J Neurooncol (2014) 116:221–230 DOI 10.1007/s11060-013-1292-2
LABORATORY INVESTIGATION
18beta-Glycyrrhetinic acid induces apoptosis in pituitary adenoma cells via ROS/MAPKs-mediated pathway Di Wang • Hei-Kiu Wong • Yi-Bin Feng Zhang-Jin Zhang
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Received: 15 May 2013 / Accepted: 21 October 2013 / Published online: 27 October 2013 Ó Springer Science+Business Media New York 2013
Abstract The purpose of the present study was to evaluate the anti-tumor effects of 18beta-glycyrrhetinic acid (GA), a natural compound extracted from liquorice, against pituitary adenoma and its underlying mechanisms in cultured cells and mouse model of xenografted tumor. GA induced cellular damage in rat pituitary adenoma-derived MMQ and GH3 cells, manifested as reduced cell viability, increased lactate dehydrogenase release, elevated intracellular reactive oxygen species (ROS) and Ca2? concentration. GA also caused G0/G1 phase arrest, increased apoptosis rate and increased mitochondrial membrane permeabilization by suppressing the mitochondrial membrane potential and down-regulating a ratio of B cell lymphoma 2 (Bcl-2) and Bax. GA activated calcium/calmodulin-dependent protein kinase II (CaMKII), c-Jun N-terminal kinase (JNK) and P38; but these activating effects were attenuated by pretreatment with N-acetyl-L-cysteine, a ROS inhibitor. Pretreatment with KN93, a CaMKII inhibitor, also abolished the GA activation of JNK and P38. GA remarkably inhibited growth of pituitary adenoma grafted on nude mice. These results suggest that the anti-pituitary adenoma effect of GA is associated with its apoptotic actions by activating mitochondria-mediated ROS/mitogen-activated protein kinase pathways in Electronic supplementary material The online version of this article (doi:10.1007/s11060-013-1292-2) contains supplementary material, which is available to authorized users. D. Wang H.-K. Wong Y.-B. Feng Z.-J. Zhang (&) School of Chinese Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, China e-mail: [email protected] D. Wang College of Life Science, Jilin University, Changchun 130012, Jilin, China
particular CaMKII that may serve a linkage between ROS accumulation and the activation of JNK and P38. This study provides experimental evidence in the support of further developing GA as a chemotherapeutic agent for pituitary adenoma. Keywords 18beta-Glycyrrhetinic acid Apoptosis ROS/MAPKs Mitochondria
Introduction Pituitary adenoma accounts for about 15 % intracranial neoplasms [1]. Although most pituitary adenoma is benign, it could nonetheless cause significant morbidity and mortality [1]. Previous studies have demonstrated that pituitary adenoma is associated with prolactin (PRL) hyper-secretion which may lead to amenorrhoea, galactorrhea, sexual impairments, and infertility [1]. Treatment of pituitary tumors with conventional anti-tumor agents, represented by bromocriptine (BMT) often cause broad adverse side effects, including nausea, headache, dizziness, and even psychosis and locomotor symptoms [2]. Therefore, the search for novel therapeutic agents is highly desired. Our previous studies have found that an herbal preparation called peony-glycyrrhiza decoction (PGD) made from Paeonia and Glycyrrhiza radices significantly lowered pathologically high PRL level in schizophrenic patients with hyperprolactimia [3] and in animal model [4]. 18betaGlycyrrhetinic acid (GA) is a major compound separated from Glycyrrhiza Radix. While GA has been shown to have anti-inflammatory and anti-viral effects [5–8], it also displays cytotoxic effects against human ovarian cancer [8], hepatocellular carcinoma [9] and breast cancer [10]. Our preliminary study has further revealed an effect of GA
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in lowering hyperprolactimia in rat model. These findings suggest that GA may possess the chemotherapeutic potential for pituitary adenoma and deserved to be further investigated. Apoptosis is a complex programmed cell death that is associated with various signaling pathways. Reactive oxygen species (ROS) are produced from cellular oxidative stress and can induce apoptosis via interaction with proteins related with mitochondrial function [11]. Cellular ROS accumulation may result in intracellular Ca2? overload [12] and activation of mitogen-activated protein kinase (MAPKs), including extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNK) and P38 [13]. The purpose of this study was to determine the antitumor effects of GA against pituitary adenoma and its underlying mechanisms in MMQ and GH3 cells which are derived from pituitary adenoma. The effects of GA on cell viability, lactate dehydrogenase release, intracellular ROS and Ca2? concentration and various proteins associated with apoptosis and ROS/MAPKs pathway were evaluated in these two cell lines. The anti-tumor effect of GA was also determined in nude mice grafted with GH3 tumor.
were divided into two groups (n = 3 each) randomly; 20 mg/kg of GA (treatment group) or equivalent volume of PBS (vehicle group) was administered intraperitoneally once every 2 days for 12 days. Tumor size was measured every 2 days using calipers and the tumor volume (mm3) was estimated using the equation: length 9 (width)2 9 0.5. Fold of tumor size in the curves was normalized by tumor size at day 0. Animals were sacrificed at the end of experiment with 200 mg/kg pentobarbital and tumors were carefully dissected.
Materials and methods
Released lactate dehydrogenase (LDH) analysis
Cell culture
Released LDH in cultured medium was measured using in vitro Toxicology Assay Kit (Sigma-Aldrich, USA) [16]. Following 24 h treatment, medium in each group was collected. 60 lL mixed assay solution was added into 30 lL cultured medium, after 30 min incubation at room temperature in darkness, 10 lL 1 N HCl was added to terminate the reaction. Absorbance was spectrophotometrically measured at a wavelength of 490 nm.
