Dropwort-induced metabolic reprogramming restrains YAP/TAZ/TEAD oncogenic axis in mesothelioma
Over the past decade, newly designed cancer therapies have not significantly improved the survival of patients diagnosed with Malignant Pleural Mesothelioma (MPM). Among a limited number of genes that are frequently mutated in MPM several of them encode proteins that belong to the HIPPO tumor suppressor pathway.
The anticancer effects of the top flower standardized extract of Filipendula vulgaris (Dropwort) were characterized in “in vitro” and “in vivo” models of MPM. At the molecular level, two “omic” approaches were used to investigate Dropwort anticancer mechanism of action: a metabolomic profiling and a phosphoarray analysis.
We found that Dropwort significantly reduced cell proliferation, viability, migration and in vivo tumor growth of MPM cell lines. Notably, Dropwort affected viability of tumor-initiating MPM cells and synergized with Cisplatin and Pemetrexed in vitro. Metabolomic profiling revealed that Dropwort treatment affected both glycolysis/tricarboxylic acid cycle as for the decreased consumption of glucose, pyruvate, succinate and acetate, and the lipid metabolism. We also document that Dropwort exerted its anticancer effects, at least partially, promoting YAP and TAZ protein ubiquitination.
Our findings reveal that Dropwort is a promising source of natural compound(s) for targeting the HIPPO pathway with chemo-preventive and anticancer implications for MPM management.
KeywordsHIPPO tumour suppressor pathway YAP Mesothelioma Phytonutrient Cancer metabolism
associated protein gene 1
cyclin dependent kinase inhibitor 2A gene
Focal Adhesion Kinase
human untransformed mesothelial cell
malignant pleural mesothelioma
mammalian target of rapamycin
Tricarboxylic Acid Cycle
variable importance in projection
Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising from lining of pleural or peritoneal mesothelial cell surfaces [1, 2]. Currently, the clinical outcome is poor with the median survival rate of less than 12 months. Its silent clinical progression and the aberrant resistance to current therapies underlie the poor prognosis of the disease [3, 4]. Platinum based-chemotherapy combined with the folate antagonist, Pemetrexed, represents the conventional treatment [5, 6]. At the present, there is no second line standard therapy for MPM. Consequently, there is an urgent need of new therapeutic options for such a rare if not an orphan disease .
There are three histological subtypes of MPM: the epithelioid, the spindled and the biphasic MPM . Most of the MPM incidence at population levels is explained by asbestos exposure after decades of latency period [9, 10, 11]. Thus the MPM incidence is expected to rise in the next decade with a large variability across countries due to differences in asbestos commercialization, its use and the duration of banned periods [12, 13, 14]. The pathophysiology of the disease is mostly explained by chronic inflammation due to the inhalation of nanoparticles causing mesothelial surfaces infiltration by macrophages that activate pro-inflammatory responses and a release of cytokines and chemokines to pleural and lung tissues [2, 15, 16]. Other factors, including Erionite, a natural fibrous compound that belongs to zeolite minerals, a radiation exposure, an infection by SV40 virus or various genetic factors have been considered as additional risk factors for MPM development [12, 17, 18].
Dietary phytochemicals usually do not cause adverse effects and generally have ability to target multiple signalling pathways. Therefore, medicinal plants have been considered appealing as co-adjuvants in anticancer therapies . Natural polyphenols exhibit pleiotropic anticancer effects thereby impacting on cell proliferation, apoptosis, angiogenesis, oxidation and inflammation . Collectively, it has previously been shown that some phytonutrients can exert anticancer effects in mesothelioma cell lines by interfering with specific oncogenic and/or tumor suppressor pathways [21, 22, 23, 24, 25].
