Article pubs.acs.org/JAFC
A Novel Triterpenoid Isolated from Apple Functions as an Antimammary Tumor Agent via a Mitochondrial and CaspaseIndependent Apoptosis Pathway Aimin Qiao,† Yihai Wang,§ Limin Xiang,§ Chunhua Wang,§ and Xiangjiu He*,§ †
School of Biosciences and Biopharmaceuticals and §School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China ABSTRACT: A novel triterpenoid, named 3β-trans-cinnamoyloxy-2α-hydroxy-urs-12-en-28-oic acid (CHUA), was one of the main components of apple peels and showed potent in vitro antitumor activity against human tumor cells. In vivo antitumor experiments showed that CHUA could significantly inhibit the growth of mammary tumor in a nude mouse xenograft model at a dose of 50 mg/kg/day without body weight loss and mortality. In vitro, CHUA could induce apoptosis in MDA-MB-231 cells through the detection of DNA fragments and LDH activity. Simultaneously, mitochondrial transmembrane potential was markedly reduced and the release of cytochrome c was increased after CHUA treatment. It also up-regulated the expression ratio of mitochondrial Bax/Bcl-2 regulated by SIRT1 and p53. Interestingly, z-VAD-fmk and z-DEVD-fmk augmented cell death after CHUA treatment. Other protease(s) different from caspase-3 might be responsible for the degradation of PARP. These results suggested that the pro-apoptotic activity of CHUA may be adjusted by mitochondrial and caspase-independent pathways. KEYWORDS: apple, caspase-3-independent apoptosis, MDA-MB-231 cells, 3β-trans-cinnamoyloxy-2α-hydroxy-urs-12-en-28-oic acid
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INTRODUCTION Apple (Malus pumila) is one of the most widely cultivated tree fruits. Epidemiological studies have shown that the consumption of apples may be related to a significantly reduced risk of many diseases such as carcinogenesis,1,2 cardiovascular disease,3 cognitive impairment,4 acute inflammatory disease,5 and diabetes.6 Moreover, apple extracts have been shown to protect against gastrointestinal mucosa alterations7 and have antitumor activity. Therefore, apples as an important part of consumption of plant foods could be beneficial for human health. The bioactive compounds and antitumor activities of apple were investigated in our previous study. Twenty-two triterpenoids, including eight novels, have been purified from the peels of Red Delicious apple. It was found that the triterpenoids of apples exhibited pronounced in vitro antitumor activities and may be responsible for the antitumor activities of whole apples.8,9 The total triterpenoids (ATT) of apples showed antitumor activity and could initiate apoptosis of breast cancer cells through mitochondrial pathway.10 Apoptosis is a fundamental cellular response, which plays a crucial role in regulating tissue homeostasis by removing unused cells. It is a well conserved cell death pathway executed by caspases including initiator and executioner. Caspases, a family of cysteine proteases, have the main function as central regulators of cell death during apoptosis.11,12 Cell apoptosis is mainly induced by intrinsic and extrinsic pathways involving either mitochondria or death receptors. The mitochondria-dependent apoptosis pathway is mainly regulated by pro-apoptotic factor Bax and the anti-apoptotic factor Bcl-2 family. The balance between Bax and Bcl-2 or Bcl-xL in the mitochondrial may determine cell survival or death. These proteins can also promote mitochondrial outer membrane © XXXX American Chemical Society
permeabilization by regulating Mfn1, a GTPase involved in mitochondrial fusion.13,14 p53, a known tumor suppressor, induces cell cycle arrest and apoptosis related to DNA damage. It can also activate its downstream target genes, including Bcl-2 and Bax, for apoptosis. SIRT1 is a highly conserved nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase that plays an important role in promoting cell survival through regulating p53 in response to DNA damage.15,16 A new compound named 3β-trans-cinnamoyloxy-2α-hydroxy-urs-12-en-28-oic acid (CHUA) was isolated and identified from apple in our previous study.8 CHUA is the one of main triterpenoids of apple and has been shown to significantly inhibit the growth of a number of malignant cell lines in a former preliminary study, especially breast cancer cells. In the present work, CHUA was extracted and purified from red apple, and its antitumor activities in vivo were investigated using the nude mouse xenograft model. The proposed mechanisms of CHUA-induced MDA-MB-231 cell apoptosis were studied through mitochondrial and caspaseindependent pathways in vitro.
