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Introduction

Breast cancer, one of the most common types ofcancer in women, is associated with high mortality (1). Treatment for breast cancer includeshormonal therapy, chemotherapy, radiotherapy, targeted therapy,surgery and various combinations of these strategies. However, theprognosis of certain subtypes remains poor (2).

Autophagy is a homeostatic cellular self-digestiveprocess responsible for degrading unnecessary or dysfunctionalcellular organelles and proteins in all living cells (3). Although autophagy promotes a cellsurvival response, it also serves a role in cell death (4). Previous studies have demonstratedthat autophagic cell death is triggered by numerous signalingpathways including adenosine monophosphate-activated protein kinasepathway (5), mammalian target ofrapamycin (mTOR) pathway (6), andmitogen-activated protein kinase (MAPK)/extracellularsignal-regulated kinase (ERK)1/2 pathway (7).

Medicinal plants and their extracts are commonlyused to prevent and treat numerous diseases, including cancer. TheWorld Health Organization has reported that ~4 million people (80%of the population in developing countries), depend on medicinalplants for primary healthcare (8).Pristimerin, a quinonemethide triterpenoid compound, has long beenused as an anti-inflammatory, antioxidant, antimalarial andinsecticidal agent (9).Pristimerin also possesses promising clinical potential as atherapeutic and chemopreventive agent for numerous types of cancer,including colon cancer (10),prostate cancer (11), ovariancancer (12) and breast cancer(13). Pristimerin induces celldeath via several mechanisms, including proteasome inhibition(14), caspase activation(15), inhibition of the humanepidermal growth factor receptor 2 (HER2) (13), inhibition of protein kinaseB/nuclear factor-κB/mTOR signaling (12), cell cycle arrest (12) and inhibition of migration andinvasion (10). However, theeffect of pristimerin on autophagy in human breast cancer has notbeen reported yet to the best of our knowledge.

The present study aimed to evaluate whetherpristimerin induces autophagy in human breast cancer cells. Theresults of the present study demonstrated that pristimerininhibited cell proliferation by autophagy induction. The effects ofpristimerin were enhanced when combined with paclitaxel treatmentthrough suppression of ERK1/2/p90 ribosomal S6 kinase (p90RSK)signaling, which in turn activated autophagy. These resultsindicated that pristimerin has potential to treat breast cancerthrough autophagy and combination therapy can enhancepaclitaxel-induced anticancer activities.

Materials and methods

Chemicals

Pristimerin was obtained from Sigma-Aldrich (MerckKGaA, Darmstadt, Germany) and dissolved in dimethyl sulfoxide(DMSO) to give a stock solution of 100 mM and stored at −20°C inaliquots. Paclitaxel was gifted by Boryung Co., Ltd. (Seoul,Korea). Dulbecco's modified Eagle medium (DMEM), fetal bovine serum(FBS) and penicillin/streptomycin were obtained from GE HealthcareLife Sciences HyClone (Logan, UT, USA). Trypsin/EDTA was purchasedfrom Gibco™ (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Thefollowing primary antibodies were used: Rabbit polyclonalanti-human light chain (LC) 3-I/II (1:1,000; cat. no. 4108), rabbitpolyclonal anti-human phosphorylated (phospho)-p44/p42 MAPK(ERK1/2; Thr202/Tyr204; 1:1,000; cat. no. 9101), rabbit monoclonalanti-human P-90RSK (1:1,000; cat. no. 9355), rabbit polyclonalanti-human phospho-p90RSK (Ser380; 1:1000; cat. no. 9314) werepurchased from Cell Signaling Technology, Inc. (Danvers, MA, USA)and rabbit polyclonal anti-human ERK (1:1,000; cat. no. sc-94),mouse monoclonal anti-human p62 (1:1,000; sc-48389), rabbitpolyclonal anti-human beclin1 (1:1,000; cat. no. sc-11427) andrabbit polyclonal anti-human GAPDH (1:1,000; cat. no. sc-25778)were obtained from Santa Cruz Biotechnology, Inc., (Dallas, TX,USA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbitantibodies were purchased from BD Biosciences, Pharmingen (SanDiego, CA, USA). SuperSignal® West Pico ChemiluminescentSubstrate was purchased from Pierce (Thermo Fisher Scientific,Inc.). The Cell Counting Kit-8 (CCK-8) was purchased from DojindoMolecular Technologies, Inc., (Kumamoto, Japan) and the AutophagyDetection kit (cat. no. ab139484) was purchased fromAbcam® (Cambridge, MA, USA). 3-MA, ceramide C6 and allother reagents were obtained from Sigma-Aldrich (Merck KGaA).