The HIPPO signalling pathway controls cell proliferation and organ size [26, 27, 28]. YAP and TAZ, are two major effectors of the Hippo pathway and act as sensors of the cell microenvironment and as regulators of cell stemness . Importantly, YAP and TAZ are aberrantly expressed in a wide variety of tumors and their activation drives different steps of the metastatic cascade [30, 31, 32]. Despite the fact that the Hippo pathway is deregulated in human cancers, only a few somatic mutations have been reported so far in the Hippo pathway genes. MPM is one of a few cancers that harbor mutations in Hippo pathway genes . Accordingly to the COSMIC database, the genes that are most frequently mutated in MPM are loss of function mutations of tumor suppressor genes such as cyclin dependent kinase inhibitor 2A gene (CDNKN2A), TP53, Neurofibromin 2 (NF2) and BRACA1 associated protein gene 1 (BAP1) . Transcriptome and whole-exome sequencing have revealed that NF2 is frequently mutated in tissues of MPM patients together with a copy number loss and/or loss of expression of NF2, LATS1 and LATS2 [35, 36, 37, 38, 39, 40]. NF2 is an upstream regulator of the Hippo pathway. The inactivation of NF2 and LATS2 in MPM prevents the phosphorylation of the transcriptional co-activators YAP/TAZ at Serine 127. This leads to the YAP shuttling into the cell nucleus, favouring an unregulated interaction with TEAD family transcription factors; thereby promoting tumorigenesis [33, 41, 42]. The identification of agents that inhibit YAP/TAZ oncogenic activities might represent a novel and efficacious therapeutic approach for treatment of MPM patients [43, 44, 45] Here, we screen thirty natural extracts with anti-inflammatory properties (data not shown) Among them we found that Filipendula vulgaris top flower extract exhibited the best IC50 value in MPM cell lines treatment. Filipendula hexapetala Gilib (dropwort, syn. Filipendula vulgaris Moench) belongs to genus Filipendula (Rosaceae). It is a perennial herb (up to 80 cm high) diffused in Eastern European countries with pinkish white flowers and characteristic tuberous roots . It is known for its analgesic, antirheumatic, anti-inflammatory and diuretic effects [47, 48]. Several evidences have reported other properties of the plant as antimicrobial, antigenotoxic, antioxidant [49, 50] and hepatoprotective effects .
We also show that a standardized extract from Dropwort (Filipendula vulgaris, Fil.v.), significantly impairs mesothelioma progression in “in vitro” and “in vivo” models of the disease. This impairment occurs, at least partially, through the silencing of YAP and TAZ oncogenic activities. We find that Dropworth-derived formulation promotes YAP/TAZ ubiquitination and therefore restrains the oncogenic axis of YAP/TAZ/TEAD in MPM progression.
Materials and methods
Cell cultures and treatments
Dropwort (Filipendula vulgaris) extract components. Different classes of compounds present in the Dropwort flowering tops phytocomplex are reported. The phytocomplex was derived from Filipendula vulgaris
Classes of Compounds
Levels found in Dropwort freeze-dried extract
Of which Lignins total
Of which tannins total
Of which phenols and phenolic acids total
- Gallic acid
- Protocatechuic acid
- Ellagic acid
- Gentistic acid
- Methyl gallate (methyl 3,4,5-trhydroxyphenylglycol)
- Shikimic acid
Of which flavonoids total
Of wich flavonols, flavanols, isoflavones
- Quercetin 3-b-glucoside
- Quercetin 3-D-galactoside
Of which catechins
Of which phenylpropanoid derivates
- Chlorogenic acids
- Caffeic acids
- Of which salicilates total
Organic Acids (%)
Free amino acids (%)
Of which Soluble dietary fiber
Of which Insoluble dietary fiber
Of which monosaccharides
Minerals (Oligo, micro and macroelements) (%)
RNA processing and qRT-PCR
Total RNA from mesothelioma cell lines differently treated or not was extracted by using Trizol Reagent following manufacturer’s instructions (Ambion). cDNA was synthesized according to the manufacturer’s instructions (M-MLV RT kit, Invitrogen). Gene expression was measured by real-time PCR using the FastStart SYBR Green Master Mix (Applied Biosytems) on a studio 7-instrument (Applied Biosystems). Sequences of qPCR primers are ACTIN Fw: 5′-GGCATGGGTCAGAAGGATT-3′, Rv: 5′-CACACGCAGCTCATTGTAGAAG-3; YAP1 Fw: 5′-CACAGCATGTTCGAGCTCAT-3′, Rv: 5′-GATGCTGAGCTGTGGGTGTA-3′; TAZ Fw: 5′-CCATCACTAATAATAGCTCAGATC-3′, Rv: 5′-GTGATTACAGCCAGGTTAGAAAG-3′; MCM7 Fw: 5′-TCGAGGCATGAAAATCCGGG-3′, Rv: 5’CGCCAGTCGATCAATGTATGACA-3′; ANKRD1 Fw: 5′-AGTAGAGGAACTGGTCACTGG-3′, Rv: 5′-TGGGCTAGAAGTGTCTTCAGAT-3′; CTGF Fw: 5′-GCCACAAGCTGTCCAGTCTAATCG-3′, Rv: 5′-TGCATTCTCCAGCCATCAAGAGAC-3′; p21 Fw: 5′-GGGACAGCAGAGGAAGAC-3′, p21 Rv: 5′-GCGTTTGGAGTGGTAGAAATC-3′.
