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Inhibition of cytochrome P450 2J2 by tanshinone IIA induces apoptotic cell death in hepatocellular carcinoma HepG2 cells
European Journal of Pharmacology 764 (2015) 480–488
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Inhibition of cytochrome P450 2J2 by tanshinone IIA induces apoptotic cell death in hepatocellular carcinoma HepG2 cells Yu Jin Jeon a,1, Joong Sun Kim b,c,1, Geun Hye Hwang a, Zhexue Wu a, Ho Jae Han d, Soo Hyun Park c, Woochul Chang e, Lark Kyun Kim f, You-Mie Lee a, Kwang-Hyeon Liu a,n, Min Young Lee a,n a
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea Research Center, Dongnam Institute of Radiological and Medical Sciences (DIRAMS), Busan, Korea c College of Veterinary Medicine, Chonnam National University, Gwangju, Korea d College of Veterinary Medicine, Seoul National University, Seoul, Korea e Department of Biology Education, College of Education, Pusan National University, Busan, Korea f Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 March 2015 Received in revised form 17 July 2015 Accepted 20 July 2015 Available online 21 July 2015
Cytochrome P450 2J2 (CYP2J2) is highly expressed in human tumors and carcinoma cell lines, and has been implicated in the pathogenesis of human cancers. The aim of this study was to identify a compound that could inhibit the activity of CYP2J2, and to examine its anticancer activity. To identify CYP2J2 inhibitors, 10 terpenoids obtained from plants were screened using astemizole as a CYP2J2 probe substrate in human liver microsomes (HLMs). Of these, tanshinone IIA dose-dependently and non-competitively inhibited CYP2J2-mediated astemizole O-demethylation activity. Tanshinone IIA significantly decreased viability of human hepatoma HepG2 cells and SiHa cervical cancer cells; however, it was not cytotoxic against mouse hepatocytes. Furthermore, treatment of cells with tanshinone IIA significantly increased apoptotic cell death rate, as shown by the increase in Annexin V-stained cell populations, Bcl-2 associated X protein (Bax)/B-cell lymphoma 2 (Bcl-2) ratio, and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage in HepG2 cells. Furthermore, the results of this study showed that tanshinone IIA significantly decreased HepG2 cell-based tumor growth in nude mice in a dose-dependent manner. On the other hand, the tanshinone IIA-induced apoptotic cell death rate was significantly attenuated by enhanced upregulation of CYP2J2 expression. Thus, our data strongly suggest that tanshinone IIA exerts its anticancer effect by inhibiting CYP2J2 activity. & 2015 Elsevier B.V. All rights reserved.
Keywords: Cytochrome P450 2J2 Tanshinone IIA HepG2 cells Anticancer Apoptosis
1. Introduction Cancer is one of the major causes of death worldwide, and only modest progress has been made in reducing the morbidity and mortality rates of this disease (Hail, 2005). Throughout history, natural products have been well recognized as sources of potential drug candidates for the treatment of human diseases, including cancer. Several important anticancer agents have been obtained from natural sources either by structural modification of natural compounds or synthesis of novel compounds. Vincristine, irinotecan, etoposide, and paclitaxel are classic examples of plantderived compounds; actinomycin D, mitomycin C, bleomycin, n
Corresponding authors. Fax: þ 82 53 950 8557. E-mail addresses: [email protected] (K.-H. Liu), [email protected] (M.Y. Lee). 1 These authors contributed equally to this work
http://dx.doi.org/10.1016/j.ejphar.2015.07.047 0014-2999/& 2015 Elsevier B.V. All rights reserved.
doxorubicin, and L-asparaginase are drugs derived from microbial sources; and cytarabine is a drug that originated from a marine source (Nobili et al., 2009). All these drugs exhibit different intracellular mechanisms, including interaction with microtubules, inhibition of topoisomerases I or II, alkylation of DNA, and interference with tumor signal transduction. In the present study, we focused on cytochrome P450 epoxygenase 2J2 (CYP2J2) as an intracellular target for cancer. CYP2J2 is known to metabolize arachidonic acids to epoxyeicosatrienoic acids (EETs) (Xu et al., 2011). Accumulating evidence indicates that CYP2J2 is highly expressed in a variety of cancer cells and tissues. Elevated CYP2J2 mRNA and protein levels have been observed in diverse human-derived cancer cell lines and human cancer tissues but not in non-cancer cell lines or adjacent normal tissues (J.G. Jiang et al., 2007). In various tumor types, overexpression of CYP2J2 and elevated EET levels promote tumor malignancy,
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including proliferation and metastasis, while the selective inhibition of CYP2J2 attenuates these effects (Chen et al., 2009, 2011; Jiang et al., 2005; J.G. Jiang et al., 2007). Additionally, Cui et al. (2010) reported that sodium nitroprusside downregulates CYP2J2 mRNA levels and decreases cell survival, while 11,12-EET treatment attenuates sodium nitroprusside-induced effects on HepG2 cell survival. Taken together, these findings suggest that the inhibition of CYP2J2 may be a novel and effective approach for the treatment of human cancer. To identify a CYP2J2-specific inhibitor, 10 terpenoids isolated from medicinal plants were screened using astemizole as a CYP2J2 probe substrate in human liver microsomes (HLMs). The results showed that tanshinone IIA had strong inhibitory effects on CYP2J2-mediated astemizole O-demethylation activity (Liu et al., 2006; Matsumoto and Yamazoe, 2001). As one of the major lipophilic constituents of Salvia miltiorrhiza, tanshinone IIA is known to have anticancer effects against various types of cancer; however, the underlying mechanisms have not been fully elucidated. Although tanshinone IIA has been reported to exhibit various pharmacological activities, including cardioprotective, neuroprotective, hepatoprotective, antiatherosclerotic, and anticancer effects (Liu et al., 2013; Pang et al., 2014; Wei et al., 2013; Yu et al., 2014), recent studies have focused mainly on its anticancer activity and biological mechanisms. Therefore, to confirm the potential anticancer effects of tanshinone IIA, we further evaluated its cancer cell-specific cytotoxicity, and the mechanism by which it inhibited CYP2J2.
2. Materials and methods 2.1. Materials The 10 terpenoids (Table 1 and Fig. 1A) were a gift from the Institute for Korea Traditional Medical Industry (Daegu, Korea). Astemizole and hydroxyebastine were purchased from Toronto Research Chemicals (North York, Canada). Glucose-6-phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PDH), β-nicotinamide adenine dinucleotide phosphate (NADP þ ), propidium iodide (PI), and tanshinone IIA were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pooled HLMs (H161, 5 mg/ml) were purchased from BD Biosciences (San Jose, CA, USA). Eight-week-old male ICR mice were purchased from Daehan Bio Link Co. (Incheon, Korea). Six-week-old male BALB/c nu/nu mice were purchased from the Central Lab., Animal Inc., (Seoul, Korea). All animal handling procedures were conducted in accordance with the Standard Operation Protocols established by the Kyungpook National University. The HepG2 hepatocellular carcinoma cell line was purchased from the Korean Cell Line Bank (Seoul, Korea). Cell Counting Kit-8 (CCK8) was purchased from Dojindo Laboratories (Kumamoto, Japan).
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X-tremeGENE HP DNA transfection reagent was acquired from Roche Applied Science (Mannheim, Germany). CMV-human CYP2J2 cDNA expressing plasmid and Hyclone™ fetal bovine serum (FBS) were acquired from Thermo Scientific (USA). The antiBax, anti-Bcl-2, anti-PARP-1, anti-CYP2J2, goat anti-rabbit IgG, and goat anti-mouse IgG antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, USA). The fluorescein isothiocyanate (FITC)-Annexin V apoptosis assay kit was purchased from BioLegend (USA). The caspase-3/7-Glo assay kit was purchased from Promega (USA). 2.2. CYP2J2 inhibitor screening The inhibitory potential of 10 terpenoids against CYP2J2mediated astemizole O-demethylation was determined using pooled HLMs in the presence or absence of test compounds. Briefly, the incubation mixtures (final volume, 100 μl) containing pooled HLMs (0.25 mg/ml), astemizole (1 μM), and the test compound (5 μg/ml) were preincubated for 5 min at 37 °C. The reaction was initiated by the addition of an NADPH-generating system (1.3 mM NADP þ , 3.3 mM G6P, 3.3 mM MgCl2, and 500 unit/ml G6PDH) after a 5-min pre-incubation step. In order to determine the half-maximal inhibitory concentration (IC50) values of tanshinone IIA against CYP2J2-catalyzed astemizole O-demethylation in HLMs, tanshinone IIA (0–20 μM) was added to reaction mixtures containing astemizole (1 μM). After incubation at 37 °C for 20 min in a thermoshaker, the reactions were terminated by addition of 50 μl of cold methanol containing 100 nM mebendazole (internal standard, IS). After mixing and centrifuging at 13,000g for 5 min at 4 °C, aliquots of the supernatants (1 μl) were analyzed by liquid chromatography-tandem mass spectrometry (LC–MS/MS) as described previously (Lee et al., 2014). 2.3. Isolation of mouse hepatocytes Primary mouse hepatocytes were isolated from mouse livers using the two-step EDTA and collagenase perfusion method (Li et al., 2010). Briefly, after the mouse was anesthetized, the liver was perfused with Krebs–Henseleit buffer without Ca2 þ and SO42 (115 mM NaCl, 25 mM NaHCO3, 5.9 mM KCl, 1.18 mM MgCl2, 1.23 mM NaH2PO4, 6 mM glucose, 0.1 mM EDTA) via the hepatic portal vein to rinse out the blood (flow, 7–9 ml/min for 5 min). Next, the liver was perfused with Krebs–Henseleit buffer without Ca2 þ and SO42 containing 0.02% collagenase and 0.1 mM CaCl2 until the liver appeared soft. The liver was then removed, gently minced, and the cells obtained were dispersed in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, NY, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies). The solution containing the mixed cells and debris was filtered through a 100-μm filter. Subsequently,
Table 1 Compounds evaluated in the cytochrome P450 2J2 (CYP2J2) inhibition assay using human liver microsomes. No.