Cell line and cell culture

The MDA-MB-231 human breast cancer cell line waspurchased from American Type Culture Collection (Manassas, MD,USA). Cells were grown in DMEM supplemented with 10% (v/v) FBS,penicillin (100 U/ml)/streptomycin (100 µg/ml) at 37°C in ahumidified CO2 (5%)-controlled incubator.

Cell viability assay

Cells were seeded into 96-well microplates at adensity of 5×103 cells/ml and allowed to attach for 24h. Pristimerin (1, 2.5, 5 and 10 µM) and paclitaxel (6, 12, 24 and30 µM) were added to the medium at various concentrations.Following treatment, the cell cytotoxicity and/or proliferation wasassessed using the CCK-8 assay. CCK-8 (10 µl) was added to eachwell and incubated for 3 h at 37°C; cell proliferation andcytotoxicity were assessed by measuring the absorbance at awavelength of 450 nm using a microplate reader (Corning, Inc.,Corning, NY, USA). A total of three replicated wells were used perexperimental condition.

Western blotting analysis

Cells were harvested using Trypsin-EDTA, washedtwice with cold phosphate buffered saline (PBS), lysed with lysisbuffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100,0.5% NP-40, 1 mM PI, 1 mM DTT and 1 mM PMSF), placed on ice for 1 hwith occasional vortexing and centrifuged at 13,000 × g for 10 minat 4°C to collect the supernatant. A Pierce BCA Protein Assay kit(Pierce; Thermo Fisher Scientific Inc.) was used to determineprotein concentration. Cell lysates (50 µg) were subjected to 8,10, and 15% SDS-PAGE and transferred to a polyvinylidene difluoridemembrane. Blots were blocked with 5% skim milk in PBS containing0.05% Tween-20 (PBST) for 1 h at 25°C and then incubated withprimary antibodies (1:1,000) overnight at 4°C. After washing withPBST, membranes were incubated with anti-rabbit horseradishperoxidase-conjugated IgG (1:3,000) at room temperature for 2 h andvisualized with enhanced chemiluminescence. Band intensity wasquantified by densitometry using ImageJ (version 1.52) software(National Institutes of Health, Bethesda, MD, USA) and wasnormalized to loading controls. Quantification value was expressedas the fold change vs. band numbered 1.0.

Autophagy detection assay

Autophagy determination was performed using anAutophagy Detection kit according to the manufacturer's protocol.According to product overview by the company, the AutophagyDetection kit can measure autophagic vacuoles and monitorautophagic flux in live cells using a novel dye that selectivelylabels autophagic vacuoles. The dye has been optimized throughidentification of titratable functional moieties that allow forminimal staining of lysosomes while exhibiting bright fluorescenceupon incorporation into pre-autophagosomes, autophagosomes andautolysosomes (autophagolysosomes). Cells were seeded into 8-wellchamber slides at a density of 1×10 cells/ml and treated withindicated drugs. Following drug treatment, cells were washed with1X assay buffer, following incubation with 100 µl microscopy dualdetection reagent for 30 min at 37°C in the dark. Following theincubation, cells were washed with 1X assay buffer to removeunbound detection reagent and examined using a confocal microscope(LSM5; Carl Zeiss AG, Oberkochen, Germany).

Statistical analysis

All results presented were confirmed in at leastthree independent experiments. Data were presented as the mean ±standard deviation. Statistical differences were analyzed byone-way analysis of variance followed by a Tukey test usingIBM® SPSS® Statistics Version 24.0 (IBMCorp., Armonk, NY, USA). P<0.05 was considered to indicate astatistically significant difference.