MMQ and GH3, rat pituitary adenoma cells, were cultured in F12 and F10 medium respectively supplemented with 10 % horse serum (HS), 5 % fetal bovine serum (FBS), 100 units/ml penicillin and 100 lg/ml streptomycin under a humidified atmosphere containing 5 % CO2 and 95 % air at 37 °C [14]. The cultured medium was refreshed every 3 days. Cells were ready for treatment when reached 70 % confluence in the plates. Cell culture reagents were obtained from Invitrogen, USA. Mouse mode of GH3-xenografted tumor Animal experiments were conducted in accordance with the NIH Guidelines for the Committee on the Use of Live Animals in Teaching and Research (CULATR) of Li Ka Shing Faculty of Medicine of the University of Hong Kong. BALB/c athymic nude mice were housed in groups of two in clear plastic cages and maintained on a 12-h light/ dark cycle (lights on 07:00–19:00 h) at 23 ± 1 °C with water and food available ad libitum. Cultured GH3 cells (5 9 106 per mouse) were subcutaneously (s.c.) injected into right side of waist of 5-week-old male BALB/c athymic nude mice. After 3–5 days, when the largest diameter of the tumors measured 3–5 mm, the mice
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Cell viability analysis Cell viability was measured by a quantitative colorimetric assay with 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) [15]. MMQ and GH3 cells were seeded into 96-well plates and exposed to GA (Source leaf biological technology Co, LTD, Shanghai, China; purity [98.0 %) or BMT (Sigma, USA) for 24 h. Treated cells were incubated with 0.5 mg/mL MTT for 4 h at 37 °C in darkness. Purple formazan crystals were solubilized by adding 100 ll of DMSO and the absorbance was measured using a microplate reader at a wavelength of 540 nm (BioRad, USA).
Cellular and nuclear morphology analysis Nucleus morphological alterations were analyzed using Hoechst 33342 staining. Cells (1 9 106 cells per well) were seeded into 6-well plates. After 24-h treatment, cells were incubated with Hoechst 33342 (5 lg/ml, SigmaAldrich, USA) for 15 min at 37 °C in darkness. Cellular morphology was detected by normal photography. Nucleus morphology was determined by laser scanning confocal microscopy with an excitation wavelength of 350 nm and an emission wavelength of 460 nm (209, Axio Observer Z1; Carl Zeiss, Germany). Flow cytometric analysis of apoptosis Cellular apoptotic alterations were examined with a twocolor analysis of fluorescein isothiocyanate (FITC)-labeled
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annexin V binding and the uptake of propidium iodide (PI) [17]. After 24 h treatment, the density of cells collected was adjusted to 1 9 106 cells/ml. Cells were suspended in binding buffer containing annexin V-FITC (20 lg/ml) and PI (50 lg/ml), and incubated for 10 min at room temperature. The intensity of fluorescence was analyzed using flow cytometer (FC500, Beckman Coulter, USA). Assessment of cell cycle After exposure to GA for 24 h, cells were collected and fixed in 70 % ethanol at 4 °C overnight. Cells were stained using 50 mg/ml PI for 30 min at room temperature in darkness. After three washes with PBS, the cell cycle was analyzed with flow cytometry (FC500, Beckman Coulter, USA). Measurement of ROS DCFH-DA (Sigma-Aldrich, USA) staining was used to measure intracellular ROS levels. After 12 h treatment, cells were incubated with 10 lM DCFH-DA in PBS at 37 °C for 10 min. After three washes with PBS, fluorescence intensity was analyzed by flow cytometry (FC500, Beckman Coulter, USA).
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using a 12 % SDS-PAGE gel and transferred electrophoretically onto nitrocellulose membranes (Bio Basic, Inc.). The transferred membranes were then blotted with following primary antibodies against phosphorylated (P) and total proteins (T) at 4 °C overnight at dilution of 1:1000: phosphorCaMKII (P-CaMKII), total-CaMKII (T-CaMKII), phosphorERKs (P-ERKs), total ERKs (T-ERKs), phosphor-JNK (PJNK), total-JNK (T-JNK), phosphor-P38 (P-P38), total-P38 (T-P38), B-cell lymphoma 2 (Bcl-2), Bax, Cleaved Caspase3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Beverly, MA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Chemiluminescence was detected using ECL detection kits (GE Healthcare, UK). The intensity of the bands was quantified by scanning densitometry using software Quantity One-4.5.0. Statistical analysis All values were expressed as mean ± S.D. One-way analysis of variance (ANOVA) was used to detect statistical significance, followed by post hoc multiple comparisons (Dunn’s test). A value of P \ 0.05 was considered to be significant.
Measurement of intracellular Ca2? concentration Results MMQ and GH3 cells were treated with GA for 12 h were then collected and incubated 20 min with Ca2? sensitive probe Fluo-4-AM (Invitrogen, USA) with a final concentration of 5 lM at 37 °C [18]. After three washes to remove excess probe, the changes of intracellular Ca2? concentration were analyzed by flow cytometry (FC500, Beckman Coulter, USA). Measurement of mitochondrial membrane potential (MMP, Dwm) 5,50 ,6,60 -tetrachloro-1,10 ,3,30 tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Sigma-Aldrich, USA) staining was used to measure the Dwm changes [19]. After 12 h treatment, cells were incubated with 2 lM JC-1 at 37 °C for 5 min in darkness. Following three washes with PBS, changes in fluorescent color in the mitochondria were examined using fluorescent microscope (Axio Observer Z1, CCD; Carl Zeiss, Germany). Data were expressed as a ratio of red to green fluorescent intensity. Western blot analysis Western blot method used in this study has been reported previously [20]. Briefly, collected cell lysates were separated
GA exhibits potent cytotoxicity and induces apoptosis in pituitary adenoma cells BMT, a commonly used drug for pituitary adenoma, was applied as a positive control. IC50 values obtained from 24-h GA treatment were 69.6 lM in GH3 cells and 111.5 lM in MMQ cells, much lower than 233.6 lM in GH3 cells and 200.6 lM in MMQ cells treated with BMT (Fig. 1a), indicating a more efficient cytotoxic effect of GA than BMT under this experimental setting. GA treatment for 24 h also significantly increased the release of LDH in the two cell lines in a dose-dependent manner (P \ 0.01; Fig. 1b). Cellular morphologic analysis revealed that GA treatment for 24 h resulted in cell shrinkage, detachment and even gathered to a mass. Hoechst staining showed that GA significantly enhanced blue fluorescence intensity in both two cell lines, indicating an effect of GA in inducing nucleus apoptotic damage (Fig. 1c). Exposure to GA for 24 h significantly increased the early and median apoptosis rate in both cell lines in a dose-dependent manner (Fig. 1d). The accumulation of G0/G1 phase also reached a peak value of 14.9 % in GH3 and MMQ cells exposed to GA for 24 h compared to untreated cells (Fig 1e).