Compound
Origin
1 2 3 4 5 6 7 8 9 10 11
Pyrethrin I Pyrethrin II Carvacrol Costunolide Androgarpholide Ginkgolide A Ginkgolide B Hederagenin Tanshinone IIA Taraxerol Hydroxy ebastine
BioPurify Phytochemicals BioPurify Phytochemicals Sigma-Aldrich Co. Aucklandia lappa Andrographis paniculata BioPurify Phytochemicals BioPurify Phytochemicals BioPurify Phytochemicals Sigma-Aldrich Co. Styrax japonica
HPLC, high-performance liquid chromatography.
Ltd. Ltd.
Ltd. Ltd. Ltd.
Purity % (HPLC)
Concentration
% Inhibition
Reference
495 495 498 498 499 498 498 499 498 499
5 μg/ml(≒15.2 μM) 5 μg/ml (≒13.4 μM) 5 μg/ml(≒ 33.3 μM) 5 μg/ml (≒21.5 μM) 5 μg/ml(≒15.2 μM) 5 μg/ml(≒ 12.2 μM) 5 μg/ml(≒11.8 μM) 5 μg/ml(≒10.6 μM) 5 μg/ml (≒17.0 μM) 5 μg/ml(≒ 11.7 μM) 5 μg/ml (≒10.3 μM)
30 12 0 0 21 51 1 0 82 30 50
– – – Choi et al. (2008) Yang and Song (2014) – – – – Min et al. (2004)
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Fig. 1. (A) Chemical structures of 10 terpenoids. (B) Inhibitory effects of 10 terpenoids (5 μg/ml) against cytochrome P450 2J2 (CYP2J2)-mediated astemizole O-demethylation activity. (C) Inhibitory effect (IC50) of tanshinone IIA against CYP2J2-catalyzed astemizole O-demethylation activity in HLMs. IC50, half-maximal inhibitory concentration; HLMs, human liver microsomes.
the filtrate was centrifuged at 50g for 1 min at 4 °C, cells were collected and washed with DMEM three times, and then seeded in cell culture plates. Cells were maintained in DMEM high glucose (4.5 g/L) supplemented with 10% FBS, 1% penicillin/streptomycin, 1 mg/ml insulin, and 10 12 M dexamethasone for 24 h at 37 °C in a humidified atmosphere (5% CO2). 2.4. Cell culture The mouse hepatocytes and HepG2 cells were maintained in DMEM high glucose (4.5 g/L) supplemented with 10% FBS and 1% penicillin/streptomycin 100 solution at 37 °C in a humidified atmosphere (5% CO2). One day (24 h) prior to the experiments, the cells were incubated with fresh DMEM without FBS. 2.5. Cell viability assay The cell viability was detected using CCK-8. HepG2 cells were cultured in 96-well plates with three triplicate wells in each group. The cells were treated with conditioned medium as indicated. The CCK-8 solution was added to each well at a 1:10 dilution followed by further incubation at 37 °C for 3 h. Absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Inc., VT, USA). All values are expressed as the mean 7standard error (S.E. M.) of experiments performed in triplicate. The values were
converted from absolute counts to percentage of the control. 2.6. Western blot analysis The cell homogenates (20 μg of protein) were separated using 10–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The blots were then washed with Tris-buffered solution containing Tween-20 (TBST, 10 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.05% Tween-20), blocked with 5% skimmed milk powder in TBST for 1 h, and then incubated for 12 h with the appropriate primary antibody at the dilutions recommended by the supplier (1:1000). The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (1:5000) for 12 h. Protein bands were visualized using an enhanced chemiluminescence detection system (Thermo Scientific, USA) according to the manufacturer's protocol. Densitometric analysis was performed using Image J software from the National Institutes of Health (NIH). 2.7. Flow cytometry After harvesting the cells with 0.25% trypsin–EDTA, the suspended cells were washed twice with cold cell-staining buffer, and then resuspended in Annexin V binding buffer at a concentration
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groups were injected subcutaneously with 3, 10, or 30 mg/kg tanshinone IIA dissolved in 99% ethanol and 1% Tween-20, 5 days a week for 30 days. The dosage was considered optimal for anticancer effects, as reported previously. On day 30 after inoculation of HepG2 cells, body weight was measured, and the tumors were excised and weighed. All experimental procedures were conducted in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, and followed a protocol approved by the Standard Operation Protocols established by the Kyungpook National University. All animals were cared for in accordance with the National Animal Welfare Law of Korea. 2.9. Establishment of CYP2J2-overexpressing stable HepG2 cell line Lentiviral expression constructs containing cDNA encoding human CYP2J2 or green fluorescent protein (GFP) were generated for this study. These transgene cassettes use the cytomegalovirus (CMV) immediate-early promoter. Lentiviral vectors containing the puromycin-resistant gene and packaging vectors were kindly provided by Dr. Yibing Qyang (Yale Cardiovascular Research Center, Yale School of Medicine, USA). For construction, CMV-CYP2J2 and CMV-GFP were subcloned into lentiviral vectors containing the puromycin-resistance gene. In addition, CMV-CYP2J2 and CMV-GFP control viruses were generated by transfecting the lentiviral vector, and packaging them in HEK293T cells. Culture medium containing virus was concentrated by ultracentrifugation at 55,200g for 2 h (Hitachi Koki Co., Japan). HepG2 cells were exposed to concentrated virus-containing medium for 24 h followed by 2 days culturing in basal medium. The virus-infected cells were selected by treatment with puromycin (2 μg/ml) for 1 week. 2.10. Statistical analysis The results are expressed as the mean 7 S.E.M. Differences between mean values were analyzed using Student's t-test. A P value of o0.05 was considered significant. Fig. 2. Effect of tanshinone IIA on the viability of HepG2 cells and primary mouse hepatocytes. (A and B) HepG2 cells and primary mouse hepatocytes were treated with the indicated concentration of tanshinone IIA for 24 h, and cell viability was measured using a CCK-8 reduction assay. Values are expressed as the mean 7 S.E.M. of three experiments with triplicate dishes. nPo 0.05 vs. control. (C) HepG2 cells and primary mouse hepatocytes were homogenized, and the levels of CYP2J2 were determined using western blot analysis using a CYP2J2 antibody. The figure represents three independent experiments. The graph shows mean 7 S.E.M. of three experiments for each condition determined by densitometry relative to β-actin. n o 0.05 vs. control. CCK-8, Cell Counting Kit-8; CYP2J2, cytochrome P450 2J2.