Results

Pristimerin enhances cell death andactivates autophagy in MDA-MB-231 cells

Exposure to pristimerin for 24 h significantlyinhibited growth of MDA-MB-231 cells in a concentration-dependentmanner compared with control treatment with a vehicle (P<0.05;Fig. 1A). Whetherpristimerin-mediated inhibition of MDA-MB-231 cells originated fromautophagy was further examined. Autophagy can be accuratelymeasured by assessing the expression of microtubule-associatedprotein light chain 3 (LC3), namely the conversion of LC3-I toLC3-II using western blot analysis. Following treatment withpristimerin at 10 µM for 24 h, the ratio of LC3-II/LC3-I as well asLC3-II levels were increased (Fig.1B). Co-treatment with 2 mM 3-MA (an autophagy inhibitor)inhibited LC3-II accumulation induced by 10 µM pristimerin(Fig. 1C) and promoted cellviability (Fig. 1D). These resultssuggested that pristimerin exposure induced autophagic cytotoxicityin MDA-MB-231 cells whereas inhibition of pristimerin-inducedautophagy by 3-MA increased cell viability.

Figure 1. Pristimerin-induced cell death isassociated with activation of autophagy. (A) Cell viabilityfollowing treatment with pristimerin (0, 1, 2.5, 5 and 10 µM) inMDA-MB-231 human breast cancer cells. *P<0.05 vs. pristimerin (0µM). (B) MDA-MB-231 breast cancer cells were treated with 0, 1, 5and 10 µM pristimerin for 24 h and expression levels of LC3-I andLC3-II were detected by western blot analysis. GAPDH was used as aloading control. Values are expressed as the mean ± standarddeviation. *P<0.05 vs. control. (C) Cells were pre-incubatedwith or without 3-MA (2 mM) for 3 h and then incubated withpristimerin (10 µM) for 24 h, following which LC3-I and LC3-IIlevels were detected by western blotting analysis. (D) Cells werepre-incubated with or without 3-MA (2 mM) for 3 h and thenincubated with 0, 1, 5 and 10 µM pristimerin, and cell viabilitywas detected using a Cell Counting Kit-8 assay. Data are expressedas the mean ± standard deviation. *P<0.05 vs. pristimerin singletreatment. 3-MA, 3-methyladenine; LC3-I, light chain 3.

Paclitaxel induces autophagy inMDA-MB-231 cells

To examine the inhibitory effect of paclitaxel onthe proliferation of MDA-MB-231 cells breast cancer cells, variousconcentrations (0, 6, 12, 24 and 30 µM) of paclitaxel wereevaluated by CCK-8 viability assay. Paclitaxel exhibited nosignificant toxicity to MDA-MB-231 cells at any concentrationevaluated up to 24 µM (Fig. 2A).However, high concentrations of paclitaxel (over 48 µM)demonstrated strong toxicity (data not shown). Paclitaxel canmodulate autophagy (16,17), although its mode of action remainscontroversial. In the present study, the effect of variousconcentrations of paclitaxel on autophagy in MDA-MB-231 cells wasexamined. Following treatment with paclitaxel at 12, 24, or 30 µMfor 24 h, the ratio of LC3-II/LC3-I was significantly increased(P<0.05;Fig. 2B). Theseresults demonstrated that paclitaxel induced autophagy inMDA-MB-231 cells without demonstrating significantcytotoxicity.

Figure 2. Cell viability following treatmentwith paclitaxel in MDA-MB-231 human breast cancer cells. (A) Cellviability following treatment with various concentrations (0, 6,12, 24 and 30 µM) of paclitaxel. (B) Cells were treated withpaclitaxel for 24 h and the expression levels of LC3-II/LC3-I weredetected by western blot analysis. GAPDH was used as a loadingcontrol. Data are expressed as the mean ± standard deviation.*P<0.05 vs. 0 µM treatment. LC3-I, light chain 3.

Involvement of autophagy in theadditive action of pristimerin in combination with paclitaxel