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Fig. 1 GA treatment for 24 h induced apoptotic cell death in both GH3 and MMQ cells. a Various doses of GA and BMT reduced cell viability determined with MTT. b GA enhanced LDH release. c 24-h GA treatment resulted in cellular and nucleus apoptotic morphologic alteration in both cell lines which was determined by Hoechst 33342 staining. Scale bar 200 lm. d Enhanced apoptosis rate was observed
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in pituitary adenoma cells after exposure to GA for 24 h (n = 3). e G1 phase was strikingly accumulated after exposure to GA for 24 h (n = 3). Data are transformed as percentage of control values and expressed as mean ± S.D.: *P \ 0.05, **P \ 0.01 and ***P \ 0.001 versus control group
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Fig. 2 ROS-dependent mitochondrial pathway contributes to GAmediated cell apoptotic death. a The dissipation of Dwm was observed in cells exposed to GA for 12 h. b The activation of Caspase3 was strongly enhanced in cells exposed to GA for 24 h. c 24-h GA treatment resulted in a reduction of Bcl-2 expression and an increase of Bax expression. d Intracellular ROS was strikingly accumulated in
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pituitary adenoma cells after 12-h GA treatment. e GA-mediated apoptotic cell death was partially abolished by pretreatment with 1 mM NAC for 30 min, followed by co-treatment with GA for 24 h. Data are transformed as percentage of control values and expressed as mean ± S.D. (n = 3): *P \ 0.05, ***P \ 0.001 versus control group, #P \ 0.05 and ##P \ 0.01 versus GA-treated group
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Fig. 3 ROS-dependent MAPKs activation was involved in GAmediated cell apoptotic death. a GA enhanced the phosphorylation of P38 and JNK in a time-dependent manner, but had no effects on ERKs activation. GA-mediated apoptotic cell death b and activation of MAPKs (P38 and JNK) c were attenuated by pretreatment with 10 lM SP60125 and 10 lM SB203580 for 30 min, respectively.
d The activation of JNK and P38 in cells exposed to GA for 3 h was abolished by pretreatment with 1 mM NAC for 1 h. Data are transformed as percentage of control values and expressed as mean ± S.D. (n = 3): *P \ 0.05 and ***P \ 0.001 versus control group, #P \ 0.05, ##P \ 0.01 and ###P \ 0.001 versus GA-treated group
ROS-dependent mitochondrial dysfunction contributes to GA-induced cell damage
in MMQ cells (Fig. 2a). GA treatment also enhanced activation of Caspase 3 (Fig. 2b) and activated the expression of Bax, but suppressed the expression of Bcl-2 (Fig. 2c). These data indicate that mitochondrial dysfunction contributes to GA-induced pituitary adenoma cell apoptotic death. Fluorescence intensity of DCFH-DA was
GA significantly induced dissipation of Dwm with 43.8 ± 6.5 % compared to 25.6 ± 9.3 % of controls (P \ 0.05) in GH3 cells and 28.3 ± 4.7 versus 18.3 ± 2.2 % (P \ 0.05)
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Fig. 4 CaMKII serves a linkage between ROS accumulation and the activation of MAPKs. a Intracellular Ca2? concentration was enhanced after 12-h GA treatment, analyzed using Fluo-4-AM staining. b GA enhanced the phosphorylation of CaMKII in a timedependent fashion. c GA-reduced cell viability was partially abolished by pretreatment with 5 lM KN93 for 30 min. d GA-
increased the activation of JNK and P38 was suppressed by pretreatment with 5 lM KN93 for 30 min. e GA-increased activation of CaMKII was suppressed by pretreatment with 1 mM NAC for 1 h. Data are expressed as percent of values in control group and the mean ± S.D. (n = 3): **P \ 0.01 and ***P \ 0.001 versus control group, ##P \ 0.01 and ###P \ 0.001 versus GA-treated group
analyzed with flow cytometry. The increment of intracellular ROS levels were expressed as percentage changes in Fig. 2d. In the presence of GA for 12 h, intracellular ROS levels were significantly increase by 16.3 % in GH3 and 27.9 % in MMQ cells (Fig. 2d). Further experiments showed that the reduced cell viability caused by GA was strikingly abolished by pretreatment with 1 mM NAC, a ROS inhibitor, for 1 h in the two cell lines (P \ 0.05) (Fig. 2e), indicating that GA-induced apoptosis is associated with intracellular ROS accumulation.
compared to GA treatment alone. Pretreatment with 1 mM NAC for 1 h almost completely inhibited the GA-activation of JNK and P38 (Fig. 3d), indicating that GA exerts its cytotoxic effects on pituitary adenoma cells via the activation of JNK and P38 by intracellular ROS accumulation.
Activation of MAPKs is involved in GA-induced cell apoptotic death GA enhanced the phosphorylation of JNK and P38 as early as 0.5 h, and reached the peak at 3 h, but had a least effect on the activation of ERKs (Fig. 3a). Pretreatment with 10 lM SP60125, a JNK inhibitor, or 10 lM SB203580, a P38 inhibitor, for 30 min, significantly attenuated the magnitude of GA-induced decrement of cell viability and reversed the phosphorylation of JNK and p38 (Fig. 3b, c)
CaMKII serves a linkage between the activation of MAPKs and intracellular ROS accumulation Compared with control cells, the exposure to GA for 12 h resulted in a striking elevation of the intracellular Ca2? level by 32.3 % GH3 cells and 26.4 % in MMQ cells which were analyzed by flow cytometry after Fluo-4-AM staining (Fig. 4a). GA also enhanced the phosphorylation of CaMKII in both cell lines as early as 0.5 h, and this enhancement reached the peak at 3 h (Fig. 4b). Pretreatment with 5 lM KN93, a CaMKII inhibitor, for 30 min markedly attenuated GA-induced suppression of cell viability (Fig. 4c) and enhancement of JNK and P38 activation (Fig. 4d). Pretreatment with the ROS inhibitor NAC at 1 mM also pronouncedly eliminated the magnitude of GA-
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Fig. 5 Inhibitory effect of GA on GH3 cells tumor growth in xenografted mouse model. a Tumor growth in GH3-xenografted nude mice administrated with PBS or 20 mg/kg GA. b Tumor volumes were measured every other day. Fold of tumor size in the curves was normalized by tumor size measured in day 0. c Bodyweight was not
different between GA- and vehicle-treated animals (mean ± S.D). d The expression of apoptosis related protein in tumor tissues was determined by Western blot. Data are expressed as mean ± S.D. (n = 3): *P \ 0.05 versus vehicle group
induced expression of P-CaMKII (Fig. 4e), indicating that GA enhancement of phosphorylation of JNK and P38 is associated with the activation of CaMKII by intracellular ROS accumulation.
