- Primary research
- Open Access
Antiproliferative and apoptosis-inducing activity of schisandrin B against human glioma cells
- Qun Li†1,
- Xiang-he Lu†1,
- Cheng-de Wang1,
- Lin Cai1,
- Jiang-long Lu1,
- Jin-sen Wu1,
- Qi-chuan Zhuge1,
- Wei-ming Zheng1Email author and
- Zhi-peng Su1Email author
© Li et al.; licensee BioMed Central. 2015
- Received: 27 October 2014
- Accepted: 7 January 2015
- Published: 4 February 2015
Malignant glioma is the most devastating and aggressive tumour in the brain and is characterised by high morbidity, high mortality and extremely poor prognosis. The main purpose of the present study was to investigate the effects of schisandrin B (Sch B) on glioma cells both in vitro and in vivo and to explore the possible anticancer mechanism underlying Sch B-induced apoptosis and cell cycle arrest.
The anti-proliferative ability of Sch B on glioma cells were assessed by MTT and clony formation assays. Flow cytometric analysis was used to detect cell cycle changes. Apoptosis was determined by Hoechst 33342 staining and annexin V/PI double-staining assays. The mitochondrial membrane potential was detected by Rhodamine 123 staining. The in vivo efficacy of Sch B was measured using a U87 xenograft model in nude mice. The expressions of the apoptosis-related and cell cycle-related proteins were analysed by western blot. Student’s t-test was used to compare differences between treated groups and their controls.
We found that Sch B inhibited growth in a dose- and time-dependent manner as assessed by MTT assay. In U87 and U251 cells, the number of clones was strongly suppressed by Sch B. Flow cytometric analysis revealed that Sch B induced cell cycle arrest in glioma cells at the G0/G1 phase. In addition, Sch B induced glioma cell apoptosis and reduced mitochondrial membrane potential (ΔΨm) in a dose-dependent manner. Mechanically, western blot analysis indicated that Sch B induced apoptosis by caspase-3, caspase-9, PARP, and Bcl-2 activation. Moreover, Sch B significantly inhibited tumour growth in vivo following the subcutaneous inoculation of U87 cells in athymic nude mice.
In summary, Sch B can reduce cell proliferation and induce apoptosis in glioma cells and has potential as a novel anti-tumour therapy to treat gliomas.
- Glioma cells
- Schisandrin B
- Cell cycle
Glioma, which is characterised by rapid proliferation and diffuse invasion, is one of the most common malignant brain tumours in adults. In the United States, malignant gliomas account for approximately 70% of malignant primary brain tumours in adults . Because surgical treatment is unable to remove all malignant glioma cells, it is difficult to eliminate this type of malignancy. As a result, malignant gliomas are associated with high morbidity, high mortality and extremely poor prognosis [2-7]. Tumour recurrence usually recurs within 6.9 months, resulting in a median patient survival of just 12–15 months following diagnosis . Moreover, high drug resistance and the blood–brain barrier usually lower the efficacy of chemotherapy drugs . Hence, new effective treatments and drugs are urgently needed to improve outcomes for patients with malignant gliomas.
Sch B inhibits the proliferation and viability of glioma cells in a dose-dependent manner
Sch B induces G0/G1 phase arrest by regulating the expression of cell cycle-related proteins in U87 and U251 cells
Next, we assessed the levels of the G0/G1 phase-related proteins cyclin D1 and cyclin-dependent kinase 4 (CDK4) using western blot analysis. As shown in Figure 3C, Sch B treatment reduced the expression of cyclin D1 and CDK4 in a dose-dependent manner, which is consistent with G0/G1 phase arrest induced by Sch B.