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MATERIALS AND METHODS
Plant Material and Reagents. Red Fuji apples, which were harvested in October 2009, were bought from Wuhan Shahu Fruits Market (Wuhan, China). All analytical and HPLC grade chemicals were purchased from Mallinckrodt Chemicals (Phillipsburg, NJ, USA). D101 macroporus resin was purchased from Xi’an Lanxiao Resin Corp. Ltd. (Xi’an, Received: August 4, 2014 Revised: December 18, 2014 Accepted: December 18, 2014
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DOI: 10.1021/jf5053546 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry China). Cell culture medium, Dulbecco’s modified Eagle medium (DMEM), and fetal bovine serum (FBS) were products of Gibco Life Technologies (Grand Island, NY, USA). Bax, ICAD, PARP, cytochrome c, p53, Bcl-2, Bcl-XL, SIRT1, and secondary antibody were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Z-Ile-Glu-Thr-Asp-FMK (Z-IETD-FMK) and Z-Leu-Glu(OMe)-HisAsp(OMe)-FMC (Z-LEHD-FMK) were purchased from ICN (Aurora, OH, USA). Z-Asp(O-Me)-Glu(O-Me)-Val-Asp(O-Me) fluoromethyl ketone (Z-DEVD-FMK) and benzylocarbonyl-Val-AlaAsp-fluoromethyl ketone (Z-VAD-FMK) were purchased from Calbiochem (Gibbstown, NJ, USA). RNase A, rhodamine 123, proteinase K, and MTT were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Extraction and Purification of CHUA from Apples. The fresh apple peels of red Fuji variety (20 kg) were dried under vacuum at 60 °C and then smashed into powder, which was refluxed using 10 volumes of 95% ethanol for 2 h twice. The solvent was recovered under vacuum at 45 °C, and the residue was suspended in 2 L of water and subjected to a D101 macroporus resin column (100 × 1500 mm). The column was eluted with 10 L of 60% ethanol and then with 10 L of 95% ethanol, respectively. The fraction of 95% ethanol elution was concentrated and dried, getting the total triterpenoids (96.0 g). The total triterpenoids (92.0 g) was subjected to a silica gel column (230− 400 mesh, 420 × 80 mm), gradiently eluted with CH2Cl2/MeOH (the CH2Cl2/MeOH ratios were from 100:0 to 0:100). The CH2Cl2/ MeOH (50:1) elution (5.1 g) was purified by a Sephadex LH-20, followed by a HPLC (250 × 22 mm, 5 μm, Alltima C18) using 82% methanol (adding 0.1% CF3COOH, pH 2.0) as mobile phase, and CHUA (810.2 mg) were obtained. The purity of CHUA was determined according to a HPLC method using a Waters HPLC system (equipped with a 600 pump and PDA detector) (Milford, MA, USA). The column was LiChrospher 100 RP100e 5 μm (4.6 × 250 mm), and detection was at 210 nm. The mobile phase was CH3CN/H2O/CF3COOH (80:20:0.1) with a flow rate at 1.0 mL/min. The column temperature was set at 40 °C. Nude Mouse Xenograft Model. Six-week old female nude mice (Balb/c-nu/nu) were bought from Guangdong Medical Experimental Animal Center. All animal experiment protocols were confirmed by the Animal Ethics Committee of Wuhan University. CHUA was dissolved in DMSO. Mice were randomly allocated to two groups with 10 animals per group. One hundred microliters of exponential MDAMB-231 cells (3 × 106 cells) was injected subcutaneously into the backs of mice. When tumors become apparent after 9 days, 100 μL of DMSO containing CHUA was administered intraperitonelly to the therapy group at a dose of 50 mg/kg/day; the vehicle group was treated by only 100 μL of DMSO in the same way. Mice were sacrificed on day 10 post-CHUA treatment, and the tumor tissues were collected and weighed. The length and width of the tumors were measured, and the tumor volume was calculated using the following equation: volume = (length × width2)/2. Antiproliferative Effects of CHUA against Cancer Cell Line. Human breast cancer (MDA-MB-231, MCF-7), human liver cancer (HepG2), human colon cancer (Caco-2), human cervical cancer (HeLa), and human lung adenocarcinoma (A549) cell lines were purchased from ATCC. The MTT method was utilized to evaluate the bioactivity of CHUA. Briefly, tumor cells were cultured in DMEM with 10% FBS and maintained at 37 °C in 5% CO2. A total of 2.5 × 104 cells were seeded in each well of a 96-well plate before the experiment day. After incubation overnight, the cells were treated by various concentrations of CHUA for 48 h. Cell viability was determined by the MTT assay. Experiments were performed in triplicate for each sample. DNA Ladder Assay. DNA extraction and electrophoresis were performed as described previously.17 Briefly, MDA-MB-231 cells (2 × 105) were seeded into a 100 cm dish for 24 h before the experimental day, and then the cells treated by 3.5 μM CHUA at indicated times were incubated with 0.25% trypsin for 10 min at 37 °C. The enzymatic digestion was terminated by mixing the suspension with an equal volume of DMEM supplemented with 10% FBS. Then floating cells were collected by centrifugation at 1000g for 10 min. The cell pellet