of 1 106 cells/ml. Then, 100 ml of the cell suspension was transferred to a 5-ml conical tube and treated with PI/ Annexin V-FITC. After incubation in the dark for 15 min, 400 ml of Annexin V binding buffer was added to the cells in each tube, and then flow cytometric analysis was performed using a FACS Aria III (BD biosciences, USA). 2.8. In vivo tumor xenograft study HepG2 cells were cultured, detached by trypsinization, washed, and then resuspended in DMEM. Female BALB/c nu/nu mice were used after a 1-week quarantine and acclimatization. All animals were maintained in a room at 23 72 °C with relative humidity of 50 75%, artificial lighting from 08:00 to 20:00 daily, and 13–18 air changes/h. They had ad libitum access to a standard laboratory diet and water. HepG2 cells (1 106 cells per animal in 100 μl PBS) were injected subcutaneously (SC) into the right flank of the mice. After 1 week, the mice were randomly divided into four groups (n ¼6 mice per group). The control (vehicle) group was injected with the same volume of 99% ethanol and 1% Tween-20 (ethanol concentration 10%) 5 days a week for 30 days. The treatment
3. Results 3.1. Screening of inhibitory potential of 10 plant-derived terpenoids against CYP2J2 activity This study investigated the effects of natural terpenoids on the catalytic activity of human CYP2J2 in HLMs, as shown in Fig. 1A. In particular, tanshinone IIA (Fig. 1B) inhibited the CYP2J2-mediated astemizole O-demethylation activity at a concentration of 5 μg/ml (17.0 μM), while the other compounds showed weak or negligible inhibitory effects. Our previous study showed that the hydroxyebastine inhibitory potential against CYP2J2-catalyzed astemizole O-demethylation was concentration-dependent with an IC50 of 10.9 μM (Lee et al., 2014). Therefore, we used hydroxyebastine as a positive control. Since tanshinone IIA showed stronger inhibitory potential than hydroxyebastine against CYP2J2 (4 80% vs. 50% inhibition, respectively), a well-known CYP2J2 inhibitor was used in further experiments to estimate the IC50 of tanshinone IIA (Liu et al., 2006). In this study, the results showed that tanshinone IIA inhibited the CYP2J2-catalyzed astemizole O-demethylation activity with an IC50 of 2.5 μM (Fig. 1C). 3.2. Tanshinone IIA induced apoptotic cell death in HepG2 hepatocellular carcinoma cells To evaluate the cytotoxic effect of tanshinone IIA, HepG2 cells and mouse hepatocytes were treated with various concentrations of tanshinone IIA for 24 h, and cell viability was subsequently
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Fig. 3. Effect of tanshinone IIA on apoptosis of HepG2 cells. (A) HepG2 cells were cultured under the indicated conditions for 24 h. Cell lysates were subjected to western blot analysis using Bax, Bcl-2, and PARP-1-specific antibodies. Figures represent three experiments. Intensities of Bax, Bcl-2, and cleaved PARP-1 were determined by densitometry relative to β-actin, and the Bax:Bcl-2 ratio was calculated. Values are expressed as the mean 7 S.E.M. of three independent experiments. nP o 0.05 s. control. (B) Cells were cultured under the indicated conditions for 24 h, stained with FITC-Annexin V/PI, and then analyzed using conventional flow cytometry. Representative scatter plots are shown in which the upper quadrants represent Annexin V-positive (apoptotic) cells. Values are expressed as the mean7 S.E.M. of three independent experiments. nPo 0.05 vs. control. Bax, Bcl-2 associated X protein; Bcl-2, B-cell lymphoma 2; PARP-1, poly (ADP-ribose) polymerase 1.
examined using the CCK-8 assay. In this experiment, treatment of HepG2 cells with tanshinone IIA at concentrations ranging from 1– 25 μM for 24 h significantly and dose-dependently decreased cell viability (Fig. 2A). While tanshinone IIA decreased the viability of HepG2 cells, in contrast, it increased the viability of mouse hepatocytes (Fig. 2B). These results indicate that tanshinone IIA is specifically cytotoxic to HepG2 hepatocellular carcinoma cells. To investigate the selective mechanism of tanshinone IIA, we analyzed CYP2J2 expression using western blot analysis. As shown in Fig. 2C, HepG2 cells exhibited CYP2J2 protein expression while the mouse hepatocytes did not. Additional experiments were carried out to examine whether the apoptotic pathway was involved in the tanshinone IIA-induced cytotoxicity in HepG2 cells. This involved western blot analysis to examine the expression levels of apoptosis-related proteins, including Bax, Bcl-2, and PARP-1. As shown in Fig. 3A, treatment of HepG2 cells with tanshinone IIA for 24 h caused a dose-dependent increase in Bax expression, and a decrease in Bcl-2 expression. The Bax/Bcl-2 ratio significantly and dose-dependently increased. Tanshinone IIA also increased the cleavage of PARP-1. Furthermore, tanshinone IIA significantly increased the percentage of Annexin V positive cells in a dose-dependent manner (Fig. 3B). These results suggest that tanshinone IIA-induced cancer-specific cytotoxicity is mediated by the apoptotic pathway. 3.3. Tanshinone IIA decreased cell viability in SiHa cervical carcinoma cells To examine the effect of tanshinone IIA in another cell type known to express CYP2J2, we treated the SiHa cervical cancer cell
Fig. 4. Effect of tanshinone IIA on the viability of SiHa cells. SiHa cells were treated with the indicated concentration of tanshinone IIA for 24 h, and cell viability was measured using a CCK-8 reduction assay. Inset represents CYP2J2 expression in HepG2 and SiHa cells. Values are expressed as the mean 7 S.E.M. of three experiments, each with triplicate dishes. nPo 0.05 vs. control. CCK-8, Cell Counting Kit-8; CYP2J2, cytochrome P450 2J2.
line with tanshinone IIA (Jiang et al., 2005). SiHa cells were treated with various concentrations of tanshinone IIA for 24 h, and cell viability was subsequently examined using the CCK-8 assay. As shown in Fig. 4, CYP2J2 expression was observed in SiHa as well as HepG2 cells, and treatment of SiHa cells with tanshinone IIA at concentrations ranging from 1–25 μM for 24 h significantly and dose-dependently decreased cell viability.
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3.4. Tanshinone IIA suppressed HepG2 xenograft growth in nude mice To study the effect of tanshinone IIA on hepatocarcinoma development, we used an in vivo HepG2 xenograft nude mouse model. Mice were treated with the vehicle and three doses of tanshinone IIA (3, 10, and 30 mg/kg/day). Progressive tumor growth was observed in the vehicle-treated group (Fig. 5A). The body weights of the vehicle- and tanshinone IIA-treated mice were compared on day 30 after cancer inoculation. The results did not show any significant difference in body weight when mice were treated with 3, 10, and 30 mg/kg/day of tanshinone IIA for 5 days a week for 30 days (Fig. 5B). However, the 30 mg/kg/day group showed a significant suppression of tumor growth rate when compared with the vehicle group. The 3 and 10 mg/kg/day doses showed a trend to lower tumor volume and tumor weight relative to the vehicle control, although the effect was not statistically significant (Fig. 5C). 3.5. CYP2J2 was involved in tanshinone IIA-induced apoptosis in HepG2 cells To elucidate any direct relationship between CYP2J2 and the tanshinone IIA-induced apoptosis in HepG2 cells, we generated a transgenic HepG2 cell line overexpressing CYP2J2. Cells were infected with CMV-CYP2J2 or CMV-GFP lentivirus, and CYP2J2 protein expression levels were examined. The CMV-GFP virus was used as a positive control. As shown in Fig. 6A and B, CYP2J2 expression was significantly greater in CMV-CYP2J2-infected cells than in wild type HepG2 cells. Interestingly, our data showed that Bcl-2 expression significantly increased following CMV-CYP2J2 virus infection, while the expression levels of Bax and cleaved PARP-1 were not affected (Fig. 6C). Next, we compared the difference in tanshinone IIA reactivity between wild-type HepG2 and CMV-CYP2J2 virus-infected HepG2 cells. As shown in Fig. 7A, the decrease in cell viability following treatment with 10 μM tanshinone IIA was significantly attenuated by CMV-CYP2J2 virus infection. Furthermore, the percentage of Annexin V positive cells decreased more in CMV-CYP2J2 virus-infected cells that in wildtype HepG2 cells (Fig. 7B). Collectively, our results suggest that apoptotic cell death induced by treatment with tanshinone IIA is mediated by inhibition of CYP2J2.
4. Discussion Recently, CYP2J2 and its EET metabolites have been implicated in the pathological development of both solid tumors and hematologic malignancies (Chen et al. 2009, 2011; Freedman et al., 2007; Jiang et al., 2005; J.G. Jiang et al., 2007). Previous reports suggested that either CYP2J2 or its metabolites promote marked proliferation of cancer cells through increased activation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt pathways, and enhanced phosphorylation of epidermal growth factor receptor (EGFR). Additionally, CYP2J2 inhibits carcinoma cell apoptosis by upregulation of the anti-apopFig. 5. Effect of tanshinone IIA treatment on HepG2 hepatocellular carcinoma xenograft. HepG2 (5 106) cells were subcutaneously injected in the right flank of each mouse to initiate ectopic tumor growth. Vehicle or tanshinone IIA was administered at 3, 10, and 30 mg/kg/day (in the vehicle), 5 days a week for 30 days. Treatment started 7 days following tumor cell injection and continued for 30 days. Mice were euthanized on day 30 after inoculation, and then tumors were excised and weighed. (A) Representative images of xenografts. (B) After euthanization, vehicle- and tanshinone IIA-treated mice were weighed. (C) Cancer tumor tissues were weighed after excision. Values are expressed as mean 7 S.E.M. (n¼6 mice per group). nPo 0.05 vs. control.
