancer. Hesperetin (30 ,5,7-trihydroxy-4-methoxyflavanone), also referred to as hesperitin, is a member of flavanone, subclass of flavonoids, and occurs in fruit sources including citrus
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species. It has been demonstrated that hesperetin has various pharmacological activities such as antiinflammatory, antihypertensive, antiatherogenic effects, and antioxidant properties [7–9]. Previous studies found that hesperetin could inhibit proliferation or induce apoptosis on various cancer cells, including colon cancer cells, breast cancer cells, prostate cancer cells, and cervical cancer cells [10– 13]. And some researchers reported that hesperetin interfered with transforming growth factor-b (TGF-b) signaling pathway ligand–receptor interactions and then hindered TGF-b1-induced cancer cell migration and invasion [14]. Also, hesperetin inhibits insulin-induced glucose uptake through impaired cell membrane translocation of glucose transporter 4 (GLUT4) which could contribute to its anticancer effects [15]. In addition, it has been reported that hesperetin could upregulate the expression of Notch1 and its downstream effectors hairy and enhancer of split 1 (Hes1) and cause the apoptosis of cancer cells [16, 17]. Meanwhile, the actions of hesperitin including inhibition of proliferation and induction of apoptosis on various cancer cells involve oxidant/antioxidant imbalance [18, 19]. However, the precise molecular mechanisms underlying hesperetin-induced apoptosis still remain to be delineated. Much information is needed to make the anticancer effects and the mechanism of hesperitin on human gastric cancer cells clear. Therefore, this study was undertaken to address the action of hesperitin on human gastric cancer cells both in vitro and in vivo and clarify its potential mechanism.
Materials and Methods Cell Culture and Reagents Human gastric cancer cells lines (SGC-7901, MGC-803, and HGC-27) were purchased from the Shanghai Cell Collection (Shanghai, China) and cultured in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco), 1 % antibiotics (100 IU penicillin and 100 lg/mL streptomycin) in a humidified incubator at 37 °C, and 5 % CO2. Hesperetin ([98 % purity) was purchased from Sigma (Sigma-Aldrich, St. Louis, USA). The hesperetin stock solution was prepared at 200 mM in dimethyl sulfoxide (DMSO) and stored at -20 °C. Cell Proliferation Assay Cell Counting Kit-8 (CCK-8, Beyotime, China) was used to quantitatively evaluate cell viability. Cells were seeded (5 9 103 cells/well) in 96-well culture plates, and the next day the supernatant was replaced and cells were cultured in fresh medium containing hesperetin (0, 20, 40, 60, 80, 120,
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150, 200, 300, 400 lM) diluted from the stock solution for 0, 12, 24, 36, 48, 60, and 72 h or pretreated with 1 mM H2O2 or 5 mM N-acetyl-L-cysteine (NAC, Beyotime, China) for 2 h and cultured with hesperetin. Then 10 ml of the CCK-8 solution was added to each well, and cells were incubated for an additional 2 h. The absorbance at 450 nm was determined using a microplate reader (Victor3 1420 Multilabel Counter, Perkin Elmer, USA). DMEM containing 10 % CCK-8 was used as a control. Colony formation assay was also used to evaluate cell proliferation. Cells were seeded (1 9 103 cells/well) in 6-well culture plates, and the next day the supernatant was replaced and cells were cultured in fresh medium containing hesperetin (0, 20, 40, 80 lM) or pretreated with H2O2 (1 mM) or NAC (5 mM) for 2 h and then cultured with hesperetin (40 lM). Every 2–3 days, the supernatant was replaced and the above processes were repeated. Fourteen days later, the cells were fixed by paraformaldehyde and stained by Giemsa and colony numbers were counted. Measurement of Reactive Oxygen Species (ROS) Levels of intracellular ROS were detected using a 20 ,70 dichlorofluorescin diacetate (DCFH-DA, Sigma-Aldrich, St. Louis, USA). Gastric cancer cells were seeded (5 9 103 cells/well) in 96-well culture plates or (1 9 105 cells/well) in 6-well culture plates. After 24 h, the supernatant was replaced, and with or without pretreatment with H2O2 (1 mM) or NAC (5 mM) for 2 h, cells in 96-well culture plates were cultured in fresh medium containing hesperetin (0, 100, 200, 400 lM) for 24, 48, and 72 h. Cells in 6-well culture plates were only cultured for 24 h. Then supernatant was removed, and the cells were washed with PBS for three times. Cells were maintained with DCFH-DA (10 lM) in DMEM (without FBS) for 20 min and then washed with PBS for another three times. After that, the levels of intracellular ROS of cells in 96-well culture plates were determined by the fluorescent absorbance value using a microplate reader (Victor3 1420 Multilabel Counter, Perkin Elmer, USA). Meanwhile, the levels of intracellular ROS of cells in 6-well culture plates were determined by a fluorescence microscope (BX51, Olympus, Japan). Hoechst 33258 Staining for Apoptotic Cells Gastric cancer cells in exponential growth were placed at a final concentration of 1 9 105 cells per well in 6-well plates. Twenty-four hours later, cells were divided into two groups: In one group, cells were pretreated with H2O2 (1 mM) or NAC (5 mM) for 2 h, while in the other group cells were not pretreated. Then they were cultured in fresh medium containing hesperetin (0, 100, 200, 400 lM) for 24 h. The cells were subsequently fixed, washed three
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times with PBS, and stained with Hoechst 33258 (SigmaAldrich, St. Louis, USA) according to the manufacturer’s protocol. Apoptotic features were assessed by analyzing chromatin condensation and by staining the fragments under a fluorescent microscope (BX51, Olympus, Japan). Under the magnification of 2009, three random fields per well were examined and the average apoptotic rate was calculated. Measurement of Mitochondrial Membrane Potential The mitochondrial membrane potential (4wm) was examined using JC-1 kit (C2006, Beyotime, China). Gastric cancer cells were seeded (1 9 105 cells/well) in 6-well culture plates. After 24 h, with or without pretreatment with H2O2 (1 mM) or NAC (5 mM) for 2 h, the supernatant was replaced and cells were cultured in fresh medium containing hesperetin (0, 100, 200, 400 lM) for 24 h. Then cells were washed with PBS and maintained with JC-1 (1 ml) in DMEM (1 ml) for 20 min at 37 °C. After that, cells were washed twice with buffer solution (4 °C) and covered with DMEM to be examined by a laser confocal fluorescence microscopy (FV1200, Olympus, Japan). Western Blot Analysis Gastric cancer cell lysates were prepared with RIPA lysis buffer (Beyotime Institute of Biotechnology) and phenylmethylsulfonyl fluoride (PMSF; Beyotime Institute of Biotechnology). The extracted cellular proteins were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), and blocked with 5 % nonfat milk in TBST. Then, the membranes were immunoblotted with several rabbit polyclonal antibodies (all from Cell Signaling Technology, Beverly, MA, USA) including anti-Bcl-2, anti-Bax, anti-caspase-9, anti-caspase-3, anti-apoptosis-inducing factor (AIF), antiapoptosis protease activating factor-1(Apaf-1), anti-cytochrome C (Cyt C), or anti-GAPDH, overnight at 4 °C. After washing with TBST for three times, the membranes were incubated with a 1:10,000 diluted goat anti-rabbit secondary antibody (LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature before they were washed with TBST for another three times. Finally, the membranes were scanned using a two-color infrared imaging system (Odyssey, Lincoln, NE, USA). Membranes were also probed for GAPDH as an additional loading control. Xenograft Tumor Model Male BALB/c-nu/nu nude mice (4–6 weeks old) were purchased from the Center for Animal Experiment of Wuhan University. Gastric cancer cells, harvested from
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subconfluent cultures, washed in serum-free medium, and suspended in 100 ll PBS, were subcutaneously inoculated into the dorsal area of the nude mice. When the tumors reached approximately 150 mm3 in size, the nude mice were divided into four groups (six in each group): a control group, a low-dose group (10 mg/kg), a mid-dose group (20 mg/kg), and a high-dose group (40 mg/kg). Hesperetin was administrated via intraperitoneal injection three times per week. Next, the mice were weighed, and the size of each tumor and its central necrotic area were monitored using calipers every 3 days. At the end of the experiment, tumors were harvested and weighed and were analyzed by HE staining and TUNEL assay. All experiments were performed according to the recommendations of the Institutional Animal Care and Use Committee, and the study protocol was approved by the Ethics Committee for Animal Research of Wuhan University, China. HE Staining and TUNEL Assay For histologic analysis, tumor tissues were fixed in 4 % formaldehyde, dehydrated with an ethanol gradient, and embedded in paraffin, and the paraffin tumor tissue sections (4 lm) were stained with hematoxylin and eosin (H&E). The TUNEL assay for apoptosis was performed using an apoptosis detection kit (Roche Diagnostics, Branchburg, NJ, USA) according to the manufacturer’s instructions. Positive cells were identified, counted (six random fields per slides), and analyzed by light microscopy (BX51, Olympus, Japan). Statistical Analysis Data are expressed as the mean ± SD. The difference among groups was determined by ANOVA, and the difference between groups was analyzed by the Student’s t test using SPSS 17.0 for Microsoft Windows (SPSS Inc., Chicago, IL, USA). A value of P \ 0.05 was considered to indicate a statistically significant result.