pathway. This finding is consistent with previous studies, showing that GA induces apoptosis in several cancer cell lines [8, 10, 21]. In the current study, the incremental intracellular ROS level was observed after the exposure to GA. Moreover, GA depleted the mitochondrial membrane potential in cells and down-regulated Bcl-2 expression, but up-regulated Bax expression. It is known that mitochondria are the main source of ROS in the cell and intracellular ROS accumulation contributes to various types of cancer cell apoptosis [22]. ROS accumulation directly causes the opening of the mitochondrial permeability transition pore, altering the mitochondrial membrane potential [23]. Mitochondrial dysfunction plays a central role in chemotherapeutic agentinduced apoptosis [10, 24] and increases the ratio of Bax/ Bcl-2 which is a hallmark used to detect cell death caused by mitochondria dysfunction [25]. It appears that increased ROS accumulation that immediately results in mitochondria-related cell death is an initial upstream mechanism responsible for GA-induced apoptosis in pituitary adenoma cells. It is known that increased ROS production in a cell leads to the activation MAPKs and then in turn influences mitochondrial function and activates caspases [26–28]. In the present study, we found that, while GA enhanced the activation of JNK and P38, the two key kinases of MAPKs, these GA enhancing effects were suppressed by the ROS
GA inhibits GH3 cells xenografted tumor growth in nude mice Repeated treatment with 20 mg/kg GA significantly inhibited the tumor growth in nude mice (Fig. 5a). The tumor size only grew by 18.3 ± 6.6 folds in GA-treated mice, significantly smaller than 32.6 ± 6.3 folds in vehicle-treated mice (P \ 0.05) (Fig. 5b). Bodyweight of GAtreated mice was not significantly different from that of vehicle-treated mice (Fig. 5c). GA chronic treatment also significantly suppressed the expression of the anti-apoptosis protein Bcl-2 in tumor cells, but enhanced the expressions of the pro-apoptosis protein Bax and Cleaved Caspase 3 in tumor cells compared to vehicle-treated mice (P \ 0.05) (Fig. 5d).
Discussion The principal finding of the present study is that GA exerts the anti-tumor effects against pituitary adenoma by inducing apoptotic cell death via ROS/MAPKs-mediated
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inhibitor NAC, suggesting that the activation of MARK pathway is also involved in GA-induced pituitary adenoma cell death and ROS may serve a linkage between MAPKs and mitochondrial apoptotic pathway. The present study showed that, while GA exposure increased intracellular ROS accumulation and Ca2? influx, it also facilitated the phosphorylation of CaMKII, a conduit for translation of cytosolic Ca2? influx, but the ROS inhibitor NAC suppressed GA-enhanced phosphorylation of CaMKII. CaMKII is known to be activated by intracellular Ca2? overload [29]. Increased ROS accumulation enhances intracellular Ca2? concentration, resulting in the activation of CaMKII [30–32]. These data suggest a link between CaMKII activation and ROS accumulation and that the two events are involved in the anti-tumor effects of GA. We further revealed that the addition of CaMKII inhibitor KN93 robustly inhibited GA-induced JNK and P38 activation, confirming the linkage between the activation of CaMKII and MAPKs. CaMKII-dependent apoptosis has been observed in human and mouse cells associated with endoplasmic reticulum stress-induced pathology; the activation of JNK is involved in this effect [33]. The activation of JNK and P38 are dependent on Ca2? influx and this activation are partially reduced by calmodulin-dependent kinase inhibitors, such as KN93 [34]. Taken together, CaMKII may be an intermediate between ROS accumulation and the activation of MAPKs (JNK and P38). We noticed that the concentration of 50–100 lM GA used in our culture cell experiments was comparable to a previous study [35], where *21–170 lM was used in hepatocellular carcinoma culture cells, but the dose used in mice with GH3-xenografted tumor was much lower, with 20 mg/kg of GA once every 2 days, than the previous study with nearly 50 mg/kg once daily [35], suggesting that the GA may be more potent in inhibiting pituitary adenoma growth. In addition, GH3 cells seemed to be more sensitive to GA than MMQ cells. The main difference between the two cell lines is that MMQ expresses active dopamine D2 receptors but GH3 cells do not. In a separate experiment, we found that GA enhanced the expressions of the D2 receptors and dopamine transporters in rat adrenal pheochromocytoma (PC12) cells and suppressed prolactin synthesis and release in MMQ, but not in GH3 cells (supplemental data). GA also suppressed abnormally higher serum level of prolactin in D2 receptor blockerinduced hyperprolactimia in rats. Since pituitary adenoma is a major pathological factor contributing to prolactin over-secretion [1], the relationship among GA-induced pituitary adenoma cell apoptosis, dopaminergic neuronal system and prolactin secretion deserves further exploration. Collectively, GA-induced apoptosis in pituitary adenoma cells is associated with its modulation of ROS/ MAPK-dependent pathway. Increased ROS accumulation
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is an initial upstream mechanism by which CaMKII and MARKs are activated, leading to mitochondrial dysfunction. CaMKII serves a linkage between the enhancement of ROS accumulation and the activation of MARKs. These results confirm the anti-pituitary adenoma effects of GA and provide experimental evidences in the support of further developing GA as a chemotherapeutic agent for pituitary adenoma. Acknowledgments This study was supported by General Research Fund (GRF) of Research Grant Council of HKSAR (Project Reference No.: 785813) and HKU intramural Seed Funding Program for Basic Research (Project Reference No.: 201210159051). Conflict of interest conflict of interest.