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Fig. 6. Effect of overexpression of cytochrome P450 2J2 (CYP2J2) in HepG2 cells. (A) Representative images are shown for HepG2 cells infected with CMV-CYP2J2 lentivirus, CMV-GFP lentivirus, or uninfected control. Images were obtained by fluorescence microscopy (Leica DMIL Fluo, 100 ). Scale bar, 100 μm. (B) Cells were infected with either CMV-CYP2J2 or CMV-GFP lentivirus, and cell lysates were subjected to western blot analysis using a CYP2J2-specific antibody. Charts represent three experiments. Values are expressed relative to β-actin levels. Error bars denote the mean 7 S.E.M. for three independent experiments levels. nP o0.05 vs. control. (C) Cells were infected with either CMV-CYP2J2 or CMV-GFP lentivirus, and cell lysates were subjected to western blot analysis using Bax, Bcl-2, and PARP-1-specific antibodies. Charts represent three experiments. Intensities of Bax, Bcl-2, and cleaved PARP-1 were determined using densitometry and expressed relative to β-actin, and the Bax:Bcl-2 ratio was calculated. Values are expressed as the mean 7S.E.M. of three independent experiments. nPo 0.05 vs. control. CMV-CYP2J2, cytomegalovirus-cytochrome P450 2J2; GFP, green fluorescent protein; Bax, Bcl-2 associated X protein; Bcl-2, B-cell lymphoma 2; PARP-1, poly (ADP-ribose) polymerase 1.
totic proteins Bcl-2 and Bcl-xL and down-regulation of the proapoptotic protein Bax (Jiang et al., 2005). Furthermore, C. Jiang et al. (2007); J.G. Jiang et al. (2007) reported that CYP2J2-derived
EET might play an important role in promoting the invasion and metastasis of human cancers through down-regulation of metastatic suppressor genes and up-regulation of metastasis-enhancing
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Fig. 7. Effect of overexpression of cytochrome P450 2J2 (CYP2J2) on tanshinone IIA-induced apoptosis. (A) Wild-type HepG2 or CMV-CYP2J2 lentivirus-infected HepG2 cells were cultured with the indicated concentrations of tanshinone IIA for 24 h, and cell viability was measured using a CCK-8 reduction assay. Values are expressed as the mean 7 S.E.M. for three experiments, each with triplicate dishes. n P o0.05 vs. control. (B) Cells were cultured under the indicated conditions for 24 h, then stained with FITC-Annexin V/PI, and analyzed using conventional flow cytometry. Representative scatter plots are shown, in which the upper quadrants represent Annexin V-positive (apoptotic) cells. Values are expressed as the mean 7 S.E.M. of three independent experiments. nPo 0.05 vs. control. CMVCYP2J2, cytomegalovirus-cytochrome P450 2J2; CCK-8, Cell Counting Kit-8, FITC; fluorescein isothiocyanate; PI, propidium iodide.
genes (C. Jiang et al., 2007; J.G. Jiang et al., 2007). Therefore, CYP2J2 might be a potential target for human cancer therapy. However, limited information is available on CYP2J2 enzyme inhibitors (Lafite et al., 2007; Wu et al., 2013). In the present study, we screened the inhibitory potential of 10
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plant-derived terpenoids against the CYP2J2 enzyme. The effects of these natural products on the catalytic activity of human CYP2J2 were investigated in HLMs. Our results showed that tanshinone IIA inhibited CYP2J2-mediated astemizole O-demethylation activity at a concentration of 5 μg/ml, while the IC50 was 2.5 μM. Additional experiments were carried out to evaluate the effect of tanshinone IIA, which showed an inhibitory effect against CYP2J2 enzyme in vitro and in vivo. Tanshinone IIA showed significant cytotoxicity against HepG2 cells but not against mouse hepatocytes. Previously, it was reported that in humans, the CYP2J subfamily comprises a single gene CYP2J2, which is not expressed in mice (Nelson et al., 2004; Zhang and Ding, 2008). Indeed, in our study, HepG2 cells showed increased CYP2J2 expression while mouse hepatocytes did not. The anticancer effect of tanshinone IIA was also observed in the cervical cancer cell line, SiHa, which expresses CYP2J2. Because mouse hepatocytes can survive without CYP2J2, the target of tanshinone IIA, we hypothesized that survival of mouse hepatocytes was not affected by tanshinone IIA because of the absence of a target for TIIA. Therefore, the different effects of tanshinone IIA in different species, and the protective effect of tanshinone IIA in mouse hepatocytes, might be owing to differential CYP2J2 expression. Furthermore, tanshinone IIA has been reported to have protective effects via additional mechanisms in various organs, including the heart, brain, and liver (Liu et al., 2013; Pang et al., 2014; Park et al., 2009; Wang et al., 2014; Zhang et al., 2012). For example, tanshinone IIA protects ischemic cardiomyocytes from apoptosis through increased miR-133 expression regulated by the MAPK pathway. It was also reported that tanshinone IIA protects hepatocytes from CCl4-induced hepatotoxicity through inhibition of the mitochondrial permeability transition (Zhu et al., 2010). Collectively, our results strongly suggest that tanshinone IIA could be used as a cancer-specific therapeutic agent. A variety of anticancer agents inhibit cancer cell proliferation through an apoptotic mechanism of action (Luo et al., 2014). The Bcl-2 family comprises important regulators of various apoptotic pathways (Sarada et al., 2008). Apoptotic signals upregulate the expression of pro-apoptotic Bcl-2 family members, such as Bax, and downregulate the expression of anti-apoptotic Bcl-2 family members, such as Bcl-2 (Mazumder et al., 2008). In addition, PARP-1, an enzyme necessary for the repair of damaged DNA, is considered a hallmark of apoptosis (Chaitanya et al. 2010). PARP-1 is one of several known cellular substrates of caspases that play central roles in apoptosis. Cleavage of PARP-1 allows the degradation of DNA, a process necessary for apoptosis (Li and Darzynkiewicz, 2000). In this study, tanshinone IIA increased the Bax/ Bcl-2 ratio, levels of cleaved PARP-1, and Annexin V-positive cell population in a dose-dependent manner. Therefore, our results indicate that apoptotic cell death is a major mechanism of cytotoxicity induced by tanshinone IIA. The anticancer effect of tanshinone IIA was also observed in in vivo experiments. The effects of tanshinone IIA treatment on tumor formation were assessed in a mouse xenograft model, which can be used to evaluate potential anticancer drug candidates (Kelland, 2004). In this experiment, tumor formation and size significantly reduced following tanshinone IIA treatment. Based on our results, we hypothesized that if the tanshinone IIA-induced apoptosis of HepG2 cells was owing to the inhibition of CYP2J2, then up-regulation of CYP2J2 should attenuate the effect of tanshinone IIA. In order to obtain direct evidence for the role of CYP2J2 in tanshinone IIA-induced apoptosis, we established a transgenic HepG2 cell line overexpressing CYP2J2, and examined the effect of tanshinone IIA. Our results showed that CMV-CYP2J2 lentivirus infection significantly upregulated CYP2J2 protein expression in transfected cells compared to that in wild type HepG2 cells. Furthermore, up-regulation of CYP2J2 significantly attenuated the tanshinone IIA-dependent reduction in cell viability,
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and diminished the tanshinone IIA-dependent increases in Annexin V-positive cells. These results indicate that CYP2J2 overexpression protects HepG2 cells from apoptosis induced by tanshinone IIA treatment. Therefore, we conclude that tanshinone IIA exerts its anticancer effect directly via inhibition of CYP2J2 activity. Additionally, CYP2J2-overexpressing HepG2 cells showed a significant increase in Bcl-2 expression, while Bax expression and cleaved PARP-1 levels were not changed. These results are supported by a previous study in which elevated expression of CYP2J2 was shown to render carcinoma cells more resistant to apoptotic cell death (Jiang et al., 2005). Thus, our results highlight the anticancer activity of tanshinone IIA and the significance of functional CYP2J2 as a potential antitumor therapeutic target. In conclusion, we have demonstrated that tanshinone IIA has an anticancer effect that is directly due to inhibition of CYP2J2 activity, and that CYP2J2 could be a potential target for various human cancers. Further studies on the roles of CYP2J2 in carcinogenesis would be required to diagnose CYP2J2-dependent cancers, and to develop novel and improved treatments.
Conflicts of interest The authors declare no conflicts of interest.
Acknowledgments We thank Dr. Yibing Qyang for providing the lentiviral vectors. This study was supported by a National Research Foundation of Korea (NRF) Grant (NRF-2012R1A4A1028835) funded by the Korean Government (MSIP).