Results Hesperetin Inhibits the Proliferation of Gastric Cancer Cells The results of CCK-8 assay showed that hesperetin could inhibit the proliferation of gastric cancer cells and was in a dose- and time-dependent manner (Fig. 1a). Meanwhile, pretreatment with H2O2 (1 mM) augmented the inhibition of hesperetin on human gastric cancer cells, while NAC (5 mM) attenuated this inhibition. In addition, it was observed that of the three gastric cancer cell lines, SGC-7901
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was the most sensitive to hesperetin. Thus, SGC-7901 was chosen for the following experiment (Fig. 1a–c). In the colony formation assay, the numbers of colony in control group, low-hesperetin group (20 lM), middlehesperetin group (40 lM), high-hesperetin group (80 lM), H2O2-pretreated group, and NAC-pretreated group were 147 ± 13.5, 103 ± 11.9, 82 ± 10.7, 57 ± 8.3, 35 ± 7.1, and 163 ± 21.5, respectively (Fig. 2). The results indicated that hesperetin could inhibit the proliferation of gastric cancer cells and it may be associated with the changed levels of intracellular ROS. Hesperetin Induces the Generation of Intracellular ROS To determine whether the growth-inhibitory effect of hesperetin on human gastric cancer cells is related to the deregulation of cellular redox status, the levels of
Fig. 1 Dose- and time-dependent effects of hesperetin on gastric cancer cell viability (SGC-7901, HGC-27, and MGC-803). a Hesperetin inhibits the viability of gastric cancer cells in a dose- and time-dependent manner; b the pretreatment of H2O2 (1 mM) can
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intracellular ROS were detected using DCFH-DA. It was shown that the relative fluorescence value varied with the doses and incubation time of hesperetin (Fig. 3a), which indicated that hesperetin could interfere with the levels of intracellular ROS. Meanwhile, the fluorescence intensity detected by a fluorescence microscope also showed that hesperetin could increase the levels of intracellular ROS in human gastric cancer cells and this increase was enhanced when the dose was increased (Fig. 3b). Hesperetin Induces the Apoptosis of Gastric Cancer Cells By using Hoechst 33258 staining, we assessed the effect of hesperetin on the apoptosis of human gastric cancer cells. In the control groups, the nuclei were stained a weak homogeneous blue, while in the groups treated with hesperetin, bright chromatin condensation and nuclear
augment the inhibition of hesperetin on the viability of gastric cancer cells; c the pretreatment of NAC (5 mM) can reduce the inhibition of hesperetin on the viability of gastric cancer cells
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Fig. 2 Hesperetin inhibited the proliferation of gastric cancer cells. SGC-7901 was treated with hesperetin (0, 20, 40, 80 lM), H2O2 (1 mM) pretreated ? hesperetin (40 lM), and NAC (5 mM)
pretreated ? hesperetin (40 lM), and colony numbers were counted. *P \ 0.05 versus control, #P \ 0.05 versus hesperetin (40 lM)
Fig. 3 Level of ROS in SGC-7901 treated with hesperetin, hesperetin ? H2O2 (1 mM), and hesperetin ? NAC (5 mM). a SGC7901 cells were treated with hesperetin (0, 100, 200, 400 lM), H2O2 (1 mM) pretreated ? hesperetin (0, 100, 200, 400 lM), and NAC (5 mM) pretreated ? hesperetin (0, 100, 200, 400 lM) for different time periods (24, 48 and 72 h) followed by incubation with 10 lM DCFH-DA for 20 min and then analyzed for the generation of intracellular ROS by microplate reader. b SGC-7901 cells were
treated with hesperetin (0, 100, 200, 400 lM), H2O2 pretreated (1 mM) ? hesperetin (0, 100, 200, 400 lM), and NAC pretreated (5 mM) ? hesperetin (0, 100, 200, 400 lM) for different time periods (24, 48 and 72 h) followed by incubation with 10 lM DCFH-DA for 20 min and then analyzed for the generation of intracellular ROS by fluorescence microscope. *P \ 0.05 versus control, #P \ 0.05 versus hesperetin (0, 100, 200, 400 lM)
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fragmentation could be observed (Fig. 4). Percentages of apoptotic cells in the control group, low-dose hesperetin group (100 lM), mid-dose hesperetin group (200 lM), and high-dose hesperetin group (400 lM) were 1.3 ± 0.46, 8.1 ± 1.01, 17.6 ± 1.73, and 31.4 ± 1.52 %, respectively. While pretreated with H2O2 (1 mM) or NAC (5 mM), the percentages of apoptotic cells varied accordingly: 1.8 ± 0.43 % in the control group, 16.7 ± 0.84 % in the mid-dose hesperetin group (200 lM), 38.7 ± 1.33 % in the mid-dose hesperetin with H2O2 (1 mM)-pretreated group, and 4.6 ± 1.19 % in the mid-dose hesperetin with NAC (5 mM)-pretreated group (Fig. 4b). Hesperetin Changes the Mitochondrial Membrane Potential via Promoting the Accumulation of ROS JC-1 was used to examine the changes in mitochondrial membrane potential (4wm). The results showed that with the increase in the dose of hesperetin (0, 100, 200, 400 lM), 4wm was decreased accordingly. Meanwhile, the pretreatment with H2O2 could augment the hesperetin-induced decrease in 4wm, and the pretreatment with NAC could attenuate the hesperetin-induced decrease in 4wm (Fig. 5). Fig. 4 SGC-7901 cells were incubated with hesperetin, H2O2 (1 mM) pretreated ? hesperetin, and NAC (5 mM) pretreated ? hesperetin; apoptosis was detected after 24 h using Hoechst 33258; and apoptotic features were assessed by observing chromatin condensation and fragments staining. a SGC-7901 cells were incubated with hesperetin (0, 100, 200, 400 lM); b SGC7901 cells were incubated with hesperetin, H2O2 (1 mM) pretreated ? hesperetin, and NAC (5 mM) pretreated ? hesperetin. Scale bar represents 50 lm. Original magnification: 9200. *P \ 0.05 versus control, #P \ 0.05 versus hesperetin (200 lM)
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Hesperetin Induces the Apoptosis of Gastric Cancer Cells via ROS Triggering the Activation of the Mitochondrial Pathway To further study the detailed mechanism with respect to hesperetin-induced apoptosis, we examined the effect of hesperetin on the mitochondrial pathway using western blot. As shown in Fig. 6, hesperetin led to an increase in the levels of AIF, Apaf-1, and Cyt C, which is known as the critical roles which plays the role in activating of caspase-3 and caspase-9 and the subsequent mitochondria-mediated apoptosis. Also, hesperetin caused an increase in Bax protein levels and a decrease in Bcl-2 levels and the decrease in the antiapoptotic/proapoptotic (Bcl-2/Bax) protein ratio. In addition, pretreatment with H2O2 (1 mM) augmented the hesperetin-induced increase or decrease in the levels of mitochondrial pathway proteins above, while NAC (5 mM) attenuated these changes (Fig. 6). Antitumor Effects In Vivo On the basis of the in vitro data above, we investigated the effects of hesperetin on xenograft tumor growth in vivo. As