Cell viability assay
Cell viability of treated cells was assessed using ATPlite assay (Perkin Elmer, Massachusset, USA) accordingly to the manufacturer’s instructions. Cells (8 × 102 cells) were seeded in 96 well-plates and cultured for 24 h and treated for 72 h with Fil.v. extract (0, 3, 6, 12.5, 25, 50, 100 and 200 μg/ml). Each plate was evaluated immediately on a microplate reader (Expire Technology, Perkin Elmer). Calcusyn software was used to calculate combination index (CI) .
MPM cell lines were grown at 70% confluence and treated with Fil.v. extract or with vehicle. Sixteen hours later, cells were detached and seeded at 600 cells per 6 well into six-well dishes (Corning-Costar, Tewksbury, MA, USA) in drug-free media. Fresh media (25%) was added every three days. After 15–21 days, colonies were stained with crystal violet and colonies counted.
For propidium iodide (PI) staining, cells were seeded in 6-well plates at a density of 104 cells/ml. After 24 h cells were treated with indicated plant extract concentrations for different time intervals. Floating and attached cells were harvested, washed in PBS, fixed in ice-cold ethanol (70% v/v) and stored at − 20 °C. For the analysis, cells were washed in PBS and incubated with RNase A (1 mg/ml) and PI (40 μg/ml) was added. For PI/Annexin V double staining treated cells were harvested and suspended in binding buffer (HEPES pH 7.4, CaCl2 2.5 mM, NaCl 140 mM). Aliquots of cells were incubated for 15 min with Annexin V FITC (0.2 μg/ml) (Abcam, ab-63,556) and PI (5 mg/ml) (Invitrogen). For each FACS analysis, 3 × 103 events for each sample were analyzed. Flow cytometry analyses were carried out with Easycyte 8HT (Guava, Millipore) followed by analysis using InCyte software (Millipore).
ALDH activity assay
ALDEFLUOR kit (Stem Cell Technologies, Vancouver, Canada) was used to asses ALDH activity of MSTO-211H treated or not with Fil.v. extract. ALDH-positive cells showed greater fluorescence compared to a control staining reaction containing the ALDH inhibitor, DEAB (diethylaminobenzaldehyde), upon addition of the synthetic ALDH substrate BAAA. Flow cytometry analyses were carried out with Easycyte 8HT (Guava, Millipore) followed by analysis using InCyte software (Millipore).
Transwell invasion assay
Migration assay was performed using a 24-well Boyden chamber with a non-coated 8-mm pore size filter in the insert chamber (BD Falcon, Franklin Lakes, NJ, USA). Cells were suspended in 0.5 ml DMEM/F12 media without containing FBS and seeded into the insert chamber. Cells were allowed to migrate for 24 h into the bottom chamber containing 0.5 ml of DMEM/F12 media containing 10% FBS in a humidified incubator at 37 °C in 5% CO2. Migrated cells that attached to the outside of the filter were visualized by staining with DAPI (Thermo Fisher) and counted. The average number of cells per field was expressed as percentage of the control after normalizing for cell number.