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Inhibition of cytochrome P450 2J2 by tanshinone IIA induces apoptotic cell death in hepatocellular carcinoma HepG2 cells Yu Jin Jeon a,1, Joong Sun Kim b,c,1, Geun Hye Hwang a, Zhexue Wu a, Ho Jae Han d, Soo Hyun Park c, Woochul Chang e, Lark Kyun Kim f, You-Mie Lee a, Kwang-Hyeon Liu a,n, Min Young Lee a,n a
College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea Research Center, Dongnam Institute of Radiological and Medical Sciences (DIRAMS), Busan, Korea c College of Veterinary Medicine, Chonnam National University, Gwangju, Korea d College of Veterinary Medicine, Seoul National University, Seoul, Korea e Department of Biology Education, College of Education, Pusan National University, Busan, Korea f Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 March 2015 Received in revised form 17 July 2015 Accepted 20 July 2015 Available online 21 July 2015
Cytochrome P450 2J2 (CYP2J2) is highly expressed in human tumors and carcinoma cell lines, and has been implicated in the pathogenesis of human cancers. The aim of this study was to identify a compound that could inhibit the activity of CYP2J2, and to examine its anticancer activity. To identify CYP2J2 inhibitors, 10 terpenoids obtained from plants were screened using astemizole as a CYP2J2 probe substrate in human liver microsomes (HLMs). Of these, tanshinone IIA dose-dependently and non-competitively inhibited CYP2J2-mediated astemizole O-demethylation activity. Tanshinone IIA significantly decreased viability of human hepatoma HepG2 cells and SiHa cervical cancer cells; however, it was not cytotoxic against mouse hepatocytes. Furthermore, treatment of cells with tanshinone IIA significantly increased apoptotic cell death rate, as shown by the increase in Annexin V-stained cell populations, Bcl-2 associated X protein (Bax)/B-cell lymphoma 2 (Bcl-2) ratio, and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage in HepG2 cells. Furthermore, the results of this study showed that tanshinone IIA significantly decreased HepG2 cell-based tumor growth in nude mice in a dose-dependent manner. On the other hand, the tanshinone IIA-induced apoptotic cell death rate was significantly attenuated by enhanced upregulation of CYP2J2 expression. Thus, our data strongly suggest that tanshinone IIA exerts its anticancer effect by inhibiting CYP2J2 activity. & 2015 Elsevier B.V. All rights reserved.
Keywords: Cytochrome P450 2J2 Tanshinone IIA HepG2 cells Anticancer Apoptosis
1. Introduction Cancer is one of the major causes of death worldwide, and only modest progress has been made in reducing the morbidity and mortality rates of this disease (Hail, 2005). Throughout history, natural products have been well recognized as sources of potential drug candidates for the treatment of human diseases, including cancer. Several important anticancer agents have been obtained from natural sources either by structural modification of natural compounds or synthesis of novel compounds. Vincristine, irinotecan, etoposide, and paclitaxel are classic examples of plantderived compounds; actinomycin D, mitomycin C, bleomycin, n
Corresponding authors. Fax: þ 82 53 950 8557. E-mail addresses: [email protected] (K.-H. Liu), [email protected] (M.Y. Lee). 1 These authors contributed equally to this work
http://dx.doi.org/10.1016/j.ejphar.2015.07.047 0014-2999/& 2015 Elsevier B.V. All rights reserved.
doxorubicin, and L-asparaginase are drugs derived from microbial sources; and cytarabine is a drug that originated from a marine source (Nobili et al., 2009). All these drugs exhibit different intracellular mechanisms, including interaction with microtubules, inhibition of topoisomerases I or II, alkylation of DNA, and interference with tumor signal transduction. In the present study, we focused on cytochrome P450 epoxygenase 2J2 (CYP2J2) as an intracellular target for cancer. CYP2J2 is known to metabolize arachidonic acids to epoxyeicosatrienoic acids (EETs) (Xu et al., 2011). Accumulating evidence indicates that CYP2J2 is highly expressed in a variety of cancer cells and tissues. Elevated CYP2J2 mRNA and protein levels have been observed in diverse human-derived cancer cell lines and human cancer tissues but not in non-cancer cell lines or adjacent normal tissues (J.G. Jiang et al., 2007). In various tumor types, overexpression of CYP2J2 and elevated EET levels promote tumor malignancy,
Y.J. Jeon et al. / European Journal of Pharmacology 764 (2015) 480–488
including proliferation and metastasis, while the selective inhibition of CYP2J2 attenuates these effects (Chen et al., 2009, 2011; Jiang et al., 2005; J.G. Jiang et al., 2007). Additionally, Cui et al. (2010) reported that sodium nitroprusside downregulates CYP2J2 mRNA levels and decreases cell survival, while 11,12-EET treatment attenuates sodium nitroprusside-induced effects on HepG2 cell survival. Taken together, these findings suggest that the inhibition of CYP2J2 may be a novel and effective approach for the treatment of human cancer. To identify a CYP2J2-specific inhibitor, 10 terpenoids isolated from medicinal plants were screened using astemizole as a CYP2J2 probe substrate in human liver microsomes (HLMs). The results showed that tanshinone IIA had strong inhibitory effects on CYP2J2-mediated astemizole O-demethylation activity (Liu et al., 2006; Matsumoto and Yamazoe, 2001). As one of the major lipophilic constituents of Salvia miltiorrhiza, tanshinone IIA is known to have anticancer effects against various types of cancer; however, the underlying mechanisms have not been fully elucidated. Although tanshinone IIA has been reported to exhibit various pharmacological activities, including cardioprotective, neuroprotective, hepatoprotective, antiatherosclerotic, and anticancer effects (Liu et al., 2013; Pang et al., 2014; Wei et al., 2013; Yu et al., 2014), recent studies have focused mainly on its anticancer activity and biological mechanisms. Therefore, to confirm the potential anticancer effects of tanshinone IIA, we further evaluated its cancer cell-specific cytotoxicity, and the mechanism by which it inhibited CYP2J2.
2. Materials and methods 2.1. Materials The 10 terpenoids (Table 1 and Fig. 1A) were a gift from the Institute for Korea Traditional Medical Industry (Daegu, Korea). Astemizole and hydroxyebastine were purchased from Toronto Research Chemicals (North York, Canada). Glucose-6-phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PDH), β-nicotinamide adenine dinucleotide phosphate (NADP þ ), propidium iodide (PI), and tanshinone IIA were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pooled HLMs (H161, 5 mg/ml) were purchased from BD Biosciences (San Jose, CA, USA). Eight-week-old male ICR mice were purchased from Daehan Bio Link Co. (Incheon, Korea). Six-week-old male BALB/c nu/nu mice were purchased from the Central Lab., Animal Inc., (Seoul, Korea). All animal handling procedures were conducted in accordance with the Standard Operation Protocols established by the Kyungpook National University. The HepG2 hepatocellular carcinoma cell line was purchased from the Korean Cell Line Bank (Seoul, Korea). Cell Counting Kit-8 (CCK8) was purchased from Dojindo Laboratories (Kumamoto, Japan).
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X-tremeGENE HP DNA transfection reagent was acquired from Roche Applied Science (Mannheim, Germany). CMV-human CYP2J2 cDNA expressing plasmid and Hyclone™ fetal bovine serum (FBS) were acquired from Thermo Scientific (USA). The antiBax, anti-Bcl-2, anti-PARP-1, anti-CYP2J2, goat anti-rabbit IgG, and goat anti-mouse IgG antibodies were supplied by Santa Cruz Biotechnology (Santa Cruz, USA). The fluorescein isothiocyanate (FITC)-Annexin V apoptosis assay kit was purchased from BioLegend (USA). The caspase-3/7-Glo assay kit was purchased from Promega (USA). 2.2. CYP2J2 inhibitor screening The inhibitory potential of 10 terpenoids against CYP2J2mediated astemizole O-demethylation was determined using pooled HLMs in the presence or absence of test compounds. Briefly, the incubation mixtures (final volume, 100 μl) containing pooled HLMs (0.25 mg/ml), astemizole (1 μM), and the test compound (5 μg/ml) were preincubated for 5 min at 37 °C. The reaction was initiated by the addition of an NADPH-generating system (1.3 mM NADP þ , 3.3 mM G6P, 3.3 mM MgCl2, and 500 unit/ml G6PDH) after a 5-min pre-incubation step. In order to determine the half-maximal inhibitory concentration (IC50) values of tanshinone IIA against CYP2J2-catalyzed astemizole O-demethylation in HLMs, tanshinone IIA (0–20 μM) was added to reaction mixtures containing astemizole (1 μM). After incubation at 37 °C for 20 min in a thermoshaker, the reactions were terminated by addition of 50 μl of cold methanol containing 100 nM mebendazole (internal standard, IS). After mixing and centrifuging at 13,000g for 5 min at 4 °C, aliquots of the supernatants (1 μl) were analyzed by liquid chromatography-tandem mass spectrometry (LC–MS/MS) as described previously (Lee et al., 2014). 2.3. Isolation of mouse hepatocytes Primary mouse hepatocytes were isolated from mouse livers using the two-step EDTA and collagenase perfusion method (Li et al., 2010). Briefly, after the mouse was anesthetized, the liver was perfused with Krebs–Henseleit buffer without Ca2 þ and SO42 (115 mM NaCl, 25 mM NaHCO3, 5.9 mM KCl, 1.18 mM MgCl2, 1.23 mM NaH2PO4, 6 mM glucose, 0.1 mM EDTA) via the hepatic portal vein to rinse out the blood (flow, 7–9 ml/min for 5 min). Next, the liver was perfused with Krebs–Henseleit buffer without Ca2 þ and SO42 containing 0.02% collagenase and 0.1 mM CaCl2 until the liver appeared soft. The liver was then removed, gently minced, and the cells obtained were dispersed in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, NY, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies). The solution containing the mixed cells and debris was filtered through a 100-μm filter. Subsequently,
Table 1 Compounds evaluated in the cytochrome P450 2J2 (CYP2J2) inhibition assay using human liver microsomes. No.