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Fig. 5 Mitochondrial membrane potential (4wm) was detected by JC-1 kit. SGC-7901 cells were incubated with hesperetin (0, 100, 200, 400 lM) or with hesperetin (200 lM), H2O2 (1 mM) pretreated ? hesperetin (200 lM), and NAC (5 mM) pretreated ? hesperetin
(200 lM). After being stained by JC-1, 4wm was detected using a laser confocal fluorescence microscopy. *P \ 0.05 versus control, # P \ 0.05 versus hesperetin (200 lM)
shown in Fig. 7, tumors grew rapidly in the control group (2132 ± 219.5 mm3). However, tumor growth was significantly suppressed in the treatment groups in a dosedependent manner (1350 ± 89.8 mm3 in low-dose group, 921.3 ± 99.6 mm3 in mid-dose group, and 612.2 ± 87.1 mm3 in high-dose group). Meanwhile, at the end of the experiment, the tumors were harvested. The weight of the tumors in control group was heavier than that of the tumors in low-dose group, mid-dose group, and high-dose group (P \ 0.05). Tumor tissues isolated from the xenograft mice of four groups were processed for HE staining (not shown) and TUNEL assay. It demonstrated that hesperetin resulted in obvious cell apoptosis in the tumor mass (Fig. 7c), whereas little apoptosis was observed in the control group (P \ 0.05).
categories: Firstly, there are those biological processes, mainly the mitochondrial oxidative metabolism, that release ROS as a byproduct, or a waste product, of various other necessary reactions; and secondly, there are those processes, in cellular response to xenobiotics, cytokines, and bacterial invasion, that generate ROS intentionally, either in molecular synthesis or in breakdown, as part of a signal transduction pathway or as part of a cell defense mechanism [22–24]. Cumulative evidences have demonstrated that the imbalance of intracellular ROS plays critical roles in the pathogenesis of series of diseases or pathological state, such as atherosclerosis [25], diabetes [26], cardiovascular disease [27], cancer [28, 29], neurodegeneration [30], and inflammation [31]. Directly or indirectly, ROS interact with critical signaling molecules to initiate signal pathway including NF-jB signaling pathway, mitogen-activated protein kinase (MAPK), Keap1Nrf2-ARE signaling pathway, and mitochondrial apoptosis pathway and then involve in the occurrence and development of these diseases above. Hesperetin, one of the most abundant flavonoids found in citrus fruits, exhibits various pharmacological activities. It has been reported that hesperetin could decrease activities of antioxidants such as superoxide dismutase
Discussion ROS, generated through a variety of extracellular and intracellular actions, have drawn attention as novel signal mediators which are involved in growth, differentiation, progression, and death of the cell [20, 21]. The resource of cellular ROS could be broadly divided into two main
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Fig. 6 Western blots showing the expression of apoptosis-related proteins in vitro. The levels of Bcl-2, Bax, caspase-3, caspase-9, AIF, Apaf-1, and Cyt C proteins in SGC-7901 cells were detected by western blots. SGC-7901 cells were incubated with hesperetin (0, 100, 200, 400 lM) or with hesperetin (200 lM), H2O2 (1 mM) pretreated ? hesperetin (200 lM), and NAC (5 mM) pretreated ? hesperetin
Fig. 7 In vivo antitumor effect of treatment groups in a gastric tumor xenograft mouse model. a Each time point represents the mean tumor volume for each group. b Tumor weight was obtained at the end of the experiment. Error bars represent the standard error of the mean (SEM). c Detection of apoptotic cells in tumor tissue by TUNEL assay. Scale bar represents 50 lm. Original magnification: 9200. *P \ 0.05 versus control
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(200 lM). The relative expression levels of Bcl-2, Bax, caspase-3, caspase-9, AIF, Apaf-1, and Cyt C proteins in SGC-7901 cells were compared in different groups. GAPDH expression was used as internal control. A representative blot is shown from three independent experiments with identical results. *P \ 0.05 versus control, # P \ 0.05 versus hesperetin (0, 100, 200, 400 lM)
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(SOD), catalase (CAT), and glutathione peroxidase (GPx) and lead to oxidant/antioxidant imbalance [32]. Previous studies have shown neuroprotective effects with continual administration in mice, prevention of DMBA-induced mammary cancer in female rats, and suppression of the formation of aberrant crypt foci in rats with induced colon cancer [33, 34]. Researchers also reported that hesperetin exerted potent antiproliferative effects on SiHa cells in a time- and dose-dependent manner, with an IC50 value of 650 lM [12]. However, the anticancer effects of hesperitin and the specific mechanism in gastric cancer have not been examined. In this study, we examined the antiproliferation ability of hesperetin against three gastric cancer cell lines (SGC7901, MGC-803, and HGC-27). We found that hesperetin effectively suppressed the proliferation of gastric cancer cells in a dose- and time-dependent manner, and SGC-7901 cells were more sensitive to hesperetin than MGC-803 and HGC-27 cells. When pretreated with H2O2 (1 mM), which has been proved to be able to upregulate the levels of intracellular ROS, and then cultured with hesperetin, we found that the inhibition of hesperetin on gastric cancer cells was augmented remarkably. In contrast, after being pretreated with NAC (5 mM), inhibition of hesperetin on gastric cancer cells was weakened obviously. The results above indicated that hesperetin could effectively inhibit the proliferation of gastric cancer cells, especially SGC-7901, and the inhibition of hesperetin on gastric cancer cells may be concerned with the changes in the levels of intracellular ROS. In order to determine whether hesperetin could induce the generation of intracellular ROS, we examined the levels of intracellular ROS in SGC-7901 cells treated with hesperetin of different dose and incubation time. We found that the levels of intracellular ROS in hesperetin-treating groups are higher than that in control group. It confirmed our speculation that hesperetin inhibited the proliferation of gastric cancer cells by increasing the levels of intracellular ROS. Meanwhile, it was found that hesperetin induced the apoptosis of SGC-7901 cells. Hoechst 33258 staining showed that the percentage of apoptotic cells in the control group was significantly lower that of three hesperetin groups. H2O2 or NAC pretreatment altered the percentages of hesperetin-induced apoptosis of gastric cancer cells which revealed that the increased levels of intracellular ROS contributed to the apoptosis of gastric cancer cells induced by hesperetin. Although still controversial, the loss of 4wm is suggested to be an early event in the apoptotic process [35–37]. In recent years, fluorescent dyes (TMRM, Rhod123, JC-1, and DiOC63) for measuring the 4wm have become common and they vary in their degrees of toxicity to mitochondria and cells and degrees of binding to mitochondria [35, 36]. In our study, we chose JC-1, which has