As illustrated inFig.3A, combination treatment with 10 µM pristimerin and 24 µMpaclitaxel additively inhibited cell viability. Further experimentswere performed to observe whether paclitaxel influencedautophagic-cell death following treatment combined withpristimerin. Cells were pretreated with paclitaxel for 2 h and thentreated with pristimerin for another 24 h. It was observed that theratio of LC3-II/LC3-I as well as LC3-II levels were significantlyadditively increased (P<0.05;Fig.3B). Chemical inhibition of autophagy using 3-MA significantlyinhibited LC3-II accumulation induced by the combination ofpristimerin and paclitaxel (P<0.05;Fig. 3B). Autophagy was further assessedby a detection assay where a population of green detectionreagent-labeled vesicles co-localized with LC3, a specificautophagosome marker. It was revealed that paclitaxel orpristimerin treatment for 24 h resulted in the appearance of greendetection reagent while their combination exhibited stronger greenfluorescence (Fig. 3C). Autophagyinhibitor (3-MA) inhibited the autophagy reaction (Fig. 3C). Co-treatment with 3-MAsignificantly increased cell viability, even in the presence of acombination of pristimerin and paclitaxel (P<0.05;Fig. 3D). These results suggested thatpristimerin enhanced the paclitaxel-induced growth inhibition ofMDA-MB-231 cells by enhancing cytotoxic autophagic cell death.

Figure 3. Involvement of autophagy in theadditive action of pristimerin and paclitaxel. (A) Cell viabilityfollowing treatment with pristimerin (0, 1, 2.5, 5 and 10 µM) andpaclitaxel (24 µM) in MDA-MB-231 human breast cancer cells.*P<0.05 vs. pristimerin (10 µM) single treatment. (B) Cells weretreated with paclitaxel and pristimerin for 24 h and expressionlevels of LC3-II/LC3-I were detected by western blot analysis.GAPDH was used as a loading control. Data are expressed as the mean± standard deviation. *P<0.05 vs. paclitaxel and&P<0.05 vs. pristimerin. #P<0.05vs. paclitaxel and pristimerin combined treatment. (C) An autophagydetection kit was used for the detection of autophagy in cellsfollowing treatment with indicated drugs. (D) Cells werepre-incubated with or without 3-MA (2 mM) for 3 h and thenincubated with pristimerin (10 µM), paclitaxel, and a combinationof the two for 24 h. Cell viability was then measured. Data areexpressed as the mean ± standard deviation. *P<0.05 vs. combinedtreatment with pristimerin and paclitaxel. #P<0.05vs. pristimerin (10 µM) single treatment. LC3-I, light chain 3;3-MA, 3-methyladenine.

Regulation of ERK1/2 signalingcontributes to pristimerin-induced autophagy in MDA-MB-231cells

It is well known that ERK1/2 and autophagy areclosely linked (18). Whetherpristimerin treatment of MDA MB-231 cells could affect the ERK 1/2signaling pathway was investigated. Cells were treated with variousconcentrations of pristimerin for 24 h, following which,phospho-ERK 1/2 and p90RSK, one of the potentially importantsubstrates of ERK, were assessed by western blot analysis.Pristimerin dose-dependently inhibited the phosphorylation ofERK1/2 and p90RSK (Fig. 4A). Itwas also observed that beclin 1 expression and p62 degradation,both autophagic proteins, were increased by pristimerin treatment(Fig. 4A). Paclitaxel treatment(24 µM) alone also inhibited ERK1/2/p90RSK phosphorylation levelsand increased the beclin 1 expression and p62 degradation (Fig. 4A). Combined treatment ofpristimerin and paclitaxel additively inhibited ERK1/2phosphorylation and significantly increased the ratio ofLC3-II/LC3-I (P<0.05;Fig. 4B).Co-treatment with an ERK activator (ceramide C6) increasedphosphorylation of ERK and inhibited LC3-II accumulation incombination treatment of pristimerin and paclitaxel (Fig. 4B). These results suggested thatpristimerin-induced autophagy was associated with ERK1/2 signalingand paclitaxel additively enhanced pristimerin-induced autophagiccell death in MDA-MB-231 cells.

Figure 4. Pristimerin-induced autophagy isregulated by ERK1/2 signaling in MDA-MB-231 cells. (A) Cells weretreated with pristimerin and paclitaxel for 24 h and expressionlevels of p-ERK1/2, ERK1/2, p-p90RSK, p90RSK, beclin 1, and p62were detected by western blot analysis. GAPDH was used as a loadingcontrol. *P<0.05 vs. pristimerin (0 µM) (Lane no. 1) and#P<0.05 vs. paclitaxel (0 µM) (Lane no. 5). (B) Cellswere treated with pristimerin and paclitaxel and expression levelsof p-ERK1/2, ERK1/2, LC3-II/LC3-I were detected by western blotanalysis. Cells were pretreated with ERK activator ceramide C6 for3 h prior to combined treatment with pristimerin and paclitaxel.GAPDH was used as a loading control. Data are expressed as the mean± standard deviation. *P<0.05 vs. paclitaxel (lane no. 3),&P<0.05 vs. pristimerin (lane no. 2) and##P<0.01 vs. pristimerin and paclitaxel combinedtreatment without ceramide C6 (lane no. 4). p-ERK, phosphorylatedextracellular signal-regulated kinase; LC3, light chain 3; p90RSK,p90 ribosomal S6 kinase.