Compound
Origin
1 2 3 4 5 6 7 8 9 10 11
Pyrethrin I Pyrethrin II Carvacrol Costunolide Androgarpholide Ginkgolide A Ginkgolide B Hederagenin Tanshinone IIA Taraxerol Hydroxy ebastine
BioPurify Phytochemicals BioPurify Phytochemicals Sigma-Aldrich Co. Aucklandia lappa Andrographis paniculata BioPurify Phytochemicals BioPurify Phytochemicals BioPurify Phytochemicals Sigma-Aldrich Co. Styrax japonica
HPLC, high-performance liquid chromatography.
Ltd. Ltd.
Ltd. Ltd. Ltd.
Purity % (HPLC)
Concentration
% Inhibition
Reference
495 495 498 498 499 498 498 499 498 499
5 μg/ml(≒15.2 μM) 5 μg/ml (≒13.4 μM) 5 μg/ml(≒ 33.3 μM) 5 μg/ml (≒21.5 μM) 5 μg/ml(≒15.2 μM) 5 μg/ml(≒ 12.2 μM) 5 μg/ml(≒11.8 μM) 5 μg/ml(≒10.6 μM) 5 μg/ml (≒17.0 μM) 5 μg/ml(≒ 11.7 μM) 5 μg/ml (≒10.3 μM)
30 12 0 0 21 51 1 0 82 30 50
– – – Choi et al. (2008) Yang and Song (2014) – – – – Min et al. (2004)
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Fig. 1. (A) Chemical structures of 10 terpenoids. (B) Inhibitory effects of 10 terpenoids (5 μg/ml) against cytochrome P450 2J2 (CYP2J2)-mediated astemizole O-demethylation activity. (C) Inhibitory effect (IC50) of tanshinone IIA against CYP2J2-catalyzed astemizole O-demethylation activity in HLMs. IC50, half-maximal inhibitory concentration; HLMs, human liver microsomes.
the filtrate was centrifuged at 50g for 1 min at 4 °C, cells were collected and washed with DMEM three times, and then seeded in cell culture plates. Cells were maintained in DMEM high glucose (4.5 g/L) supplemented with 10% FBS, 1% penicillin/streptomycin, 1 mg/ml insulin, and 10 12 M dexamethasone for 24 h at 37 °C in a humidified atmosphere (5% CO2). 2.4. Cell culture The mouse hepatocytes and HepG2 cells were maintained in DMEM high glucose (4.5 g/L) supplemented with 10% FBS and 1% penicillin/streptomycin 100 solution at 37 °C in a humidified atmosphere (5% CO2). One day (24 h) prior to the experiments, the cells were incubated with fresh DMEM without FBS. 2.5. Cell viability assay The cell viability was detected using CCK-8. HepG2 cells were cultured in 96-well plates with three triplicate wells in each group. The cells were treated with conditioned medium as indicated. The CCK-8 solution was added to each well at a 1:10 dilution followed by further incubation at 37 °C for 3 h. Absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Inc., VT, USA). All values are expressed as the mean 7standard error (S.E. M.) of experiments performed in triplicate. The values were
converted from absolute counts to percentage of the control. 2.6. Western blot analysis The cell homogenates (20 μg of protein) were separated using 10–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. The blots were then washed with Tris-buffered solution containing Tween-20 (TBST, 10 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.05% Tween-20), blocked with 5% skimmed milk powder in TBST for 1 h, and then incubated for 12 h with the appropriate primary antibody at the dilutions recommended by the supplier (1:1000). The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (1:5000) for 12 h. Protein bands were visualized using an enhanced chemiluminescence detection system (Thermo Scientific, USA) according to the manufacturer's protocol. Densitometric analysis was performed using Image J software from the National Institutes of Health (NIH). 2.7. Flow cytometry After harvesting the cells with 0.25% trypsin–EDTA, the suspended cells were washed twice with cold cell-staining buffer, and then resuspended in Annexin V binding buffer at a concentration
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groups were injected subcutaneously with 3, 10, or 30 mg/kg tanshinone IIA dissolved in 99% ethanol and 1% Tween-20, 5 days a week for 30 days. The dosage was considered optimal for anticancer effects, as reported previously. On day 30 after inoculation of HepG2 cells, body weight was measured, and the tumors were excised and weighed. All experimental procedures were conducted in accordance with NIH Guidelines for the Care and Use of Laboratory Animals, and followed a protocol approved by the Standard Operation Protocols established by the Kyungpook National University. All animals were cared for in accordance with the National Animal Welfare Law of Korea. 2.9. Establishment of CYP2J2-overexpressing stable HepG2 cell line Lentiviral expression constructs containing cDNA encoding human CYP2J2 or green fluorescent protein (GFP) were generated for this study. These transgene cassettes use the cytomegalovirus (CMV) immediate-early promoter. Lentiviral vectors containing the puromycin-resistant gene and packaging vectors were kindly provided by Dr. Yibing Qyang (Yale Cardiovascular Research Center, Yale School of Medicine, USA). For construction, CMV-CYP2J2 and CMV-GFP were subcloned into lentiviral vectors containing the puromycin-resistance gene. In addition, CMV-CYP2J2 and CMV-GFP control viruses were generated by transfecting the lentiviral vector, and packaging them in HEK293T cells. Culture medium containing virus was concentrated by ultracentrifugation at 55,200g for 2 h (Hitachi Koki Co., Japan). HepG2 cells were exposed to concentrated virus-containing medium for 24 h followed by 2 days culturing in basal medium. The virus-infected cells were selected by treatment with puromycin (2 μg/ml) for 1 week. 2.10. Statistical analysis The results are expressed as the mean 7 S.E.M. Differences between mean values were analyzed using Student's t-test. A P value of o0.05 was considered significant. Fig. 2. Effect of tanshinone IIA on the viability of HepG2 cells and primary mouse hepatocytes. (A and B) HepG2 cells and primary mouse hepatocytes were treated with the indicated concentration of tanshinone IIA for 24 h, and cell viability was measured using a CCK-8 reduction assay. Values are expressed as the mean 7 S.E.M. of three experiments with triplicate dishes. nPo 0.05 vs. control. (C) HepG2 cells and primary mouse hepatocytes were homogenized, and the levels of CYP2J2 were determined using western blot analysis using a CYP2J2 antibody. The figure represents three independent experiments. The graph shows mean 7 S.E.M. of three experiments for each condition determined by densitometry relative to β-actin. n o 0.05 vs. control. CCK-8, Cell Counting Kit-8; CYP2J2, cytochrome P450 2J2.