was washed twice in ice-cold PBS. To detect the DNA fragments, an apoptotic DNA Laddering Kit (Shanghai Xinle Institute of Biotechnology, China) was used according to the manufacturer’s protocol. The DNA fragments were separated by 2% agarose gel containing 0.2 mg/mL EB. The gel was photographed using an ultraviolet gel documentation system (UVI Tec Ltd., Cambridge, UK). Lactate Dehydrogenase (LDH) Activity-Based Cytotoxicity Assays.17 The MDA-MB-231 cells (2 × 105) were seeded into a 6well plate for 24 h before CHUA was added. On the next day, the prepared cells were treated by 3.5 μM CHUA at different time courses or the cells were treated with or without z-VAD-fmk, z-DEVD-fmk, zIETD-fmk, z-LEHD-fmk at given concentrations for 1 h and then treated with CHUA for 24 h. After treatment, floating cells were collected from cultured medium by centrifugation at 4 °C for 5 min. The LDH released in the cultured supernatant was used as an index of necrotic death, the LDH content in the cell pellets lysis was used for the apoptotic death, and the LDH present in the adherent viable cells was used as intracellular LDH. The LDH activities of the cell lysis and the cultured supernatant were measured according to the reference with a slight modification. Measurement of Mitochondrial Transmembrane Potential (ΔΨm). To measure the mitochondrial transmembrane potential (ΔΨm), MDA-MB-231 cells (2 × 105) were seeded into a 100 cm dish for 24 h before the experimental day, and then the cells treated by 3.5 μM CHUA at the indicated time were incubated with 0.25% trypsin for 10 min at 37 °C. The enzymatic digestion was terminated by mixing the suspension with an equal volume of DMEM. The cells were collected by centrifugation (240g) at 4 °C for 5 min, then the cells were gently washed with 1 mL of cold PBS one time, and the cells were resuspended in rhodamine 123 staining solution (1 μg/mL) in the dark for 30 min. The samples were analyzed by flow cytometry. Effects of Caspase Inhibitor on MDA-MB-231 Cells Treated by CHUA. The effects of caspase inhibitor on MDA-MB-231 cells were measured by MTT assay described above. The cultured cells were seeded in a 96-well plate with a density of 2.5 × 104 cells/well without or with z-VAD-fmk, z-DEVD-fmk, z-LEHD-fmk, and z-IETDfmk at given concentrations for 1 h and then treated with 3.5 μM CHUA for another 24 h. After the treatment, the cell viability was measured by MTT assay. Western Blot Analysis. MDA-MB-231 cells were treated with 3.5 μM CHUA for different time courses as indicated. All of the cells were harvested according to the method described by Laetitia et al.18 Briefly, the cells were gently washed with ice-cold PBS and lysated by RIRA buffer containing proteinase inhibitor. Protein concentrations were determined by the Bio-Rad DC protein assay. Fifteen mirograms of proteins for each channel was separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with appropriately diluted primary antibodies, followed by horseradish peroxidase-conjugated secondary antibodies against the corresponding species. Labeling was detected using the ECL system (Amersham Biosciences). Statistical Analysis. Experimental values were expressed as the mean ± SEM. The differences between groups were assessed by one way-analysis of variance followed by multiple comparisons. Statistical significance is expressed as follows: ∗, p < 0.05; ∗∗, p < 0.01.
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RESULTS AND DISCUSSION Structure Identification of CHUA. CHUA was a white powder. The ESI-MS gave the [M − H]− ion at m/z 601, and its molecular formula was drawn as C39H54O5 from HR-MS and 13 C NMR. In its 1H NMR, there were three groups of characteristic signals of monosubstituted benzene, which showed at 8.12 (2H, br d, J = 8.6 Hz), 7.53 (2H, br t, J = 8.6 Hz), and 7.17 ppm (H, br t, J = 8.6 Hz), respectively. There was a pair of trans-conjugated olefinic protons, which appeared at 8.00 (H, d, J = 16.0 Hz) and 6.65 ppm (H, d, J = 16.0 Hz). There were 12 sp2-hybrid carbons in the lower field of 13C NMR, which included 2 carbonyl carbons. From its 1H and 13C B
DOI: 10.1021/jf5053546 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry NMR and DEPT spectra, a trans-cinnamic acid part could be deduced. In the 13C NMR spectra, there were a carbonyl signal at 180.1 ppm and two olefinic carbons at 139.7 and 126.1 ppm, which implied there was a carbon−carbon double bond in the molecule. The results showed that CHUA was a triterpene possessing an ursane skeleton. Compared with our former study,8 the triterpenoid was identified as 3β-trans-cinnamoyloxy-2α-hydroxy-urs-12-en-28-oic acid (CHUA), which was a novel triterpenoid isolated from apple. The chemical structure of CHUA is shown in Figure 1.
Figure 1. Chemical structure of CHUA.
The purity of CHUA was 97.3 ± 5.1%, determined by HPLC method. CHUA was used for in vivo and in vitro antitumor studies. It was dissolved in DMSO, and the stock solution was diluted with cell culture medium to keep the concentration of DMSO