Discussion

Breast cancer is a complicated and heterogeneousdisease with a number of biomarkers, including estrogen receptor,progesterone receptor, HER2 and triple-negative breast cancer(19). Each of them has differenttreatment strategies and prognosis (19). A high mortality rate is associatedwith breast cancer patients (19).Increasing effort has been focused on the identification of novelanti-breast cancer agents. Medicinal plants and their extracts arecommonly used to prevent and treat a number of diseases, includingcancer. Developing novel therapeutic agents from plants with fewerside-effects and high efficacy is a promising strategy to reducethe mortality rate of breast cancer.

Pristimerin, a quinonemethide triterpenoid, has beenisolated from several plants includingMaytenus chuchuhuascaandM. ilicifolia in South Africa (20). Promising anticancer activities ofpristimerin have been emphasized in terms of its therapeuticpotential for breast cancer (21).Previous studies have demonstrated that pristimerin is involved inapoptotic cell death of MDA-MB-231 (15) and SKBR3 human breast cancer cells(13). It was demonstrated thatthe apoptotic activity of pristimerin and pristimerin inducedapoptosis in MDA-MB-231 cells, as expected. However, the effect ofpristimerin on autophagy in human breast cancer has not been fullyunderstood. Certain studies have reported that triterpenoids cancause cell death by autophagy, including cimigenol (KY17) (22), 2α, 3α,24-thrihydroxyurs-12-en-28-oicacid (23), ursolic acid (24) and cucurbitane (25). In the present study, the autophagiceffect of pristimerin on MDA-MB-231 human breast cancer cells wasexamined.

Autophagy has been established as a type ofprogrammed cell death involving self-destruction characterized bydistinct morphological and biochemical features. Autophagy isgenerally considered to be pro-survival associated, orcytoprotective under stressful conditions such as g-radiation andchemotherapy (26). However, it isfrequently activated in response to a number of environmentalstresses, thereby leading to cell death (27). LC3 is considered to be a strongmarker of autophagy. The conversion of LC3-I to LC3-II and LC3puncta usually demonstrate an activation of autophagy (28). In the present study,pristimerin-induced autophagy in MDA-MB-231 human breast cancercells was examined using western blot analysis. As demonstrated inthe results, LC3-II/LC3-I levels were increased, which indicatedthat induction of autophagy was concentration-dependent. Thisautophagy induction has the same pattern as pristimerin-inducedcell death. Furthermore, it was observed that autophagy inhibitionby 3-MA partially decreased pristimerin-induced cytotoxicity andundermined LC3-II levels. These data suggested thatpristimerin-induced autophagy can serve as a cell deathpathway.