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been reported to be a more reliable indicator of 4wm than other dyes, to measure the changes in 4wm. And the results indicated that ROS-mediated changes in 4wm may contribute to hesperetin-induced apoptosis. It has been demonstrated that Bcl-2 family proteins were one of the components of mitochondrial permeability transition pore (mPTP) [38, 39]. If the Bcl-2/Bax protein ratio is reduced, the structure and permeability of mPTP will be changed which initiates mitochondrial-driven death. To clarify whether the increasing ROS induced by hesperetin led to the apoptosis of gastric cancer cells via mitochondrial pathway, we detected the levels of Bcl-2, Bax, caspase-9, caspase-3, AIF, Apaf-1, and Cyt C using western bolt. It was found that hesperetin caused an increase in the levels of AIF, Apaf-1, Cyt C, caspase-3, caspase-9, and Bax and a decrease in Bcl-2 levels. In addition, when pretreated by H2O2 or NAC, the hesperetin-induced increase or decrease in the levels of mitochondrial pathway proteins above changed. So, it could be considered that hesperetin could upregulate the levels of intracellular ROS, and excessive levels of intracellular ROS may change the structure and permeability of mPTP by decreasing the expression of Bcl2 and increasing the expression of Bax. That in turn releases Cyt C, AIF, and Apaf-1 which then form apoptosome that activates the caspase-9 and caspase-3 and induces apoptosis (Fig. 8) [40]. So far, the views concerning that whether hesperetin induces the apoptosis of gastric cancer cells by decreasing or increasing the generation and accumulation of ROS were inconsistent. Several researchers reported that hesperetin had an antioxidant potential by upregulating the levels of GSH and the expressions of antioxidant enzymes, inactivating NF-kB pathway, downregulating the expressions of iNOS and COX-2, and eliminating the levels of ONOO- [19, 41–44]. However, many studies indicated that hesperetin was an oxidant which could activate ASK1/JNK pathway, downregulate the levels of antioxidants, and upregulate the levels of oxidants [32, 45, 46]. Regarding the different opinions, we think that there are two reasons which could explain that: firstly, the generation and elimination of ROS are highly complex processes. The balance of ROS levels is influenced by various biological processes and cellular response to internal or external stimuluses, and the imbalance of ROS levels has impacts on a lot of signaling pathways and plays crucial roles in a series of cellular processes, because hesperetin, which has various pharmacological activities, may have effects on several different processes of the generation and elimination of ROS which may both increase and decrease the levels of ROS. Secondly, the actions of hesperetin in cancer cells and normal cells as well as in different kinds of cancer cells are diverse. Thus, in a cell line, hesperetininduced generation of ROS may cover up the elimination
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Fig. 8 Proposed schema for hesperetin-induced apoptosis in gastric cancer cell
and then oxidant potential is shown. In turn, in other cell lines, hesperetin-induced elimination of ROS may be greater and the antioxidant potential could be observed. In conclusion, these results provide strong molecular evidence in support of our hypothesis that hesperetin inhibits the proliferation and induces the apoptosis of gastric cancer cells via triggering the activation of the mitochondrial pathway by increasing levels of intracellular ROS. This study identifies the potential usefulness of hesperetin, a non-toxic and safe anti-tumor agent, in the management of gastric cancer.
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Dig Dis Sci (2015) 60:2985–2995 16. Patel PN, Yu XM, Jaskula-Sztul R, Chen H. Hesperetin activates the Notch1 signaling cascade, causes apoptosis, and induces cellular differentiation in anaplastic thyroid cancer. Ann Surg Oncol. 2014;21:S497–S504. 17. Zarebczan B, Pinchot SN, Kunnimalaiyaan M, Chen H. Hesperetin, a potential therapy for carcinoid cancer. Am J Surg. 2011;201:329–332; discussion 333. 18. Haidari F, Ali Keshavarz S, Reza Rashidi M, Mohammad Shahi M. Orange juice and hesperetin supplementation to hyperuricemic rats alter oxidative stress markers and xanthine oxidoreductase activity. J Clin Biochem Nutr. 2009;45:285–291. 19. Yang HL, Chen SC, Senthil Kumar KJ, et al. Antioxidant and anti-inflammatory potential of hesperetin metabolites obtained from hesperetin-administered rat serum: an ex vivo approach. J Agric Food Chem. 2012;60:522–532. 20. Zhang H, Gomez AM, Wang X, Yan Y, Zheng M, Cheng H. ROS regulation of microdomain Ca(2?) signalling at the dyads. Cardiovasc Res. 2013;98:248–258. 21. Sena LA, Chandel NS. Physiological roles of mitochondrial reactive oxygen species. Mol Cell. 2012;48:158–167. 22. Finkel T. Signal transduction by reactive oxygen species. J Cell Biol. 2011;194:7–15. 23. Grivennikova VG, Vinogradov AD. Mitochondrial production of reactive oxygen species. Biochemistry (Mosc). 2013;78: 1490–1511. 24. Starkov AA. The role of mitochondria in reactive oxygen species metabolism and signaling. Ann NY Acad Sci. 2008;1147:37–52. 25. Chang MC, Chang HH, Chan CP, et al. p-Cresol affects reactive oxygen species generation, cell cycle arrest, cytotoxicity and inflammation/atherosclerosis-related modulators production in endothelial cells and mononuclear cells. PLoS One. 2014;9: e114446. 26. Rehman AU, Dugic E, Benham C, Lione L, Mackenzie LS. Selective inhibition of NADPH oxidase reverses the over contraction of diabetic rat aorta. Redox Biol. 2013;2C:61–64. 27. Prasad K, Dhar I. Oxidative stress as a mechanism of added sugar-induced cardiovascular disease. Int J Angiol. 2014;23: 217–226. 28. Dhillon H, Chikara S, Reindl KM. Piperlongumine induces pancreatic cancer cell death by enhancing reactive oxygen species and DNA damage. Toxicol Rep. 2014;1:309–318. 29. Udensi UK, Tchounwou PB. Dual effect of oxidative stress on leukemia cancer induction and treatment. J Exp Clin Cancer Res. 2014;33:106. 30. Wang P, Xie K, Wang C, Bi J. Oxidative stress induced by lipid peroxidation is related with inflammation of demyelination and neurodegeneration in multiple sclerosis. Eur Neurol. 2014;72: 249–254. 31. Nijland PG, Witte ME, van Het Hof B, et al. Astroglial PGC1alpha increases mitochondrial antioxidant capacity and suppresses inflammation: implications for multiple sclerosis. Acta Neuropathol Commun. 2014;2:170.