The authors have declared that there is no
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LABORATORY INVESTIGATION
18beta-Glycyrrhetinic acid induces apoptosis in pituitary adenoma cells via ROS/MAPKs-mediated pathway Di Wang • Hei-Kiu Wong • Yi-Bin Feng Zhang-Jin Zhang
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Received: 15 May 2013 / Accepted: 21 October 2013 / Published online: 27 October 2013 Ó Springer Science+Business Media New York 2013
Abstract The purpose of the present study was to evaluate the anti-tumor effects of 18beta-glycyrrhetinic acid (GA), a natural compound extracted from liquorice, against pituitary adenoma and its underlying mechanisms in cultured cells and mouse model of xenografted tumor. GA induced cellular damage in rat pituitary adenoma-derived MMQ and GH3 cells, manifested as reduced cell viability, increased lactate dehydrogenase release, elevated intracellular reactive oxygen species (ROS) and Ca2? concentration. GA also caused G0/G1 phase arrest, increased apoptosis rate and increased mitochondrial membrane permeabilization by suppressing the mitochondrial membrane potential and down-regulating a ratio of B cell lymphoma 2 (Bcl-2) and Bax. GA activated calcium/calmodulin-dependent protein kinase II (CaMKII), c-Jun N-terminal kinase (JNK) and P38; but these activating effects were attenuated by pretreatment with N-acetyl-L-cysteine, a ROS inhibitor. Pretreatment with KN93, a CaMKII inhibitor, also abolished the GA activation of JNK and P38. GA remarkably inhibited growth of pituitary adenoma grafted on nude mice. These results suggest that the anti-pituitary adenoma effect of GA is associated with its apoptotic actions by activating mitochondria-mediated ROS/mitogen-activated protein kinase pathways in Electronic supplementary material The online version of this article (doi:10.1007/s11060-013-1292-2) contains supplementary material, which is available to authorized users. D. Wang H.-K. Wong Y.-B. Feng Z.-J. Zhang (&) School of Chinese Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, China e-mail: [email protected] D. Wang College of Life Science, Jilin University, Changchun 130012, Jilin, China
particular CaMKII that may serve a linkage between ROS accumulation and the activation of JNK and P38. This study provides experimental evidence in the support of further developing GA as a chemotherapeutic agent for pituitary adenoma. Keywords 18beta-Glycyrrhetinic acid Apoptosis ROS/MAPKs Mitochondria
Introduction Pituitary adenoma accounts for about 15 % intracranial neoplasms [1]. Although most pituitary adenoma is benign, it could nonetheless cause significant morbidity and mortality [1]. Previous studies have demonstrated that pituitary adenoma is associated with prolactin (PRL) hyper-secretion which may lead to amenorrhoea, galactorrhea, sexual impairments, and infertility [1]. Treatment of pituitary tumors with conventional anti-tumor agents, represented by bromocriptine (BMT) often cause broad adverse side effects, including nausea, headache, dizziness, and even psychosis and locomotor symptoms [2]. Therefore, the search for novel therapeutic agents is highly desired. Our previous studies have found that an herbal preparation called peony-glycyrrhiza decoction (PGD) made from Paeonia and Glycyrrhiza radices significantly lowered pathologically high PRL level in schizophrenic patients with hyperprolactimia [3] and in animal model [4]. 18betaGlycyrrhetinic acid (GA) is a major compound separated from Glycyrrhiza Radix. While GA has been shown to have anti-inflammatory and anti-viral effects [5–8], it also displays cytotoxic effects against human ovarian cancer [8], hepatocellular carcinoma [9] and breast cancer [10]. Our preliminary study has further revealed an effect of GA
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in lowering hyperprolactimia in rat model. These findings suggest that GA may possess the chemotherapeutic potential for pituitary adenoma and deserved to be further investigated. Apoptosis is a complex programmed cell death that is associated with various signaling pathways. Reactive oxygen species (ROS) are produced from cellular oxidative stress and can induce apoptosis via interaction with proteins related with mitochondrial function [11]. Cellular ROS accumulation may result in intracellular Ca2? overload [12] and activation of mitogen-activated protein kinase (MAPKs), including extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNK) and P38 [13]. The purpose of this study was to determine the antitumor effects of GA against pituitary adenoma and its underlying mechanisms in MMQ and GH3 cells which are derived from pituitary adenoma. The effects of GA on cell viability, lactate dehydrogenase release, intracellular ROS and Ca2? concentration and various proteins associated with apoptosis and ROS/MAPKs pathway were evaluated in these two cell lines. The anti-tumor effect of GA was also determined in nude mice grafted with GH3 tumor.
were divided into two groups (n = 3 each) randomly; 20 mg/kg of GA (treatment group) or equivalent volume of PBS (vehicle group) was administered intraperitoneally once every 2 days for 12 days. Tumor size was measured every 2 days using calipers and the tumor volume (mm3) was estimated using the equation: length 9 (width)2 9 0.5. Fold of tumor size in the curves was normalized by tumor size at day 0. Animals were sacrificed at the end of experiment with 200 mg/kg pentobarbital and tumors were carefully dissected.
Materials and methods
Released lactate dehydrogenase (LDH) analysis
Cell culture
Released LDH in cultured medium was measured using in vitro Toxicology Assay Kit (Sigma-Aldrich, USA) [16]. Following 24 h treatment, medium in each group was collected. 60 lL mixed assay solution was added into 30 lL cultured medium, after 30 min incubation at room temperature in darkness, 10 lL 1 N HCl was added to terminate the reaction. Absorbance was spectrophotometrically measured at a wavelength of 490 nm.