Sch B induces apoptosis in human glioma cells
Sch B decreases mitochondrial membrane potential (ΔΨm) in glioma cells
Sch B-induced apoptosis via the regulation of caspase-3 and Bcl-2 family members in glioma cells
Sch B reduced tumour growth in vivo
It has been reported that Sch B possesses anti-neoplastic ability in a broad range of human cancers by promoting apoptosis [10-12]. However, the effect of Sch B on glioma cells and its underlying mechanisms remain largely unknown. In the present study, we demonstrated that Sch B inhibited proliferation, induced apoptosis and G0/G1 phase arrest in glioma cells and strongly restrained the tumour growth in vivo. Thus, Sch B may be a promising drug to treat gliomas.
Results of cell cycle distribution experiment in the presence of Sch B demonstrated that it induced G0/G1 phase arrest after 48 h of treatment and that prompted us to further investigate the mechanism of cell cycle arrest. Cyclins and CDKs play key roles in cell cycle regulation . Cyclin D1, one of the key cell cycle regulatory molecules [20,21], can form a complex with CDK4 to promote the transition of cells from G0/G1 to S phase by catalysing the phosphorylation of retinoblastoma (Rb) [22-24]. Indeed, we observed that Sch B downregulated the expression of cyclin D1 and CDK4 in both U87 and U251 cells (Figure 3C). Based on these data, we believe that Sch B arrests the cell cycle in G0/G1-phase to suppress the proliferation of glioma cells by reducing the expression of cyclin D1 and CDK4.
The deregulation of apoptosis has frequently been observed in various cancers; thus, the induction of apoptosis has been proposed as an important strategy to treat cancer [25,26]. Generally, there are two major pathways that control the initiation of apoptosis: the death-receptor-induced extrinsic pathway and the mitochondria-apoptosome-mediated intrinsic pathway, both of which ultimately activate a series of effector caspases and apoptosis effector molecules . In the extrinsic pathway, the binding of ligands to membrane death-receptors leads to the activation of caspase-8; In the intrinsic pathway, Bax induces the release of cytochrome c from the mitochondria, leading to the apoptosome formation and activation of caspase 9, and subsequent caspase 3 activation. Caspase-3 is one of the most important executioner caspases and is capable of cleaving many important cellular substrates, such as PARP, resulting in apoptosis progression, morphological changes, and DNA fragmentation . In our study, Sch B treatment of U87 and U251 cells activated caspase-9 and caspase-3, accompanied by increased PARP cleavage, suggesting that the mitochondrial pathway was involved in Sch B-induced apoptosis. Proteins in the Bcl-2 family, including the apoptosis-promoting protein Bax and anti-apoptotic protein Bcl-2, are the key regulators of the mitochondria-mediated apoptosis pathway [29,30]. It has been reported that a low Bcl-2 to Bax ratio can cause ΔΨm collapse, the release of cytochrome c, and subsequent apoptosis . In the present study, we found that the ratio of Bcl-2 to Bax was reduced by 15.6- and 10.24-fold after treatment with 100 μmol/L Sch B in U87 and U251 cells, respectively, compared with that of the control group. As a result, a significant decrease of ΔΨm was observed in giloma cells.
To further address the antitumor activity of Sch B against glioma, the animal work should be taken entirely. Due to the prolonged tissue culture, the genetic and morphologic characteristics of established cell lines often differ from the primary human tumors. To overcome these shortcomings, Sarkaria lab  have developed a model of primary glioblastoma multiforme (GBM) xenografts that were established by engraftment of patient tumor specimens directly into the flank of nude mice. These xenograft lines remains key genetic features of the corresponding patient tumor, this is highly useful for both basic and translational studies. We can use this model to test the effect of Sch B on patient tumor specimens. The extension of animal numbers is also needed to further confirm the clinical significance of Sch B on the treatment of glioma.
In summary, our study suggests that Sch B attenuates the proliferation of U87 and U251 cells and arrests cell cycle in the G0/G1 phase. Sch B induces cell apoptosis by increasing Bax, cleaved caspase-9, cleaved caspase-3, and cleaved PARP and reducing Bcl-2 protein expression in glioma cells. Furthermore, Sch B dramatically inhibited tumour growth in vivo. All of the above results indicate that Sch B may be a promising drug for glioma treatment. However, further studies are needed to understand the fundamental mechanism underlying the apoptosis and cell cycle arrest induced by Sch B in glioma cells.