Wound–healing migration assay
MPM cells were grown to 80% of confluence in 6-well tissue culture plates and wounded with a sterile 10-ml pipet tip to remove cells. PBS washing was used to remove loosely attached cells. The progression of migration was photographed at different under a light microscope. The number of cells migrated into the scratched area was calculated.
Western blot analysis and protein immunoprecipitation
Cell lysis was performed on ice for 30 min in NP40 lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EGTA, 1 mM EDTA) supplemented with protease and phosphatase inhibitors (5 mM PMSF, 3 mM NaF, 1 mM DTT, 1 mM NaVO4). Equal amounts of total proteins extracts (10–30 μg) were resolved by 8–12% denaturing SDS polyacrylamide gel electrophoresis (SDS-PAGE), and transferred for 1 h and 30 min to polyvinylidene difluoride membrane. Membranes were blocked in 5% milk-TBS-0.05% Tween 20 for 1 h and incubated overnight with the specific primary antibodies. In the co-immunoprecipitation the lysis buffer was modified accordingly the protein isoelectric point. Protein concentrations were determined by colorimetric assay (Bio-Rad, Hercules, CA, USA). For each immunoprecipitation, 1 μg of rabbit YAP (Santa Cruz, sc-15,407) or mouse TAZ antibody (Sigma Aldrich, T4077) and 1 μg of rabbit or mouse IgG (Santa Cruz Biotech, sc66931 and sc69786) as control were used. Pre-cleared extracts were incubated with protein A/G-Agarose beads (Thermo Fisher Scientific, Rockford, IL, USA) in lysis buffer containing 0.05% BSA and antibodies, under constant shaking at 4 °C for 3 h. After incubation, agarose bead-bound immunocomplexes were rinsed with lysis buffer and eluted in 50 ml of SDS sample buffer for western blotting.
The following primary antibodies were used: anti phospho-AMPKα (Thr-172) (Cell Signaling, #2531); anti-phospho-mTOR (Ser-2448) (Cell Signaling, #2971); anti- β Actin (Santa Cruz, sc-81,178); anti-phospho p70 S6 Kinase (thr-389) (Cell Signaling, #9234); anti-phospho S6 (ser235/236) (Cell Signaling, #2211); PARP (Cell Signaling, # 9542); anti-caspase 7 (Cell Signaling, #9492); anti-caspase 3 (Enzo life Science, #31A1067); anti YAP (Santa Cruz, sc-15,407); anti TAZ (Sigma Aldrich, T4077); anti GAPDH (Santa Cruz, sc-47,724); anti-TEAD1 (BD, cat.n.-610,923); anti H1 (Cell Signaling, #41328) anti Tubulin (Abcam, Ab44928); anti MCM7 (Cell Signaling, #3735); anti HA (Santa Cruz, sc-57,592). All the indicated antibodies were used at the minimum dilutions suggested by the manufacturer. Secondary horseradish peroxidase-conjugated was purchased from Santa Cruz. ECL reagent (Amersham, GE Healthcare, Piscataway, NJ, USA) was employed for the chemo-luminescence detection. The Uvitec Alliance software (Eppendorf) was used to quantify the obtained data.
Nucleo/cytosol extracts preparation
MSTO-211H treated cells were washed in cold PBS. After pelleting cells were lysed in 10 mM HEPES-KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride for 10 min. After centrifugation, supernatants were removed and represented cytosolic fraction, while the pellet (nuclear fraction) was treated in a high salt buffer (20 mM HEPES-KOH pH 7.9; 25% glycerol; 0.42 M NaCl; 1.5 mM MgCl2; 0.2 mM EDTA; 0.5 mM dithiothreitol; 0.2 mM phenylmethylsulfonyl fluoride) for 20 min to extract nuclear proteins .
A number of 1.6 × 106 cells was transfected with a pCMV vector carrying hemagglutinin (HA)-tagged ubiquitin (Ub-HA) . After 18 h, cells were treated with 25 μM MG-132 for a further 6 h. Protein extracts were immunoprecipitated as described and subjected to Western Blotting.