MMQ and GH3, rat pituitary adenoma cells, were cultured in F12 and F10 medium respectively supplemented with 10 % horse serum (HS), 5 % fetal bovine serum (FBS), 100 units/ml penicillin and 100 lg/ml streptomycin under a humidified atmosphere containing 5 % CO2 and 95 % air at 37 °C [14]. The cultured medium was refreshed every 3 days. Cells were ready for treatment when reached 70 % confluence in the plates. Cell culture reagents were obtained from Invitrogen, USA. Mouse mode of GH3-xenografted tumor Animal experiments were conducted in accordance with the NIH Guidelines for the Committee on the Use of Live Animals in Teaching and Research (CULATR) of Li Ka Shing Faculty of Medicine of the University of Hong Kong. BALB/c athymic nude mice were housed in groups of two in clear plastic cages and maintained on a 12-h light/ dark cycle (lights on 07:00–19:00 h) at 23 ± 1 °C with water and food available ad libitum. Cultured GH3 cells (5 9 106 per mouse) were subcutaneously (s.c.) injected into right side of waist of 5-week-old male BALB/c athymic nude mice. After 3–5 days, when the largest diameter of the tumors measured 3–5 mm, the mice
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Cell viability analysis Cell viability was measured by a quantitative colorimetric assay with 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) [15]. MMQ and GH3 cells were seeded into 96-well plates and exposed to GA (Source leaf biological technology Co, LTD, Shanghai, China; purity [98.0 %) or BMT (Sigma, USA) for 24 h. Treated cells were incubated with 0.5 mg/mL MTT for 4 h at 37 °C in darkness. Purple formazan crystals were solubilized by adding 100 ll of DMSO and the absorbance was measured using a microplate reader at a wavelength of 540 nm (BioRad, USA).
Cellular and nuclear morphology analysis Nucleus morphological alterations were analyzed using Hoechst 33342 staining. Cells (1 9 106 cells per well) were seeded into 6-well plates. After 24-h treatment, cells were incubated with Hoechst 33342 (5 lg/ml, SigmaAldrich, USA) for 15 min at 37 °C in darkness. Cellular morphology was detected by normal photography. Nucleus morphology was determined by laser scanning confocal microscopy with an excitation wavelength of 350 nm and an emission wavelength of 460 nm (209, Axio Observer Z1; Carl Zeiss, Germany). Flow cytometric analysis of apoptosis Cellular apoptotic alterations were examined with a twocolor analysis of fluorescein isothiocyanate (FITC)-labeled
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annexin V binding and the uptake of propidium iodide (PI) [17]. After 24 h treatment, the density of cells collected was adjusted to 1 9 106 cells/ml. Cells were suspended in binding buffer containing annexin V-FITC (20 lg/ml) and PI (50 lg/ml), and incubated for 10 min at room temperature. The intensity of fluorescence was analyzed using flow cytometer (FC500, Beckman Coulter, USA). Assessment of cell cycle After exposure to GA for 24 h, cells were collected and fixed in 70 % ethanol at 4 °C overnight. Cells were stained using 50 mg/ml PI for 30 min at room temperature in darkness. After three washes with PBS, the cell cycle was analyzed with flow cytometry (FC500, Beckman Coulter, USA). Measurement of ROS DCFH-DA (Sigma-Aldrich, USA) staining was used to measure intracellular ROS levels. After 12 h treatment, cells were incubated with 10 lM DCFH-DA in PBS at 37 °C for 10 min. After three washes with PBS, fluorescence intensity was analyzed by flow cytometry (FC500, Beckman Coulter, USA).
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using a 12 % SDS-PAGE gel and transferred electrophoretically onto nitrocellulose membranes (Bio Basic, Inc.). The transferred membranes were then blotted with following primary antibodies against phosphorylated (P) and total proteins (T) at 4 °C overnight at dilution of 1:1000: phosphorCaMKII (P-CaMKII), total-CaMKII (T-CaMKII), phosphorERKs (P-ERKs), total ERKs (T-ERKs), phosphor-JNK (PJNK), total-JNK (T-JNK), phosphor-P38 (P-P38), total-P38 (T-P38), B-cell lymphoma 2 (Bcl-2), Bax, Cleaved Caspase3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Beverly, MA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Chemiluminescence was detected using ECL detection kits (GE Healthcare, UK). The intensity of the bands was quantified by scanning densitometry using software Quantity One-4.5.0. Statistical analysis All values were expressed as mean ± S.D. One-way analysis of variance (ANOVA) was used to detect statistical significance, followed by post hoc multiple comparisons (Dunn’s test). A value of P \ 0.05 was considered to be significant.
Measurement of intracellular Ca2? concentration Results MMQ and GH3 cells were treated with GA for 12 h were then collected and incubated 20 min with Ca2? sensitive probe Fluo-4-AM (Invitrogen, USA) with a final concentration of 5 lM at 37 °C [18]. After three washes to remove excess probe, the changes of intracellular Ca2? concentration were analyzed by flow cytometry (FC500, Beckman Coulter, USA). Measurement of mitochondrial membrane potential (MMP, Dwm) 5,50 ,6,60 -tetrachloro-1,10 ,3,30 tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Sigma-Aldrich, USA) staining was used to measure the Dwm changes [19]. After 12 h treatment, cells were incubated with 2 lM JC-1 at 37 °C for 5 min in darkness. Following three washes with PBS, changes in fluorescent color in the mitochondria were examined using fluorescent microscope (Axio Observer Z1, CCD; Carl Zeiss, Germany). Data were expressed as a ratio of red to green fluorescent intensity. Western blot analysis Western blot method used in this study has been reported previously [20]. Briefly, collected cell lysates were separated
GA exhibits potent cytotoxicity and induces apoptosis in pituitary adenoma cells BMT, a commonly used drug for pituitary adenoma, was applied as a positive control. IC50 values obtained from 24-h GA treatment were 69.6 lM in GH3 cells and 111.5 lM in MMQ cells, much lower than 233.6 lM in GH3 cells and 200.6 lM in MMQ cells treated with BMT (Fig. 1a), indicating a more efficient cytotoxic effect of GA than BMT under this experimental setting. GA treatment for 24 h also significantly increased the release of LDH in the two cell lines in a dose-dependent manner (P \ 0.01; Fig. 1b). Cellular morphologic analysis revealed that GA treatment for 24 h resulted in cell shrinkage, detachment and even gathered to a mass. Hoechst staining showed that GA significantly enhanced blue fluorescence intensity in both two cell lines, indicating an effect of GA in inducing nucleus apoptotic damage (Fig. 1c). Exposure to GA for 24 h significantly increased the early and median apoptosis rate in both cell lines in a dose-dependent manner (Fig. 1d). The accumulation of G0/G1 phase also reached a peak value of 14.9 % in GH3 and MMQ cells exposed to GA for 24 h compared to untreated cells (Fig 1e).