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ORIGINAL ARTICLE
Hesperetin Induces the Apoptosis of Gastric Cancer Cells via Activating Mitochondrial Pathway by Increasing Reactive Oxygen Species Jixiang Zhang1 • Dandan Wu1 • Vikash1 • Jia Song1 • Jing Wang1 Jiasheng Yi1 • Weiguo Dong1
•
Received: 5 January 2015 / Accepted: 29 April 2015 / Published online: 14 May 2015 Ó Springer Science+Business Media New York 2015
Abstract Background Hesperetin, has been shown to exert biological activities on various types of human cancers. However, few related studies on gastric cancer are available. Aim In this study, we sought to investigate the effect of hesperetin on gastric cancer and clarify its specific mechanism. Materials and Methods Cell Counting Kit-8, 20 ,70 dichlorofluorescin diacetate, JC-1, Hoechst 33258 staining, and western bolt were used to detect cell viability, levels of intracellular reactive oxygen species (ROS), changes in mitochondrial membrane potential (4wm), cell apoptosis, and expressions of mitochondrial pathway proteins, respectively. Meanwhile, xenograft tumor models in nude mice were made to evaluate the effect of hesperetin on gastric cancer in vivo. Results Compared with the control group, the proliferation of gastric cancer cells in hesperetin groups was significantly inhibited (P \ 0.05), and dose- and time-dependent effects were observed. Pretreatment with H2O2 (1 mM) or N-acetyl-L-cysteine (5 mM) enhanced or attenuated the hesperetin-induced inhibition of cell viability (P \ 0.05). Percentages of apoptotic cells, levels of intracellular ROS, and 4wm varied with the dose and treatment time of hesperetin (P \ 0.05), and hesperetin caused an
& Weiguo Dong [email protected] Jixiang Zhang [email protected] 1
Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
increase in the levels of AIF, Apaf-1, Cyt C, caspase-3, caspase-9, and Bax and a decrease in Bcl-2 levels (P \ 0.05). Meanwhile, hesperetin significantly inhibited the growth of xenograft tumors (P \ 0.05). Conclusion Our study suggests that hesperetin could inhibit the proliferation and induce the apoptosis of gastric cancer cells via activating the mitochondrial pathway by increasing the ROS. Keywords Hesperetin Apoptosis Gastric cancer Mitochondria Reactive oxygen species
Introduction Gastric cancer is the fourth most prevalent malignant disease and the second leading cause of cancer-related mortality worldwide [1]. Based on the data of GLOBOCAN 2012, about 951 thousand gastric cancer cases and 723 thousand gastric cancer deaths are estimated to have occurred in 2012 [2]. Currently, main therapeutic modalities involve surgery, radiation, and chemotherapy. However, about 70 % gastric cancer patients have a locally advanced or metastatic disease at the time of initial diagnosis which compromises the effects of surgery and radiation greatly [3, 4]. Chemotherapeutic drugs, including 5-fluorouracil, cisplatin, and adriamycin, are used commonly. But chemotherapeutic options have become restricted due to drug resistance and cell toxicity [5, 6]. Therefore, it is of great value to discover the natural anticancer compounds that have high efficacy and low toxicity in the treatment of gastric cancer. Hesperetin (30 ,5,7-trihydroxy-4-methoxyflavanone), also referred to as hesperitin, is a member of flavanone, subclass of flavonoids, and occurs in fruit sources including citrus
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species. It has been demonstrated that hesperetin has various pharmacological activities such as antiinflammatory, antihypertensive, antiatherogenic effects, and antioxidant properties [7–9]. Previous studies found that hesperetin could inhibit proliferation or induce apoptosis on various cancer cells, including colon cancer cells, breast cancer cells, prostate cancer cells, and cervical cancer cells [10– 13]. And some researchers reported that hesperetin interfered with transforming growth factor-b (TGF-b) signaling pathway ligand–receptor interactions and then hindered TGF-b1-induced cancer cell migration and invasion [14]. Also, hesperetin inhibits insulin-induced glucose uptake through impaired cell membrane translocation of glucose transporter 4 (GLUT4) which could contribute to its anticancer effects [15]. In addition, it has been reported that hesperetin could upregulate the expression of Notch1 and its downstream effectors hairy and enhancer of split 1 (Hes1) and cause the apoptosis of cancer cells [16, 17]. Meanwhile, the actions of hesperitin including inhibition of proliferation and induction of apoptosis on various cancer cells involve oxidant/antioxidant imbalance [18, 19]. However, the precise molecular mechanisms underlying hesperetin-induced apoptosis still remain to be delineated. Much information is needed to make the anticancer effects and the mechanism of hesperitin on human gastric cancer cells clear. Therefore, this study was undertaken to address the action of hesperitin on human gastric cancer cells both in vitro and in vivo and clarify its potential mechanism.
Materials and Methods Cell Culture and Reagents Human gastric cancer cells lines (SGC-7901, MGC-803, and HGC-27) were purchased from the Shanghai Cell Collection (Shanghai, China) and cultured in DMEM (Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco), 1 % antibiotics (100 IU penicillin and 100 lg/mL streptomycin) in a humidified incubator at 37 °C, and 5 % CO2. Hesperetin ([98 % purity) was purchased from Sigma (Sigma-Aldrich, St. Louis, USA). The hesperetin stock solution was prepared at 200 mM in dimethyl sulfoxide (DMSO) and stored at -20 °C. Cell Proliferation Assay Cell Counting Kit-8 (CCK-8, Beyotime, China) was used to quantitatively evaluate cell viability. Cells were seeded (5 9 103 cells/well) in 96-well culture plates, and the next day the supernatant was replaced and cells were cultured in fresh medium containing hesperetin (0, 20, 40, 60, 80, 120,
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150, 200, 300, 400 lM) diluted from the stock solution for 0, 12, 24, 36, 48, 60, and 72 h or pretreated with 1 mM H2O2 or 5 mM N-acetyl-L-cysteine (NAC, Beyotime, China) for 2 h and cultured with hesperetin. Then 10 ml of the CCK-8 solution was added to each well, and cells were incubated for an additional 2 h. The absorbance at 450 nm was determined using a microplate reader (Victor3 1420 Multilabel Counter, Perkin Elmer, USA). DMEM containing 10 % CCK-8 was used as a control. Colony formation assay was also used to evaluate cell proliferation. Cells were seeded (1 9 103 cells/well) in 6-well culture plates, and the next day the supernatant was replaced and cells were cultured in fresh medium containing hesperetin (0, 20, 40, 80 lM) or pretreated with H2O2 (1 mM) or NAC (5 mM) for 2 h and then cultured with hesperetin (40 lM). Every 2–3 days, the supernatant was replaced and the above processes were repeated. Fourteen days later, the cells were fixed by paraformaldehyde and stained by Giemsa and colony numbers were