Plasmids and transfections
Transfections were performed with Lipofectamine 2000 or Lipofectamine RNAiMax (Life Technologies) according to manufacturer’s recommendations. The following siRNAs (Eurofins MWG) were used to inhibit YAP, TAZ and TEAD expression in MSTO-211H cells: siYAP: 5′-GACAUCUUCUGGUCAGAGA-3′, siGFP: 5′-AAGUUCAGCGUGUCCGGGGAG-3′, siTAZ is a pool of two independent siRNAs mixed in equal amount: 5′-AAAGUUCCUAAGUCAACGU-3′ and 5′- AGGUACUUCCUCAAUCACA-3′, si-TEAD: 5′- CGAUUUGUAUACCGAAUAA. The following vectors were used to over express the same genes in MSTO-211H cells: pCDNA3-YAP-Flag, pCS2-TAZ-Flag, pQCXIH-myc-TEAD kindly gifted by prof. Georg Halder.
MSTO-211H cells were seeded into eight-chamber culture slides (BD Falcon). The next day, cells were rinsed with ice-cold PBS buffer and fixed with 4% paraformaldehyde for 10′ at room temperature and then permeabilized with 1% Triton X-100. Cells were incubated overnight with the indicated antibody. The day after, cells were washed with cold PBS three times for 3 min each and stained for 2 h with a secondary antibody Alexa 488-conjugated goat anti-mouse IgG or anti-rabbit IgG (Molecular Probes Cells) and counterstained with DAPI (40,6-diamidino-2-phenylindole dihydrochloride). Cells were examined under a Zeiss LSM 510 laser scanning fluorescence confocal microscope (Zeiss, Wetzlar, Germany).
Sample preparation for NMR spectroscopy
Each medium sample (2 ml) was lyophilized then dissolved in 700 μl of 1 mM TSP [sodium salt of 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid], 10 mM sodium azide D2O phosphate buffer solution (pH = 7.4) and finally homogenized by vortex mixing for 1 min. After centrifugation (10 min, 10.000 RCF at 22 °C), 600 μl of each resulting supernatant was transferred to a 5-mm NMR tube and used for the NMR analysis. Cell pellets were lyophilized, weighted and cryogenically grounded using Cryomill (Retsch GmbH Germany) before methanol/chloroform/water extraction (2/2/1.8) to extract polar and non-polar metabolites. Polar extracts were dissolved in 600 μl of 1 mM TSP ((trimethylsilyl)-propionic-2,2,3,3-d4 acid) and 10 mM NaN3 solution in D2O 0.1 M phosphate buffer (pH = 7.4), while nonpolar extracts were dissolved in 600 μl of CDCl3 containing 0.03% TMS (tetramethylsilane)/CD3OD solution (2:1, v/v).
All 2D 1H J-resolved (JRES) NMR spectra were acquired on a 500 MHz VNMRS Varian/Agilent spectrometer (Agilent, Santa Clara, CA) at 25 °C using a double spin echo sequence with pre-saturation for water suppression and 16 transients per increment for a total of 32 increments. These were collected into 16 k data points using spectral widths of 8 kHz in F2 and 64 Hz in F1. Each free induction decay (FID) was Fourier transformed after a multiplication with sine-bell window functions in both dimensions. JRES spectra were tilted by 45°, symmetrized about F1, referenced to lactic acid at δH = 1.33 ppm and the proton-decoupled skyline projections (p-JRES) exported using Agilent VNMRJ 3.2 software. The exported p-JRES were aligned, corrected for baseline offset and then reduced into spectral bins with widths ranging from 0.02 to 0.06 ppm by using the ACD intelligent bucketing method (1D NMR Manager software, ACD/Labs, Toronto, Canada). This method sets the bucket divisions at local minima (within the spectra) to ensure that each resonance is in the same bin throughout all spectra. The area within each spectral bin was integrated and, to compare the spectra, the integrals derived from the bucketing procedure were normalized to the total integral region. Metabolites were identified using an in-house NMR database and literature data and confirmed by 2D homo- and hetero-nuclear NMR spectroscopy.