of 1 106 cells/ml. Then, 100 ml of the cell suspension was transferred to a 5-ml conical tube and treated with PI/ Annexin V-FITC. After incubation in the dark for 15 min, 400 ml of Annexin V binding buffer was added to the cells in each tube, and then flow cytometric analysis was performed using a FACS Aria III (BD biosciences, USA). 2.8. In vivo tumor xenograft study HepG2 cells were cultured, detached by trypsinization, washed, and then resuspended in DMEM. Female BALB/c nu/nu mice were used after a 1-week quarantine and acclimatization. All animals were maintained in a room at 23 72 °C with relative humidity of 50 75%, artificial lighting from 08:00 to 20:00 daily, and 13–18 air changes/h. They had ad libitum access to a standard laboratory diet and water. HepG2 cells (1 106 cells per animal in 100 μl PBS) were injected subcutaneously (SC) into the right flank of the mice. After 1 week, the mice were randomly divided into four groups (n ¼6 mice per group). The control (vehicle) group was injected with the same volume of 99% ethanol and 1% Tween-20 (ethanol concentration 10%) 5 days a week for 30 days. The treatment
3. Results 3.1. Screening of inhibitory potential of 10 plant-derived terpenoids against CYP2J2 activity This study investigated the effects of natural terpenoids on the catalytic activity of human CYP2J2 in HLMs, as shown in Fig. 1A. In particular, tanshinone IIA (Fig. 1B) inhibited the CYP2J2-mediated astemizole O-demethylation activity at a concentration of 5 μg/ml (17.0 μM), while the other compounds showed weak or negligible inhibitory effects. Our previous study showed that the hydroxyebastine inhibitory potential against CYP2J2-catalyzed astemizole O-demethylation was concentration-dependent with an IC50 of 10.9 μM (Lee et al., 2014). Therefore, we used hydroxyebastine as a positive control. Since tanshinone IIA showed stronger inhibitory potential than hydroxyebastine against CYP2J2 (4 80% vs. 50% inhibition, respectively), a well-known CYP2J2 inhibitor was used in further experiments to estimate the IC50 of tanshinone IIA (Liu et al., 2006). In this study, the results showed that tanshinone IIA inhibited the CYP2J2-catalyzed astemizole O-demethylation activity with an IC50 of 2.5 μM (Fig. 1C). 3.2. Tanshinone IIA induced apoptotic cell death in HepG2 hepatocellular carcinoma cells To evaluate the cytotoxic effect of tanshinone IIA, HepG2 cells and mouse hepatocytes were treated with various concentrations of tanshinone IIA for 24 h, and cell viability was subsequently
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Fig. 3. Effect of tanshinone IIA on apoptosis of HepG2 cells. (A) HepG2 cells were cultured under the indicated conditions for 24 h. Cell lysates were subjected to western blot analysis using Bax, Bcl-2, and PARP-1-specific antibodies. Figures represent three experiments. Intensities of Bax, Bcl-2, and cleaved PARP-1 were determined by densitometry relative to β-actin, and the Bax:Bcl-2 ratio was calculated. Values are expressed as the mean 7 S.E.M. of three independent experiments. nP o 0.05 s. control. (B) Cells were cultured under the indicated conditions for 24 h, stained with FITC-Annexin V/PI, and then analyzed using conventional flow cytometry. Representative scatter plots are shown in which the upper quadrants represent Annexin V-positive (apoptotic) cells. Values are expressed as the mean7 S.E.M. of three independent experiments. nPo 0.05 vs. control. Bax, Bcl-2 associated X protein; Bcl-2, B-cell lymphoma 2; PARP-1, poly (ADP-ribose) polymerase 1.
examined using the CCK-8 assay. In this experiment, treatment of HepG2 cells with tanshinone IIA at concentrations ranging from 1– 25 μM for 24 h significantly and dose-dependently decreased cell viability (Fig. 2A). While tanshinone IIA decreased the viability of HepG2 cells, in contrast, it increased the viability of mouse hepatocytes (Fig. 2B). These results indicate that tanshinone IIA is specifically cytotoxic to HepG2 hepatocellular carcinoma cells. To investigate the selective mechanism of tanshinone IIA, we analyzed CYP2J2 expression using western blot analysis. As shown in Fig. 2C, HepG2 cells exhibited CYP2J2 protein expression while the mouse hepatocytes did not. Additional experiments were carried out to examine whether the apoptotic pathway was involved in the tanshinone IIA-induced cytotoxicity in HepG2 cells. This involved western blot analysis to examine the expression levels of apoptosis-related proteins, including Bax, Bcl-2, and PARP-1. As shown in Fig. 3A, treatment of HepG2 cells with tanshinone IIA for 24 h caused a dose-dependent increase in Bax expression, and a decrease in Bcl-2 expression. The Bax/Bcl-2 ratio significantly and dose-dependently increased. Tanshinone IIA also increased the cleavage of PARP-1. Furthermore, tanshinone IIA significantly increased the percentage of Annexin V positive cells in a dose-dependent manner (Fig. 3B). These results suggest that tanshinone IIA-induced cancer-specific cytotoxicity is mediated by the apoptotic pathway. 3.3. Tanshinone IIA decreased cell viability in SiHa cervical carcinoma cells To examine the effect of tanshinone IIA in another cell type known to express CYP2J2, we treated the SiHa cervical cancer cell
Fig. 4. Effect of tanshinone IIA on the viability of SiHa cells. SiHa cells were treated with the indicated concentration of tanshinone IIA for 24 h, and cell viability was measured using a CCK-8 reduction assay. Inset represents CYP2J2 expression in HepG2 and SiHa cells. Values are expressed as the mean 7 S.E.M. of three experiments, each with triplicate dishes. nPo 0.05 vs. control. CCK-8, Cell Counting Kit-8; CYP2J2, cytochrome P450 2J2.
line with tanshinone IIA (Jiang et al., 2005). SiHa cells were treated with various concentrations of tanshinone IIA for 24 h, and cell viability was subsequently examined using the CCK-8 assay. As shown in Fig. 4, CYP2J2 expression was observed in SiHa as well as HepG2 cells, and treatment of SiHa cells with tanshinone IIA at concentrations ranging from 1–25 μM for 24 h significantly and dose-dependently decreased cell viability.
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3.4. Tanshinone IIA suppressed HepG2 xenograft growth in nude mice To study the effect of tanshinone IIA on hepatocarcinoma development, we used an in vivo HepG2 xenograft nude mouse model. Mice were treated with the vehicle and three doses of tanshinone IIA (3, 10, and 30 mg/kg/day). Progressive tumor growth was observed in the vehicle-treated group (Fig. 5A). The body weights of the vehicle- and tanshinone IIA-treated mice were compared on day 30 after cancer inoculation. The results did not show any significant difference in body weight when mice were treated with 3, 10, and 30 mg/kg/day of tanshinone IIA for 5 days a week for 30 days (Fig. 5B). However, the 30 mg/kg/day group showed a significant suppression of tumor growth rate when compared with the vehicle group. The 3 and 10 mg/kg/day doses showed a trend to lower tumor volume and tumor weight relative to the vehicle control, although the effect was not statistically significant (Fig. 5C). 3.5. CYP2J2 was involved in tanshinone IIA-induced apoptosis in HepG2 cells To elucidate any direct relationship between CYP2J2 and the tanshinone IIA-induced apoptosis in HepG2 cells, we generated a transgenic HepG2 cell line overexpressing CYP2J2. Cells were infected with CMV-CYP2J2 or CMV-GFP lentivirus, and CYP2J2 protein expression levels were examined. The CMV-GFP virus was used as a positive control. As shown in Fig. 6A and B, CYP2J2 expression was significantly greater in CMV-CYP2J2-infected cells than in wild type HepG2 cells. Interestingly, our data showed that Bcl-2 expression significantly increased following CMV-CYP2J2 virus infection, while the expression levels of Bax and cleaved PARP-1 were not affected (Fig. 6C). Next, we compared the difference in tanshinone IIA reactivity between wild-type HepG2 and CMV-CYP2J2 virus-infected HepG2 cells. As shown in Fig. 7A, the decrease in cell viability following treatment with 10 μM tanshinone IIA was significantly attenuated by CMV-CYP2J2 virus infection. Furthermore, the percentage of Annexin V positive cells decreased more in CMV-CYP2J2 virus-infected cells that in wildtype HepG2 cells (Fig. 7B). Collectively, our results suggest that apoptotic cell death induced by treatment with tanshinone IIA is mediated by inhibition of CYP2J2.
4. Discussion Recently, CYP2J2 and its EET metabolites have been implicated in the pathological development of both solid tumors and hematologic malignancies (Chen et al. 2009, 2011; Freedman et al., 2007; Jiang et al., 2005; J.G. Jiang et al., 2007). Previous reports suggested that either CYP2J2 or its metabolites promote marked proliferation of cancer cells through increased activation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt pathways, and enhanced phosphorylation of epidermal growth factor receptor (EGFR). Additionally, CYP2J2 inhibits carcinoma cell apoptosis by upregulation of the anti-apopFig. 5. Effect of tanshinone IIA treatment on HepG2 hepatocellular carcinoma xenograft. HepG2 (5 106) cells were subcutaneously injected in the right flank of each mouse to initiate ectopic tumor growth. Vehicle or tanshinone IIA was administered at 3, 10, and 30 mg/kg/day (in the vehicle), 5 days a week for 30 days. Treatment started 7 days following tumor cell injection and continued for 30 days. Mice were euthanized on day 30 after inoculation, and then tumors were excised and weighed. (A) Representative images of xenografts. (B) After euthanization, vehicle- and tanshinone IIA-treated mice were weighed. (C) Cancer tumor tissues were weighed after excision. Values are expressed as mean 7 S.E.M. (n¼6 mice per group). nPo 0.05 vs. control.