counted. Measurement of Reactive Oxygen Species (ROS) Levels of intracellular ROS were detected using a 20 ,70 dichlorofluorescin diacetate (DCFH-DA, Sigma-Aldrich, St. Louis, USA). Gastric cancer cells were seeded (5 9 103 cells/well) in 96-well culture plates or (1 9 105 cells/well) in 6-well culture plates. After 24 h, the supernatant was replaced, and with or without pretreatment with H2O2 (1 mM) or NAC (5 mM) for 2 h, cells in 96-well culture plates were cultured in fresh medium containing hesperetin (0, 100, 200, 400 lM) for 24, 48, and 72 h. Cells in 6-well culture plates were only cultured for 24 h. Then supernatant was removed, and the cells were washed with PBS for three times. Cells were maintained with DCFH-DA (10 lM) in DMEM (without FBS) for 20 min and then washed with PBS for another three times. After that, the levels of intracellular ROS of cells in 96-well culture plates were determined by the fluorescent absorbance value using a microplate reader (Victor3 1420 Multilabel Counter, Perkin Elmer, USA). Meanwhile, the levels of intracellular ROS of cells in 6-well culture plates were determined by a fluorescence microscope (BX51, Olympus, Japan). Hoechst 33258 Staining for Apoptotic Cells Gastric cancer cells in exponential growth were placed at a final concentration of 1 9 105 cells per well in 6-well plates. Twenty-four hours later, cells were divided into two groups: In one group, cells were pretreated with H2O2 (1 mM) or NAC (5 mM) for 2 h, while in the other group cells were not pretreated. Then they were cultured in fresh medium containing hesperetin (0, 100, 200, 400 lM) for 24 h. The cells were subsequently fixed, washed three
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times with PBS, and stained with Hoechst 33258 (SigmaAldrich, St. Louis, USA) according to the manufacturer’s protocol. Apoptotic features were assessed by analyzing chromatin condensation and by staining the fragments under a fluorescent microscope (BX51, Olympus, Japan). Under the magnification of 2009, three random fields per well were examined and the average apoptotic rate was calculated. Measurement of Mitochondrial Membrane Potential The mitochondrial membrane potential (4wm) was examined using JC-1 kit (C2006, Beyotime, China). Gastric cancer cells were seeded (1 9 105 cells/well) in 6-well culture plates. After 24 h, with or without pretreatment with H2O2 (1 mM) or NAC (5 mM) for 2 h, the supernatant was replaced and cells were cultured in fresh medium containing hesperetin (0, 100, 200, 400 lM) for 24 h. Then cells were washed with PBS and maintained with JC-1 (1 ml) in DMEM (1 ml) for 20 min at 37 °C. After that, cells were washed twice with buffer solution (4 °C) and covered with DMEM to be examined by a laser confocal fluorescence microscopy (FV1200, Olympus, Japan). Western Blot Analysis Gastric cancer cell lysates were prepared with RIPA lysis buffer (Beyotime Institute of Biotechnology) and phenylmethylsulfonyl fluoride (PMSF; Beyotime Institute of Biotechnology). The extracted cellular proteins were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), and blocked with 5 % nonfat milk in TBST. Then, the membranes were immunoblotted with several rabbit polyclonal antibodies (all from Cell Signaling Technology, Beverly, MA, USA) including anti-Bcl-2, anti-Bax, anti-caspase-9, anti-caspase-3, anti-apoptosis-inducing factor (AIF), antiapoptosis protease activating factor-1(Apaf-1), anti-cytochrome C (Cyt C), or anti-GAPDH, overnight at 4 °C. After washing with TBST for three times, the membranes were incubated with a 1:10,000 diluted goat anti-rabbit secondary antibody (LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature before they were washed with TBST for another three times. Finally, the membranes were scanned using a two-color infrared imaging system (Odyssey, Lincoln, NE, USA). Membranes were also probed for GAPDH as an additional loading control. Xenograft Tumor Model Male BALB/c-nu/nu nude mice (4–6 weeks old) were purchased from the Center for Animal Experiment of Wuhan University. Gastric cancer cells, harvested from
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subconfluent cultures, washed in serum-free medium, and suspended in 100 ll PBS, were subcutaneously inoculated into the dorsal area of the nude mice. When the tumors reached approximately 150 mm3 in size, the nude mice were divided into four groups (six in each group): a control group, a low-dose group (10 mg/kg), a mid-dose group (20 mg/kg), and a high-dose group (40 mg/kg). Hesperetin was administrated via intraperitoneal injection three times per week. Next, the mice were weighed, and the size of each tumor and its central necrotic area were monitored using calipers every 3 days. At the end of the experiment, tumors were harvested and weighed and were analyzed by HE staining and TUNEL assay. All experiments were performed according to the recommendations of the Institutional Animal Care and Use Committee, and the study protocol was approved by the Ethics Committee for Animal Research of Wuhan University, China. HE Staining and TUNEL Assay For histologic analysis, tumor tissues were fixed in 4 % formaldehyde, dehydrated with an ethanol gradient, and embedded in paraffin, and the paraffin tumor tissue sections (4 lm) were stained with hematoxylin and eosin (H&E). The TUNEL assay for apoptosis was performed using an apoptosis detection kit (Roche Diagnostics, Branchburg, NJ, USA) according to the manufacturer’s instructions. Positive cells were identified, counted (six random fields per slides), and analyzed by light microscopy (BX51, Olympus, Japan). Statistical Analysis Data are expressed as the mean ± SD. The difference among groups was determined by ANOVA, and the difference between groups was analyzed by the Student’s t test using SPSS 17.0 for Microsoft Windows (SPSS Inc., Chicago, IL, USA). A value of P \ 0.05 was considered to indicate a statistically significant result.
Results Hesperetin Inhibits the Proliferation of Gastric Cancer Cells The results of CCK-8 assay showed that hesperetin could inhibit the proliferation of gastric cancer cells and was in a dose- and time-dependent manner (Fig. 1a). Meanwhile, pretreatment with H2O2 (1 mM) augmented the inhibition of hesperetin on human gastric cancer cells, while NAC (5 mM) attenuated this inhibition. In addition, it was observed that of the three gastric cancer cell lines, SGC-7901
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was the most sensitive to hesperetin. Thus, SGC-7901 was chosen for the following experiment (Fig. 1a–c). In the colony formation assay, the numbers of colony in control group, low-hesperetin group (20 lM), middlehesperetin group (40 lM), high-hesperetin group (80 lM), H2O2-pretreated group, and NAC-pretreated group were 147 ± 13.5, 103 ± 11.9, 82 ± 10.7, 57 ± 8.3, 35 ± 7.1, and 163 ± 21.5, respectively (Fig. 2). The results indicated that hesperetin could inhibit the proliferation of gastric cancer cells and it may be associated with the changed levels of intracellular ROS. Hesperetin Induces the Generation of Intracellular ROS To determine whether the growth-inhibitory effect of hesperetin on human gastric cancer cells is related to the deregulation of cellular redox status, the levels of