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Fig. 1 GA treatment for 24 h induced apoptotic cell death in both GH3 and MMQ cells. a Various doses of GA and BMT reduced cell viability determined with MTT. b GA enhanced LDH release. c 24-h GA treatment resulted in cellular and nucleus apoptotic morphologic alteration in both cell lines which was determined by Hoechst 33342 staining. Scale bar 200 lm. d Enhanced apoptosis rate was observed
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in pituitary adenoma cells after exposure to GA for 24 h (n = 3). e G1 phase was strikingly accumulated after exposure to GA for 24 h (n = 3). Data are transformed as percentage of control values and expressed as mean ± S.D.: *P \ 0.05, **P \ 0.01 and ***P \ 0.001 versus control group
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Fig. 2 ROS-dependent mitochondrial pathway contributes to GAmediated cell apoptotic death. a The dissipation of Dwm was observed in cells exposed to GA for 12 h. b The activation of Caspase3 was strongly enhanced in cells exposed to GA for 24 h. c 24-h GA treatment resulted in a reduction of Bcl-2 expression and an increase of Bax expression. d Intracellular ROS was strikingly accumulated in
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pituitary adenoma cells after 12-h GA treatment. e GA-mediated apoptotic cell death was partially abolished by pretreatment with 1 mM NAC for 30 min, followed by co-treatment with GA for 24 h. Data are transformed as percentage of control values and expressed as mean ± S.D. (n = 3): *P \ 0.05, ***P \ 0.001 versus control group, #P \ 0.05 and ##P \ 0.01 versus GA-treated group
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Fig. 3 ROS-dependent MAPKs activation was involved in GAmediated cell apoptotic death. a GA enhanced the phosphorylation of P38 and JNK in a time-dependent manner, but had no effects on ERKs activation. GA-mediated apoptotic cell death b and activation of MAPKs (P38 and JNK) c were attenuated by pretreatment with 10 lM SP60125 and 10 lM SB203580 for 30 min, respectively.
d The activation of JNK and P38 in cells exposed to GA for 3 h was abolished by pretreatment with 1 mM NAC for 1 h. Data are transformed as percentage of control values and expressed as mean ± S.D. (n = 3): *P \ 0.05 and ***P \ 0.001 versus control group, #P \ 0.05, ##P \ 0.01 and ###P \ 0.001 versus GA-treated group
ROS-dependent mitochondrial dysfunction contributes to GA-induced cell damage
in MMQ cells (Fig. 2a). GA treatment also enhanced activation of Caspase 3 (Fig. 2b) and activated the expression of Bax, but suppressed the expression of Bcl-2 (Fig. 2c). These data indicate that mitochondrial dysfunction contributes to GA-induced pituitary adenoma cell apoptotic death. Fluorescence intensity of DCFH-DA was
GA significantly induced dissipation of Dwm with 43.8 ± 6.5 % compared to 25.6 ± 9.3 % of controls (P \ 0.05) in GH3 cells and 28.3 ± 4.7 versus 18.3 ± 2.2 % (P \ 0.05)
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Fig. 4 CaMKII serves a linkage between ROS accumulation and the activation of MAPKs. a Intracellular Ca2? concentration was enhanced after 12-h GA treatment, analyzed using Fluo-4-AM staining. b GA enhanced the phosphorylation of CaMKII in a timedependent fashion. c GA-reduced cell viability was partially abolished by pretreatment with 5 lM KN93 for 30 min. d GA-
increased the activation of JNK and P38 was suppressed by pretreatment with 5 lM KN93 for 30 min. e GA-increased activation of CaMKII was suppressed by pretreatment with 1 mM NAC for 1 h. Data are expressed as percent of values in control group and the mean ± S.D. (n = 3): **P \ 0.01 and ***P \ 0.001 versus control group, ##P \ 0.01 and ###P \ 0.001 versus GA-treated group
analyzed with flow cytometry. The increment of intracellular ROS levels were expressed as percentage changes in Fig. 2d. In the presence of GA for 12 h, intracellular ROS levels were significantly increase by 16.3 % in GH3 and 27.9 % in MMQ cells (Fig. 2d). Further experiments showed that the reduced cell viability caused by GA was strikingly abolished by pretreatment with 1 mM NAC, a ROS inhibitor, for 1 h in the two cell lines (P \ 0.05) (Fig. 2e), indicating that GA-induced apoptosis is associated with intracellular ROS accumulation.
compared to GA treatment alone. Pretreatment with 1 mM NAC for 1 h almost completely inhibited the GA-activation of JNK and P38 (Fig. 3d), indicating that GA exerts its cytotoxic effects on pituitary adenoma cells via the activation of JNK and P38 by intracellular ROS accumulation.
Activation of MAPKs is involved in GA-induced cell apoptotic death GA enhanced the phosphorylation of JNK and P38 as early as 0.5 h, and reached the peak at 3 h, but had a least effect on the activation of ERKs (Fig. 3a). Pretreatment with 10 lM SP60125, a JNK inhibitor, or 10 lM SB203580, a P38 inhibitor, for 30 min, significantly attenuated the magnitude of GA-induced decrement of cell viability and reversed the phosphorylation of JNK and p38 (Fig. 3b, c)
CaMKII serves a linkage between the activation of MAPKs and intracellular ROS accumulation Compared with control cells, the exposure to GA for 12 h resulted in a striking elevation of the intracellular Ca2? level by 32.3 % GH3 cells and 26.4 % in MMQ cells which were analyzed by flow cytometry after Fluo-4-AM staining (Fig. 4a). GA also enhanced the phosphorylation of CaMKII in both cell lines as early as 0.5 h, and this enhancement reached the peak at 3 h (Fig. 4b). Pretreatment with 5 lM KN93, a CaMKII inhibitor, for 30 min markedly attenuated GA-induced suppression of cell viability (Fig. 4c) and enhancement of JNK and P38 activation (Fig. 4d). Pretreatment with the ROS inhibitor NAC at 1 mM also pronouncedly eliminated the magnitude of GA-
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Fig. 5 Inhibitory effect of GA on GH3 cells tumor growth in xenografted mouse model. a Tumor growth in GH3-xenografted nude mice administrated with PBS or 20 mg/kg GA. b Tumor volumes were measured every other day. Fold of tumor size in the curves was normalized by tumor size measured in day 0. c Bodyweight was not
different between GA- and vehicle-treated animals (mean ± S.D). d The expression of apoptosis related protein in tumor tissues was determined by Western blot. Data are expressed as mean ± S.D. (n = 3): *P \ 0.05 versus vehicle group
induced expression of P-CaMKII (Fig. 4e), indicating that GA enhancement of phosphorylation of JNK and P38 is associated with the activation of CaMKII by intracellular ROS accumulation.