Drugs and antibodies
Sch B was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to obtain a 100 mM stock solution, which was then diluted in the culture medium to yield the desired Sch B concentration. An equal volume of DMSO in complete culture medium was used as the vehicle control. To eliminate the cytotoxicity of DMSO, the final concentration of DMSO for all experiments was maintained at less than 0.1%. Rhodamine 123 and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. Primary antibodies against Bcl-2, Bax, cleaved caspase-9, cleaved PARP, cleaved caspase-3, cdc-2, cyclin D1, and GAPDH and secondary antibodies (goat anti-rabbit) were all purchased from Cell Signaling Technology (Danvers, MA, USA).
Cell lines and culture
The human glioma U87 and U251 cell lines were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (CAS, Shanghai, China). All cells were cultured in high-glucose DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (Gibco), 100 μg/mL streptomycin, and 100 U/mL penicillin (HyClone, USA).The cells were cultured in an atmosphere containing 5% CO2 at 37°C.
Cell viability assay
The viability of cells treated with Sch B was measured as previously described . Briefly, cells were seeded into 96-well culture plates at 5,000 cells/well, incubated overnight, then treated with 0, 20, 40, 60, 80 and 160 μmol/L Sch B for 24, 48 and 72 h. Next, added 20 μL MTT solution (5 mg/mL) to each well for another 4 h after treatment. In order to solubilise the formazan crystals, 100 μL DMSO was added to each well. The absorbance was determined at 490 nm using a microplate reader (Bio-Tek, Winooski, VT, USA). The cell survival rate was measured as the absorbance compared with that of untreated controls. The results represent the average of 3 independent experiments.
Colony formation assay
Briefly, cells in the logarithmic growth phase were seeded into 6-well plates (Corning, Corning, NY, USA) at a density of 400 cells/well and allowed to grow for 24 h. The cells were then treated with Sch B (0, 4, 8 and 16 μmol/L) for another 48 h. The Sch B-containing medium was then discarded, and the cells were washed with PBS and cultured to form colonies in complete medium for another 14 days. Next, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with 0.1% crystal violet (Sigma-Aldrich) for 30 min. After washing, the total number of colonies (>50 cells/colony) was counted manually and the stained colonies were photographed using a microscope (Leica, Wetzlar, Germany). The results were expressed as the average of three independent experiments.
Cell cycle analysis
U87 and U251 cells were treated with various concentrations of Sch B for 48 h. Both adherent and floating cells were harvested, washed twice in cold PBS, and fixed in 70% ethanol at 4°C overnight. After fixation, the cells were washed and incubated in staining buffer (10 mg/mL RNase and 1 mg/mL propidium iodide (Sigma-Aldrich)) at 37°C in the dark for 30 min. Subsequently, the samples were analysed using flow cytometry (BD Biosciences, San Diego, CA, USA). The percentages of cells in the G0/G1, S, and G2/M phases were calculated using Cell Quest acquisition software (BD Biosciences).
Cell apoptosis assay
Apoptosis was analysed using the annexin V/PI apoptosis kit according to the manufacturer’s recommendations (BD Biosciences, San Diego, CA, USA). Briefly, Two hundred thousand cells (2 × 105) were first seeded into 6-well plates and treated with Sch B (0, 25, 50, and 100 μmol/L) for 48 h. The cells were then harvested and resuspended at a density of 1 × 106 cells/mL in binding buffer. Then 5 μL annexin V-FITC and 5 μL PI (100 μg/mL) were added to the suspension cells. After incubation for 15 min in the dark at room temperature, 400 μL of the binding buffer was added to the cell suspension. The stained cells were then immediately analysed using flow cytometry (BD Biosciences, San Diego, CA, USA). Each sample was assayed in duplicate, and the experiment was repeated three times.