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Fig. 6. Effect of overexpression of cytochrome P450 2J2 (CYP2J2) in HepG2 cells. (A) Representative images are shown for HepG2 cells infected with CMV-CYP2J2 lentivirus, CMV-GFP lentivirus, or uninfected control. Images were obtained by fluorescence microscopy (Leica DMIL Fluo, 100 ). Scale bar, 100 μm. (B) Cells were infected with either CMV-CYP2J2 or CMV-GFP lentivirus, and cell lysates were subjected to western blot analysis using a CYP2J2-specific antibody. Charts represent three experiments. Values are expressed relative to β-actin levels. Error bars denote the mean 7 S.E.M. for three independent experiments levels. nP o0.05 vs. control. (C) Cells were infected with either CMV-CYP2J2 or CMV-GFP lentivirus, and cell lysates were subjected to western blot analysis using Bax, Bcl-2, and PARP-1-specific antibodies. Charts represent three experiments. Intensities of Bax, Bcl-2, and cleaved PARP-1 were determined using densitometry and expressed relative to β-actin, and the Bax:Bcl-2 ratio was calculated. Values are expressed as the mean 7S.E.M. of three independent experiments. nPo 0.05 vs. control. CMV-CYP2J2, cytomegalovirus-cytochrome P450 2J2; GFP, green fluorescent protein; Bax, Bcl-2 associated X protein; Bcl-2, B-cell lymphoma 2; PARP-1, poly (ADP-ribose) polymerase 1.
totic proteins Bcl-2 and Bcl-xL and down-regulation of the proapoptotic protein Bax (Jiang et al., 2005). Furthermore, C. Jiang et al. (2007); J.G. Jiang et al. (2007) reported that CYP2J2-derived
EET might play an important role in promoting the invasion and metastasis of human cancers through down-regulation of metastatic suppressor genes and up-regulation of metastasis-enhancing
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Fig. 7. Effect of overexpression of cytochrome P450 2J2 (CYP2J2) on tanshinone IIA-induced apoptosis. (A) Wild-type HepG2 or CMV-CYP2J2 lentivirus-infected HepG2 cells were cultured with the indicated concentrations of tanshinone IIA for 24 h, and cell viability was measured using a CCK-8 reduction assay. Values are expressed as the mean 7 S.E.M. for three experiments, each with triplicate dishes. n P o0.05 vs. control. (B) Cells were cultured under the indicated conditions for 24 h, then stained with FITC-Annexin V/PI, and analyzed using conventional flow cytometry. Representative scatter plots are shown, in which the upper quadrants represent Annexin V-positive (apoptotic) cells. Values are expressed as the mean 7 S.E.M. of three independent experiments. nPo 0.05 vs. control. CMVCYP2J2, cytomegalovirus-cytochrome P450 2J2; CCK-8, Cell Counting Kit-8, FITC; fluorescein isothiocyanate; PI, propidium iodide.
genes (C. Jiang et al., 2007; J.G. Jiang et al., 2007). Therefore, CYP2J2 might be a potential target for human cancer therapy. However, limited information is available on CYP2J2 enzyme inhibitors (Lafite et al., 2007; Wu et al., 2013). In the present study, we screened the inhibitory potential of 10
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plant-derived terpenoids against the CYP2J2 enzyme. The effects of these natural products on the catalytic activity of human CYP2J2 were investigated in HLMs. Our results showed that tanshinone IIA inhibited CYP2J2-mediated astemizole O-demethylation activity at a concentration of 5 μg/ml, while the IC50 was 2.5 μM. Additional experiments were carried out to evaluate the effect of tanshinone IIA, which showed an inhibitory effect against CYP2J2 enzyme in vitro and in vivo. Tanshinone IIA showed significant cytotoxicity against HepG2 cells but not against mouse hepatocytes. Previously, it was reported that in humans, the CYP2J subfamily comprises a single gene CYP2J2, which is not expressed in mice (Nelson et al., 2004; Zhang and Ding, 2008). Indeed, in our study, HepG2 cells showed increased CYP2J2 expression while mouse hepatocytes did not. The anticancer effect of tanshinone IIA was also observed in the cervical cancer cell line, SiHa, which expresses CYP2J2. Because mouse hepatocytes can survive without CYP2J2, the target of tanshinone IIA, we hypothesized that survival of mouse hepatocytes was not affected by tanshinone IIA because of the absence of a target for TIIA. Therefore, the different effects of tanshinone IIA in different species, and the protective effect of tanshinone IIA in mouse hepatocytes, might be owing to differential CYP2J2 expression. Furthermore, tanshinone IIA has been reported to have protective effects via additional mechanisms in various organs, including the heart, brain, and liver (Liu et al., 2013; Pang et al., 2014; Park et al., 2009; Wang et al., 2014; Zhang et al., 2012). For example, tanshinone IIA protects ischemic cardiomyocytes from apoptosis through increased miR-133 expression regulated by the MAPK pathway. It was also reported that tanshinone IIA protects hepatocytes from CCl4-induced hepatotoxicity through inhibition of the mitochondrial permeability transition (Zhu et al., 2010). Collectively, our results strongly suggest that tanshinone IIA could be used as a cancer-specific therapeutic agent. A variety of anticancer agents inhibit cancer cell proliferation through an apoptotic mechanism of action (Luo et al., 2014). The Bcl-2 family comprises important regulators of various apoptotic pathways (Sarada et al., 2008). Apoptotic signals upregulate the expression of pro-apoptotic Bcl-2 family members, such as Bax, and downregulate the expression of anti-apoptotic Bcl-2 family members, such as Bcl-2 (Mazumder et al., 2008). In addition, PARP-1, an enzyme necessary for the repair of damaged DNA, is considered a hallmark of apoptosis (Chaitanya et al. 2010). PARP-1 is one of several known cellular substrates of caspases that play central roles in apoptosis. Cleavage of PARP-1 allows the degradation of DNA, a process necessary for apoptosis (Li and Darzynkiewicz, 2000). In this study, tanshinone IIA increased the Bax/ Bcl-2 ratio, levels of cleaved PARP-1, and Annexin V-positive cell population in a dose-dependent manner. Therefore, our results indicate that apoptotic cell death is a major mechanism of cytotoxicity induced by tanshinone IIA. The anticancer effect of tanshinone IIA was also observed in in vivo experiments. The effects of tanshinone IIA treatment on tumor formation were assessed in a mouse xenograft model, which can be used to evaluate potential anticancer drug candidates (Kelland, 2004). In this experiment, tumor formation and size significantly reduced following tanshinone IIA treatment. Based on our results, we hypothesized that if the tanshinone IIA-induced apoptosis of HepG2 cells was owing to the inhibition of CYP2J2, then up-regulation of CYP2J2 should attenuate the effect of tanshinone IIA. In order to obtain direct evidence for the role of CYP2J2 in tanshinone IIA-induced apoptosis, we established a transgenic HepG2 cell line overexpressing CYP2J2, and examined the effect of tanshinone IIA. Our results showed that CMV-CYP2J2 lentivirus infection significantly upregulated CYP2J2 protein expression in transfected cells compared to that in wild type HepG2 cells. Furthermore, up-regulation of CYP2J2 significantly attenuated the tanshinone IIA-dependent reduction in cell viability,
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and diminished the tanshinone IIA-dependent increases in Annexin V-positive cells. These results indicate that CYP2J2 overexpression protects HepG2 cells from apoptosis induced by tanshinone IIA treatment. Therefore, we conclude that tanshinone IIA exerts its anticancer effect directly via inhibition of CYP2J2 activity. Additionally, CYP2J2-overexpressing HepG2 cells showed a significant increase in Bcl-2 expression, while Bax expression and cleaved PARP-1 levels were not changed. These results are supported by a previous study in which elevated expression of CYP2J2 was shown to render carcinoma cells more resistant to apoptotic cell death (Jiang et al., 2005). Thus, our results highlight the anticancer activity of tanshinone IIA and the significance of functional CYP2J2 as a potential antitumor therapeutic target. In conclusion, we have demonstrated that tanshinone IIA has an anticancer effect that is directly due to inhibition of CYP2J2 activity, and that CYP2J2 could be a potential target for various human cancers. Further studies on the roles of CYP2J2 in carcinogenesis would be required to diagnose CYP2J2-dependent cancers, and to develop novel and improved treatments.
Conflicts of interest The authors declare no conflicts of interest.
Acknowledgments We thank Dr. Yibing Qyang for providing the lentiviral vectors. This study was supported by a National Research Foundation of Korea (NRF) Grant (NRF-2012R1A4A1028835) funded by the Korean Government (MSIP).
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