Fig. 1 Dose- and time-dependent effects of hesperetin on gastric cancer cell viability (SGC-7901, HGC-27, and MGC-803). a Hesperetin inhibits the viability of gastric cancer cells in a dose- and time-dependent manner; b the pretreatment of H2O2 (1 mM) can
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intracellular ROS were detected using DCFH-DA. It was shown that the relative fluorescence value varied with the doses and incubation time of hesperetin (Fig. 3a), which indicated that hesperetin could interfere with the levels of intracellular ROS. Meanwhile, the fluorescence intensity detected by a fluorescence microscope also showed that hesperetin could increase the levels of intracellular ROS in human gastric cancer cells and this increase was enhanced when the dose was increased (Fig. 3b). Hesperetin Induces the Apoptosis of Gastric Cancer Cells By using Hoechst 33258 staining, we assessed the effect of hesperetin on the apoptosis of human gastric cancer cells. In the control groups, the nuclei were stained a weak homogeneous blue, while in the groups treated with hesperetin, bright chromatin condensation and nuclear
augment the inhibition of hesperetin on the viability of gastric cancer cells; c the pretreatment of NAC (5 mM) can reduce the inhibition of hesperetin on the viability of gastric cancer cells
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Fig. 2 Hesperetin inhibited the proliferation of gastric cancer cells. SGC-7901 was treated with hesperetin (0, 20, 40, 80 lM), H2O2 (1 mM) pretreated ? hesperetin (40 lM), and NAC (5 mM)
pretreated ? hesperetin (40 lM), and colony numbers were counted. *P \ 0.05 versus control, #P \ 0.05 versus hesperetin (40 lM)
Fig. 3 Level of ROS in SGC-7901 treated with hesperetin, hesperetin ? H2O2 (1 mM), and hesperetin ? NAC (5 mM). a SGC7901 cells were treated with hesperetin (0, 100, 200, 400 lM), H2O2 (1 mM) pretreated ? hesperetin (0, 100, 200, 400 lM), and NAC (5 mM) pretreated ? hesperetin (0, 100, 200, 400 lM) for different time periods (24, 48 and 72 h) followed by incubation with 10 lM DCFH-DA for 20 min and then analyzed for the generation of intracellular ROS by microplate reader. b SGC-7901 cells were
treated with hesperetin (0, 100, 200, 400 lM), H2O2 pretreated (1 mM) ? hesperetin (0, 100, 200, 400 lM), and NAC pretreated (5 mM) ? hesperetin (0, 100, 200, 400 lM) for different time periods (24, 48 and 72 h) followed by incubation with 10 lM DCFH-DA for 20 min and then analyzed for the generation of intracellular ROS by fluorescence microscope. *P \ 0.05 versus control, #P \ 0.05 versus hesperetin (0, 100, 200, 400 lM)
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fragmentation could be observed (Fig. 4). Percentages of apoptotic cells in the control group, low-dose hesperetin group (100 lM), mid-dose hesperetin group (200 lM), and high-dose hesperetin group (400 lM) were 1.3 ± 0.46, 8.1 ± 1.01, 17.6 ± 1.73, and 31.4 ± 1.52 %, respectively. While pretreated with H2O2 (1 mM) or NAC (5 mM), the percentages of apoptotic cells varied accordingly: 1.8 ± 0.43 % in the control group, 16.7 ± 0.84 % in the mid-dose hesperetin group (200 lM), 38.7 ± 1.33 % in the mid-dose hesperetin with H2O2 (1 mM)-pretreated group, and 4.6 ± 1.19 % in the mid-dose hesperetin with NAC (5 mM)-pretreated group (Fig. 4b). Hesperetin Changes the Mitochondrial Membrane Potential via Promoting the Accumulation of ROS JC-1 was used to examine the changes in mitochondrial membrane potential (4wm). The results showed that with the increase in the dose of hesperetin (0, 100, 200, 400 lM), 4wm was decreased accordingly. Meanwhile, the pretreatment with H2O2 could augment the hesperetin-induced decrease in 4wm, and the pretreatment with NAC could attenuate the hesperetin-induced decrease in 4wm (Fig. 5). Fig. 4 SGC-7901 cells were incubated with hesperetin, H2O2 (1 mM) pretreated ? hesperetin, and NAC (5 mM) pretreated ? hesperetin; apoptosis was detected after 24 h using Hoechst 33258; and apoptotic features were assessed by observing chromatin condensation and fragments staining. a SGC-7901 cells were incubated with hesperetin (0, 100, 200, 400 lM); b SGC7901 cells were incubated with hesperetin, H2O2 (1 mM) pretreated ? hesperetin, and NAC (5 mM) pretreated ? hesperetin. Scale bar represents 50 lm. Original magnification: 9200. *P \ 0.05 versus control, #P \ 0.05 versus hesperetin (200 lM)
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Hesperetin Induces the Apoptosis of Gastric Cancer Cells via ROS Triggering the Activation of the Mitochondrial Pathway To further study the detailed mechanism with respect to hesperetin-induced apoptosis, we examined the effect of hesperetin on the mitochondrial pathway using western blot. As shown in Fig. 6, hesperetin led to an increase in the levels of AIF, Apaf-1, and Cyt C, which is known as the critical roles which plays the role in activating of caspase-3 and caspase-9 and the subsequent mitochondria-mediated apoptosis. Also, hesperetin caused an increase in Bax protein levels and a decrease in Bcl-2 levels and the decrease in the antiapoptotic/proapoptotic (Bcl-2/Bax) protein ratio. In addition, pretreatment with H2O2 (1 mM) augmented the hesperetin-induced increase or decrease in the levels of mitochondrial pathway proteins above, while NAC (5 mM) attenuated these changes (Fig. 6). Antitumor Effects In Vivo On the basis of the in vitro data above, we investigated the effects of hesperetin on xenograft tumor growth in vivo. As
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Fig. 5 Mitochondrial membrane potential (4wm) was detected by JC-1 kit. SGC-7901 cells were incubated with hesperetin (0, 100, 200, 400 lM) or with hesperetin (200 lM), H2O2 (1 mM) pretreated ? hesperetin (200 lM), and NAC (5 mM) pretreated ? hesperetin
(200 lM). After being stained by JC-1, 4wm was detected using a laser confocal fluorescence microscopy. *P \ 0.05 versus control, # P \ 0.05 versus hesperetin (200 lM)
shown in Fig. 7, tumors grew rapidly in the control group (2132 ± 219.5 mm3). However, tumor growth was significantly suppressed in the treatment groups in a dosedependent manner (1350 ± 89.8 mm3 in low-dose group, 921.3 ± 99.6 mm3 in mid-dose group, and 612.2 ± 87.1 mm3 in high-dose group). Meanwhile, at the end of the experiment, the tumors were harvested. The weight of the tumors in control group was heavier than that of the tumors in low-dose group, mid-dose group, and high-dose group (P \ 0.05). Tumor tissues isolated from the xenograft mice of four groups were processed for HE staining (not shown) and TUNEL assay. It demonstrated that hesperetin resulted in obvious cell apoptosis in the tumor mass (Fig. 7c), whereas little apoptosis was observed in the control group (P \ 0.05).