pathway. This finding is consistent with previous studies, showing that GA induces apoptosis in several cancer cell lines [8, 10, 21]. In the current study, the incremental intracellular ROS level was observed after the exposure to GA. Moreover, GA depleted the mitochondrial membrane potential in cells and down-regulated Bcl-2 expression, but up-regulated Bax expression. It is known that mitochondria are the main source of ROS in the cell and intracellular ROS accumulation contributes to various types of cancer cell apoptosis [22]. ROS accumulation directly causes the opening of the mitochondrial permeability transition pore, altering the mitochondrial membrane potential [23]. Mitochondrial dysfunction plays a central role in chemotherapeutic agentinduced apoptosis [10, 24] and increases the ratio of Bax/ Bcl-2 which is a hallmark used to detect cell death caused by mitochondria dysfunction [25]. It appears that increased ROS accumulation that immediately results in mitochondria-related cell death is an initial upstream mechanism responsible for GA-induced apoptosis in pituitary adenoma cells. It is known that increased ROS production in a cell leads to the activation MAPKs and then in turn influences mitochondrial function and activates caspases [26–28]. In the present study, we found that, while GA enhanced the activation of JNK and P38, the two key kinases of MAPKs, these GA enhancing effects were suppressed by the ROS
GA inhibits GH3 cells xenografted tumor growth in nude mice Repeated treatment with 20 mg/kg GA significantly inhibited the tumor growth in nude mice (Fig. 5a). The tumor size only grew by 18.3 ± 6.6 folds in GA-treated mice, significantly smaller than 32.6 ± 6.3 folds in vehicle-treated mice (P \ 0.05) (Fig. 5b). Bodyweight of GAtreated mice was not significantly different from that of vehicle-treated mice (Fig. 5c). GA chronic treatment also significantly suppressed the expression of the anti-apoptosis protein Bcl-2 in tumor cells, but enhanced the expressions of the pro-apoptosis protein Bax and Cleaved Caspase 3 in tumor cells compared to vehicle-treated mice (P \ 0.05) (Fig. 5d).
Discussion The principal finding of the present study is that GA exerts the anti-tumor effects against pituitary adenoma by inducing apoptotic cell death via ROS/MAPKs-mediated
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inhibitor NAC, suggesting that the activation of MARK pathway is also involved in GA-induced pituitary adenoma cell death and ROS may serve a linkage between MAPKs and mitochondrial apoptotic pathway. The present study showed that, while GA exposure increased intracellular ROS accumulation and Ca2? influx, it also facilitated the phosphorylation of CaMKII, a conduit for translation of cytosolic Ca2? influx, but the ROS inhibitor NAC suppressed GA-enhanced phosphorylation of CaMKII. CaMKII is known to be activated by intracellular Ca2? overload [29]. Increased ROS accumulation enhances intracellular Ca2? concentration, resulting in the activation of CaMKII [30–32]. These data suggest a link between CaMKII activation and ROS accumulation and that the two events are involved in the anti-tumor effects of GA. We further revealed that the addition of CaMKII inhibitor KN93 robustly inhibited GA-induced JNK and P38 activation, confirming the linkage between the activation of CaMKII and MAPKs. CaMKII-dependent apoptosis has been observed in human and mouse cells associated with endoplasmic reticulum stress-induced pathology; the activation of JNK is involved in this effect [33]. The activation of JNK and P38 are dependent on Ca2? influx and this activation are partially reduced by calmodulin-dependent kinase inhibitors, such as KN93 [34]. Taken together, CaMKII may be an intermediate between ROS accumulation and the activation of MAPKs (JNK and P38). We noticed that the concentration of 50–100 lM GA used in our culture cell experiments was comparable to a previous study [35], where *21–170 lM was used in hepatocellular carcinoma culture cells, but the dose used in mice with GH3-xenografted tumor was much lower, with 20 mg/kg of GA once every 2 days, than the previous study with nearly 50 mg/kg once daily [35], suggesting that the GA may be more potent in inhibiting pituitary adenoma growth. In addition, GH3 cells seemed to be more sensitive to GA than MMQ cells. The main difference between the two cell lines is that MMQ expresses active dopamine D2 receptors but GH3 cells do not. In a separate experiment, we found that GA enhanced the expressions of the D2 receptors and dopamine transporters in rat adrenal pheochromocytoma (PC12) cells and suppressed prolactin synthesis and release in MMQ, but not in GH3 cells (supplemental data). GA also suppressed abnormally higher serum level of prolactin in D2 receptor blockerinduced hyperprolactimia in rats. Since pituitary adenoma is a major pathological factor contributing to prolactin over-secretion [1], the relationship among GA-induced pituitary adenoma cell apoptosis, dopaminergic neuronal system and prolactin secretion deserves further exploration. Collectively, GA-induced apoptosis in pituitary adenoma cells is associated with its modulation of ROS/ MAPK-dependent pathway. Increased ROS accumulation
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is an initial upstream mechanism by which CaMKII and MARKs are activated, leading to mitochondrial dysfunction. CaMKII serves a linkage between the enhancement of ROS accumulation and the activation of MARKs. These results confirm the anti-pituitary adenoma effects of GA and provide experimental evidences in the support of further developing GA as a chemotherapeutic agent for pituitary adenoma. Acknowledgments This study was supported by General Research Fund (GRF) of Research Grant Council of HKSAR (Project Reference No.: 785813) and HKU intramural Seed Funding Program for Basic Research (Project Reference No.: 201210159051). Conflict of interest conflict of interest.
The authors have declared that there is no
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