Hoechst 33342 staining assay
Nuclear fragmentation was determined by staining apoptotic nuclei with Hoechst 33342. U87 and U251 cells were treated with various concentrations of Sch B for 48 h, the cells were harvested, washed in cold PBS and fixed with acetic acid:ethanol (1:3) for 15 min at room temperature. The fixed cells were then washed and stained with 5 μg/mL Hoechst 33342 for 10 min. Digital images were captured under a fluorescence microscope (Leica, Germany).
Mitochondrial membrane potential (ΔΨm) assay
Rhodamine 123 (Rho123) was used to determine the mitochondrial membrane potential (ΔΨm) as previously described . Briefly, after treatment with various concentrations of Sch B for 48 h, U87 and U251 cells were collected and washed twice with cold PBS. The cells were then stained with Rhodamine 123 (Sigma-Aldrich) for 30 min at 5% CO2 and 37°C in the dark. The samples were then analysed using flow cytometry (BD Biosciences, San Diego, CA, USA). Each assay was carried out in triplicate, and the results were expressed as the mean ± SD.
Western blot analysis
U87 and U251 cells were treated with Sch B (0, 25, 50 and 100 μmol/L) for 48 h, total protein was extracted using RIPA buffer (Beyotime Institute of Biotechnology, Beijing, China) supplemented with protease inhibitor (Roche Applied Science, Indianapolis, IN, USA) at 4°C for 5 min. The protein concentration was determined using the bicinchoninic acid (BCA) assay kit (Beyotime). Total protein samples (40 μg/lane) were separated using 10% SDS-PAGE and then electrophoretically transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% skim milk for 1–2 h at room temperature, the membranes were then incubated with the indicated primary antibodies against Bcl-2, Bax, cleaved PARP, cleaved caspase-9, cleaved caspase-3, CDK4, cyclin D1, and β-actin (1:1000) at 4°C overnight. The membranes were then washed with TBST buffer for 3 × 5 minutes and incubated with the secondary antibodies (HRP-conjugated goat anti-rabbit IgG) for 1 h at room temperature. The bands were detected using enhanced chemiluminescence and visualised by a Gel Doc 2000 (BioRad, Hercules, CA, USA).
In vivo tumour xenograft study
BALB/c homozygous (nu/nu) nude mice (6–8 weeks old, 18–20 g body weight) that had been bred in house were maintained in a specific pathogen-free environment. U87 cells in log-phase growth (2.5 × 106) were suspended in 100 μL PBS and then subcutaneously injected into the left flank of the nude mice. Twenty-four hours post inoculation, the mice were randomly divided into three groups (6 mice/group). The control group was administered PBS intraperitoneally and the other two treatment groups were administered Sch B (25 and 100 mg/kg) intraperitoneally in a volume of 0.2 mL every 2 days for up to 20 days. On day 22, all mice were sacrificed, and the tumours were excised and weighed. The experimental procedure was conducted according to the institutional animal care guideline and approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University with the reference number wydw2014-0026.
All data were expressed as the mean ± SD of at least three independent experiments. Differences between groups were analyzed using the Student’s t test,. A P-value of less than 0.05 (denoted by *) was considered to be statistically significant.
This work was supported by grants from the Zhejiang medical and health science and technology plan project (No.2012RCA042) and the Wenzhou science and technology bureau (No. Y20120008).