categories: Firstly, there are those biological processes, mainly the mitochondrial oxidative metabolism, that release ROS as a byproduct, or a waste product, of various other necessary reactions; and secondly, there are those processes, in cellular response to xenobiotics, cytokines, and bacterial invasion, that generate ROS intentionally, either in molecular synthesis or in breakdown, as part of a signal transduction pathway or as part of a cell defense mechanism [22–24]. Cumulative evidences have demonstrated that the imbalance of intracellular ROS plays critical roles in the pathogenesis of series of diseases or pathological state, such as atherosclerosis [25], diabetes [26], cardiovascular disease [27], cancer [28, 29], neurodegeneration [30], and inflammation [31]. Directly or indirectly, ROS interact with critical signaling molecules to initiate signal pathway including NF-jB signaling pathway, mitogen-activated protein kinase (MAPK), Keap1Nrf2-ARE signaling pathway, and mitochondrial apoptosis pathway and then involve in the occurrence and development of these diseases above. Hesperetin, one of the most abundant flavonoids found in citrus fruits, exhibits various pharmacological activities. It has been reported that hesperetin could decrease activities of antioxidants such as superoxide dismutase
Discussion ROS, generated through a variety of extracellular and intracellular actions, have drawn attention as novel signal mediators which are involved in growth, differentiation, progression, and death of the cell [20, 21]. The resource of cellular ROS could be broadly divided into two main
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Fig. 6 Western blots showing the expression of apoptosis-related proteins in vitro. The levels of Bcl-2, Bax, caspase-3, caspase-9, AIF, Apaf-1, and Cyt C proteins in SGC-7901 cells were detected by western blots. SGC-7901 cells were incubated with hesperetin (0, 100, 200, 400 lM) or with hesperetin (200 lM), H2O2 (1 mM) pretreated ? hesperetin (200 lM), and NAC (5 mM) pretreated ? hesperetin
Fig. 7 In vivo antitumor effect of treatment groups in a gastric tumor xenograft mouse model. a Each time point represents the mean tumor volume for each group. b Tumor weight was obtained at the end of the experiment. Error bars represent the standard error of the mean (SEM). c Detection of apoptotic cells in tumor tissue by TUNEL assay. Scale bar represents 50 lm. Original magnification: 9200. *P \ 0.05 versus control
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(200 lM). The relative expression levels of Bcl-2, Bax, caspase-3, caspase-9, AIF, Apaf-1, and Cyt C proteins in SGC-7901 cells were compared in different groups. GAPDH expression was used as internal control. A representative blot is shown from three independent experiments with identical results. *P \ 0.05 versus control, # P \ 0.05 versus hesperetin (0, 100, 200, 400 lM)
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(SOD), catalase (CAT), and glutathione peroxidase (GPx) and lead to oxidant/antioxidant imbalance [32]. Previous studies have shown neuroprotective effects with continual administration in mice, prevention of DMBA-induced mammary cancer in female rats, and suppression of the formation of aberrant crypt foci in rats with induced colon cancer [33, 34]. Researchers also reported that hesperetin exerted potent antiproliferative effects on SiHa cells in a time- and dose-dependent manner, with an IC50 value of 650 lM [12]. However, the anticancer effects of hesperitin and the specific mechanism in gastric cancer have not been examined. In this study, we examined the antiproliferation ability of hesperetin against three gastric cancer cell lines (SGC7901, MGC-803, and HGC-27). We found that hesperetin effectively suppressed the proliferation of gastric cancer cells in a dose- and time-dependent manner, and SGC-7901 cells were more sensitive to hesperetin than MGC-803 and HGC-27 cells. When pretreated with H2O2 (1 mM), which has been proved to be able to upregulate the levels of intracellular ROS, and then cultured with hesperetin, we found that the inhibition of hesperetin on gastric cancer cells was augmented remarkably. In contrast, after being pretreated with NAC (5 mM), inhibition of hesperetin on gastric cancer cells was weakened obviously. The results above indicated that hesperetin could effectively inhibit the proliferation of gastric cancer cells, especially SGC-7901, and the inhibition of hesperetin on gastric cancer cells may be concerned with the changes in the levels of intracellular ROS. In order to determine whether hesperetin could induce the generation of intracellular ROS, we examined the levels of intracellular ROS in SGC-7901 cells treated with hesperetin of different dose and incubation time. We found that the levels of intracellular ROS in hesperetin-treating groups are higher than that in control group. It confirmed our speculation that hesperetin inhibited the proliferation of gastric cancer cells by increasing the levels of intracellular ROS. Meanwhile, it was found that hesperetin induced the apoptosis of SGC-7901 cells. Hoechst 33258 staining showed that the percentage of apoptotic cells in the control group was significantly lower that of three hesperetin groups. H2O2 or NAC pretreatment altered the percentages of hesperetin-induced apoptosis of gastric cancer cells which revealed that the increased levels of intracellular ROS contributed to the apoptosis of gastric cancer cells induced by hesperetin. Although still controversial, the loss of 4wm is suggested to be an early event in the apoptotic process [35–37]. In recent years, fluorescent dyes (TMRM, Rhod123, JC-1, and DiOC63) for measuring the 4wm have become common and they vary in their degrees of toxicity to mitochondria and cells and degrees of binding to mitochondria [35, 36]. In our study, we chose JC-1, which has
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been reported to be a more reliable indicator of 4wm than other dyes, to measure the changes in 4wm. And the results indicated that ROS-mediated changes in 4wm may contribute to hesperetin-induced apoptosis. It has been demonstrated that Bcl-2 family proteins were one of the components of mitochondrial permeability transition pore (mPTP) [38, 39]. If the Bcl-2/Bax protein ratio is reduced, the structure and permeability of mPTP will be changed which initiates mitochondrial-driven death. To clarify whether the increasing ROS induced by hesperetin led to the apoptosis of gastric cancer cells via mitochondrial pathway, we detected the levels of Bcl-2, Bax, caspase-9, caspase-3, AIF, Apaf-1, and Cyt C using western bolt. It was found that hesperetin caused an increase in the levels of AIF, Apaf-1, Cyt C, caspase-3, caspase-9, and Bax and a decrease in Bcl-2 levels. In addition, when pretreated by H2O2 or NAC, the hesperetin-induced increase or decrease in the levels of mitochondrial pathway proteins above changed. So, it could be considered that hesperetin could upregulate the levels of intracellular ROS, and excessive levels of intracellular ROS may change the structure and permeability of mPTP by decreasing the expression of Bcl2 and increasing the expression of Bax. That in turn releases Cyt C, AIF, and Apaf-1 which then form apoptosome that activates the caspase-9 and caspase-3 and induces apoptosis (Fig. 8) [40]. So far, the views concerning that whether hesperetin induces the apoptosis of gastric cancer cells by decreasing or increasing the generation and accumulation of ROS were inconsistent. Several researchers reported that hesperetin had an antioxidant potential by upregulating the levels of GSH and the expressions of antioxidant enzymes, inactivating NF-kB pathway, downregulating the expressions of iNOS and COX-2, and eliminating the levels of ONOO- [19, 41–44]. However, many studies indicated that hesperetin was an oxidant which could activate ASK1/JNK pathway, downregulate the levels of antioxidants, and upregulate the levels of oxidants [32, 45, 46]. Regarding the different opinions, we think that there are two reasons which could explain that: firstly, the generation and elimination of ROS are highly complex processes. The balance of ROS levels is influenced by various biological processes and cellular response to internal or external stimuluses, and the imbalance of ROS levels has impacts on a lot of signaling pathways and plays crucial roles in a series of cellular processes, because hesperetin, which has various pharmacological activities, may have effects on several different processes of the generation and elimination of ROS which may both increase and decrease the levels of ROS. Secondly, the actions of hesperetin in cancer cells and normal cells as well as in different kinds of cancer cells are diverse. Thus, in a cell line, hesperetininduced generation of ROS may cover up the elimination
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Fig. 8 Proposed schema for hesperetin-induced apoptosis in gastric cancer cell
and then oxidant potential is shown. In turn, in other cell lines, hesperetin-induced elimination of ROS may be greater and the antioxidant potential could be observed. In conclusion, these results provide strong molecular evidence in support of our hypothesis that hesperetin inhibits the proliferation and induces the apoptosis of gastric cancer cells via triggering the activation of the mitochondrial pathway by increasing levels of intracellular ROS. This study identifies the potential usefulness of hesperetin, a non-toxic and safe anti-tumor agent, in the management of gastric cancer.
6.
7.
8.
9.
10. Conflict of interest
None.
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