Paclitaxel is isolated from the bark of the yewtree. It inhibits the growth of tumor cells. It is an importanttherapeutic drug in the treatment of a number of types of cancer,including breast cancer (29). Itis known to stabilize microtubules during DNA synthesis, therebysuppressing mitosis of cancer cells. Paclitaxel is capable ofinducing mitochondria-mediated apoptosis involvingcaspase-dependent (via caspase-3) and caspase-independent pathways(via apoptosis inhibitory factor) (30). Apoptosis is frequently closelyassociated with autophagy in cancer (31). Since autophagy has a housekeepingrole in clearing damaged organelles and eliminating intracellularpathogens, autophagy is generally regarded as a survival mechanism.On the other hand, autophagy has a key role in tumorigenesis,progression and oncotherapy (30).Paclitaxel can induce autophagy in human osteosarcoma cells (MG-63)(30), non-small cell lung cancercells (A549) (16) and cervicalcancer (HeLa) (32). In thepresent study, paclitaxel treatment promoted autophagy inMDA-MB-231 cells at concentrations over 12 µM, and did notdemonstrate cytotoxicity at 12 µM. However, higher concentrationsof paclitaxel (48 and 60 µM) demonstrated strong cytotoxicity alongwith autophagy induction (data not shown). A relatively highconcentration of paclitaxel was used in the present experimentcompared with other studies. There may be certain differences indrug use. Paclitaxel was obtained for intravenous use from BoryungCo., Ltd. (Seoul, Korea). Other investigations purchased the drugfrom Sigma-Aldrich; Merck KGaA. For unknown reasons, in the presentexperiment, MDA-MB-231 cancer cell lines did not respond to lowconcentrations at all, in contrary to results of other papers. Itwas demonstrated that another previous study also used a highconcentration of paclitaxel (33).The clinical use of paclitaxel is frequently limited due toacquisition of anticancer drug resistance (34). Therefore, combined treatment isoften used to enhance the effectiveness of chemotherapy and avoidchemo-resistance to a single agent. All single anticancer agentscould similarly be used at reduced concentrations when they arecombined with others to synergistically induce cancer cell death(35). Pristimerin in combinationwith taxol can synergistically induce the death of cervical cancercells (35). In the present study,pristimerin additively enhanced paclitaxel-induced cell death byautophagic induction in MDA-MB-231 cells. To the best of ourknowledge, the present study is the first to propose that autophagyin breast cancer cells may be stimulated by pristimerin alone, aswell as in combination with paclitaxel.

In the present experimental data (not shown),pristimerin-induced apoptotic activity was increased by addition ofpaclitaxel. The mechanism involved in these effects is still beinginvestigated. There is a complex crosstalk between autophagy andapoptosis (31). It is frequentlyunclear which specific interactions may contribute to cancer celldeath. Cancer cell death in this experiment is not the effect ofonly one mechanism, namely autophagy. However, autophagy may be oneof the mechanisms that contribute to cancer cell death, which canbe detected by a number of methods.

GTPase HRas/Raf proto-oncogene serine/threonineprotein kinase/Dual specificity mitogen-activated protein kinasekinase mek/ERK pathway serves an important role in autophagy. ERKphosphorylates and inhibits TSC1/TSC2 which then activates C1 andinduces autophagy (36). Recently,it has been demonstrated that a synthetic antihepatitis drug(Bicyclol) can induce autophagy via the ERK signaling pathway inHepG2 hepatocellular carcinoma cells (37). To understand the signaling cascadethat mediates the autophagic effect of pristimerin on MDA-MB-231cells, modulation of the activation of ERK1/2 by pristimerin wasexamined. Pristimerin treatment suppressed phospho-ERK1/2 andphospho-p90RSK levels in a dose-dependent manner, although withoutaffecting total ERK1/2 and total p90RSK expression. The function ofpristimerin on ERK regulation remains controversial. A previousstudy suggested that pristimerin can decrease the level of p-ERK1/2and mTOR to induce cell death in SKBR3 breast cancer cells(13). However, another studydemonstrated that ERK phosphorylation is not altered by pristimerintreatment in HeLa cervical cancer cells (35). In the present study, ERK1/2inhibition by pristimerin, paclitaxel, or the combination wasconfirmed to induce autophagy (increased p62 degradation andincreased beclin1 expression). These effects were reversed bytreatment with an ERK activator. These results suggested thatpristimerin-induced autophagy served as a cell death pathway viaERK1/2 inhibition, and that non-toxic doses of paclitaxel canadditively enhance these activities.

In conclusion, the results of the present studyelucidated the anti-cancer mechanism of pristimerin, anddemonstrated that non-toxic paclitaxel doses induced autophagy inbreast cancer cells. The current study provides sufficient evidencethat an autophagy inducer may be used as an adjuvant modalityduring anti-cancer pharmacological treatment.

Acknowledgements

Not applicable.

Funding

The present study was supported by research fund ofChungnam National University (grant no. 2015088201).

Availability of data and materials

All data generated or analyzed during this study areincluded in this published article.

Authors' contributions

YL and JN designed the study and prepared themanuscript. ML, EC and JP performed the experiments and analyzedthe data. JS and JL were involved in the study conception anddesign and revised the manuscript.

Ethics approval and consent toparticipate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competinginterests.

References