- Wen PY, Kesari S. Malignant gliomas in adults. N Engl J Med. 2008;359(5):492–507.View ArticlePubMedGoogle Scholar
- DeAngelis LM. Brain tumors. N Engl J Med. 2001;344(2):114–23.View ArticlePubMedGoogle Scholar
- Prados MD, Levin V. Biology and treatment of malignant glioma. Semin Oncol. 2000;27(3 Suppl 6):1–10.PubMedGoogle Scholar
- Holland EC. Glioblastoma multiforme: the terminator. Proc Natl Acad Sci U S A. 2000;97(12):6242–4.View ArticlePubMed CentralPubMedGoogle Scholar
- Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352(10):987–96.View ArticlePubMedGoogle Scholar
- Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol. 2009;10(5):459–66.View ArticlePubMedGoogle Scholar
- Cheng L, Wu Q, Guryanova OA, Huang Z, Huang Q, Rich JN, et al. Elevated invasive potential of glioblastoma stem cells. Biochem Biophys Res Commun. 2011;406(4):643–8.View ArticlePubMed CentralPubMedGoogle Scholar
- Kortmann RD, Jeremic B, Weller M, Plasswilm L, Bamberg M. Radiochemotherapy of malignant glioma in adults. Clinical experiences. Strahlentherapie und Onkologie : Organ der Deutschen Rontgengesellschaft [et al.] 2003; 179(4):219–32.Google Scholar
- Liu GT. Pharmacological actions and clinical use of fructus schizandrae. Chin Med J (Engl). 1989;102(10):740–9.Google Scholar
- Li L, Wang T, Xu ZL, Yu Y, Chen W, Chen F. Effects of schisandrin B on reversing multidrug resistance in human breast cancer cells transfected with mdr1 gene. Zhonghua yi xue za zhi. 2005;85(23):1633–7.PubMedGoogle Scholar
- Liu XN, Zhang CY, Jin XD, Li YZ, Zheng XZ, Li L. Inhibitory effect of schisandrin B on gastric cancer cells in vitro. World J Gastroenterol. 2007;13(48):6506–11.View ArticlePubMedGoogle Scholar
- Wu YF, Cao MF, Gao YP, Chen F, Wang T, Zumbika EP, et al. Down-modulation of heat shock protein 70 and up-modulation of Caspase-3 during schisandrin B-induced apoptosis in human hepatoma SMMC-7721 cells. World J Gastroenterol. 2004;10(20):2944–8.PubMedGoogle Scholar
- Li L, Lu Q, Shen Y, Hu X. Schisandrin B enhances doxorubicin-induced apoptosis of cancer cells but not normal cells. Biochem Pharmacol. 2006;71(5):584–95.View ArticlePubMedGoogle Scholar
- Liu Z, Zhang B, Liu K, Ding Z, Hu X. Schisandrin B attenuates cancer invasion and metastasis via inhibiting epithelial-mesenchymal transition. PLoS One. 2012;7(7):e40480.View ArticlePubMed CentralPubMedGoogle Scholar
- Nishida H, Tatewaki N, Nakajima Y, Magara T, Ko KM, Hamamori Y, et al. Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response. Nucleic Acids Res. 2009;37(17):5678–89.View ArticlePubMed CentralPubMedGoogle Scholar
- Rodriguez-Vargas JM, Ruiz-Magana MJ, Ruiz-Ruiz C, Majuelos-Melguizo J, Peralta-Leal A, Rodriguez MI, et al. ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy. Cell Res. 2012;22(7):1181–98.View ArticlePubMed CentralPubMedGoogle Scholar
- Du L, Mei HF, Yin X, Xing YQ. Delayed growth of glioma by a polysaccharide from Aster tataricus involve upregulation of Bax/Bcl-2 ratio, activation of caspase-3/8/9, and downregulation of the Akt. Tumour Biol. 2014;35(3):1819–25.View ArticlePubMedGoogle Scholar
- She EX, Hao Z. A novel piperazine derivative potently induces caspase-dependent apoptosis of cancer cells via inhibition of multiple cancer signaling pathways. American journal of translational research. 2013;5(6):622–33.PubMed CentralPubMedGoogle Scholar
- Nigg EA. Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle. Bioessays. 1995;17(6):471–80.View ArticlePubMedGoogle Scholar
- Saab R, Bills JL, Miceli AP, Anderson CM, Khoury JD, Fry DW, et al. Pharmacologic inhibition of cyclin-dependent kinase 4/6 activity arrests proliferation in myoblasts and rhabdomyosarcoma-derived cells. Mol Cancer Ther. 2006;5(5):1299–308.View ArticlePubMedGoogle Scholar
- Marzec M, Kasprzycka M, Lai R, Gladden AB, Wlodarski P, Tomczak E, et al. Mantle cell lymphoma cells express predominantly cyclin D1a isoform and are highly sensitive to selective inhibition of CDK4 kinase activity. Blood. 2006;108(5):1744–50.View ArticlePubMed CentralPubMedGoogle Scholar
- Harbour JW, Luo RX, Dei Santi A, Postigo AA, Dean DC. Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1. Cell. 1999;98(6):859–69.View ArticlePubMedGoogle Scholar
- Ezhevsky SA, Ho A, Becker-Hapak M, Davis PK, Dowdy SF. Differential regulation of retinoblastoma tumor suppressor protein by G(1) cyclin-dependent kinase complexes in vivo. Mol Cell Biol. 2001;21(14):4773–84.View ArticlePubMed CentralPubMedGoogle Scholar
- Hinds PW, Mittnacht S, Dulic V, Arnold A, Reed SI, Weinberg RA. Regulation of retinoblastoma protein functions by ectopic expression of human cyclins. Cell. 1992;70(6):993–1006.View ArticlePubMedGoogle Scholar
- Kang N, Zhang JH, Qiu F, Tashiro S, Onodera S, Ikejima T. Inhibition of EGFR signaling augments oridonin-induced apoptosis in human laryngeal cancer cells via enhancing oxidative stress coincident with activation of both the intrinsic and extrinsic apoptotic pathways. Cancer Lett. 2010;294(2):147–58.View ArticlePubMedGoogle Scholar
- Strasser A, Cory S, Adams JM. Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. EMBO J. 2011;30(18):3667–83.View ArticlePubMed CentralPubMedGoogle Scholar
- Spencer SL, Sorger PK. Measuring and modeling apoptosis in single cells. Cell. 2011;144(6):926–39.View ArticlePubMed CentralPubMedGoogle Scholar
- Wang J, Wu A, Xu Y, Liu J, Qian X. M(2)-A induces apoptosis and G(2)-M arrest via inhibiting PI3K/Akt pathway in HL60 cells. Cancer Lett. 2009;283(2):193–202.View ArticlePubMedGoogle Scholar
- Ewings KE, Wiggins CM, Cook SJ. Bim and the pro-survival Bcl-2 proteins: opposites attract, ERK repels. Cell Cycle. 2007;6(18):2236–40.View ArticlePubMedGoogle Scholar
- Das A, Banik NL, Patel SJ, Ray SK. Dexamethasone protected human glioblastoma U87MG cells from temozolomide induced apoptosis by maintaining Bax:Bcl-2 ratio and preventing proteolytic activities. Mol Cancer. 2004;3(1):36.View ArticlePubMed CentralPubMedGoogle Scholar
- Zhang YH, Wu YL, Tashiro S, Onodera S, Ikejima T. Reactive oxygen species contribute to oridonin-induced apoptosis and autophagy in human cervical carcinoma HeLa cells. Acta Pharmacol Sin. 2011;32(10):1266–75.View ArticlePubMed CentralPubMedGoogle Scholar
- Carlson BL, Pokorny JL, Schroeder MA, Sarkaria JN. Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery. Current protocols in pharmacology/editorial board, SJ Enna 2011; Chapter 14:Unit 14 16.Google Scholar
- Hu YP, Tan ZJ, Wu XS, Liu TY, Jiang L, Bao RF, et al. Triptolide induces s phase arrest and apoptosis in gallbladder cancer cells. Molecules. 2014;19(2):2612–28.View ArticlePubMedGoogle Scholar
- Xie SQ, Wang JH, Ma HX, Cheng PF, Zhao J, Wang CJ. Polyamine transporter recognization and antitumor effects of anthracenymethyl homospermidine. Toxicology. 2009;263(2–3):127–33.View ArticlePubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.