NMR spectra pre-processing treatment
The 1D skyline projections exported were aligned and then reduced into spectral bins with ranging from 0.01 to 0.02 ppm by using the ACD intelligent bucketing method (1D NMR Manager software (ACD/Labs, Toronto, Canada). To compare the spectra, the integrals derived from the binning procedure were normalized to the total integral region, following exclusion of bins representing the residual water peak (4.33–5.17 ppm) and the TSP peak (0.5–0.5 ppm). The resulting data was used as input for multivariate analysis: Principal Component Analysis (PCA and Orthogonal projections to latent structures discriminant analysis (OPLS-DA) were performed using SIMCA-P + version 12 (Umetrics, Umea, Sweden).
The Phospho Explorer antibody microarray, which was designed and manufactured by Full Moon Biosystems, Inc. (Sunnyvale, CA), contains 1318 antibodies. Each of the antibodies has two replicates that are printed on a coated glass microscope slide, along with multiple positive and negative controls. The antibody array experiment was performed using Full Moon Biosystems, according to their established protocol. In brief, cell lysates obtained from MSTO-211H treated with the Fil.v. (50 μg/ml) or vehicle for 24 h, were biotinylated with the antibody array assay kit (Full Moon Biosystems, Inc.). The antibody microarray slides were first blocked with a blocking solution (Full Moon Biosystems, Inc.) for 30 min at room temperature, rinsed with Milli-Q grade water for 3–5 min. The slides were then incubated with the biotin-labeled cell lysates in coupling solution (Full Moon Biosystems, Inc.) at room temperature for 2 h. The array slides were washed 4 to 5 times with 1x Wash Solution (Full Moon Biosystems, Inc.) and rinsed extensively with Milli-Q grade water before detection of bound biotinylated proteins using Cy3-conjugated streptavidin. Each slide (containing six replicates) was hybridized and Cy3 fluorescence acquired by microarray scanner with a scan resolution of 10 mm (Agilent Technologies). The images were quantified using Agilent Feature Extraction (AFE) software (Agilent Technologies). The fluorescence signal of each antibody was obtained from the fluorescence intensity of this antibody spot after subtraction of the blank signal (spot in the absence of antibody) .
Bioinformatic analysis was performed with Matlab (The MathWorks Inc.). Z score transformation was used to express the background corrected spot intensity values as unit of a standard deviation from the normalized mean of zero. Features were selected basing on Z ratios calculated by taking the difference between the averages of the observed protein Z scores and dividing by the standard deviation of all the differences for that particular comparison. A Z-ratio that was higher than 1.96 was inferred as significant. Unsupervised Hierarchical Clustering was used to investigate clusters of samples. Pathway analysis was performed by DAVID program.
EnSpire® cellular label-free platform
MSTO-211H cells were seeded in specially designed 384- well plate with highly precise optical sensors able to measure changes in light refraction resulting from dynamic mass redistribution (DMR) within the cell’s monolayer. Change in the light refraction was indicated by a shift in wavelength.
CD1 mice were subcutaneously transplanted with MSTO-211H (2 × 106). At the evidence of tumor appearance (when tumor volume reached 60 mm3) animals were randomly divided into five groups. Drinking water and a complete pellet diet (GLP 4RF21, Mucedola) were supplied ad libitum. Dueto the complexity of the Fil.v. extract it was not possible to titer the effective compound but rather refer to the total extract amount administrated. Besides, treatment concentrations were translated from in vitro studies. Accordingly, three groups of mice (n = 6) were given Fil.v. extract in drinking water at the following concentrations: 25, 50 and 75 mg/ml, whereas mice of the control group (n = 6) were given vehicle (daily for three weeks). An average of 8 ml/day was estimated to be drunk by each mouse. In the last group, Pemetrexed was injected intraperitoneally at the dose of 100 mg/kg for five consecutive days . Body weight and clinical signs of the mice were checked every 3 days. After 24 days mice were euthanized while under deep anesthesia and unresponsive to all stimuli. Animals were free of pathogens according to FELASA recommendations and health status was monitored daily. All tumorigenicity assays were performed according to the guidelines set by the internal ethical committee. At the end of the experiment tumor masses were collected and fixed in 10% buffered formalin.
MSTO-211H cells were pre-treated with Fil.v. extract (50 μg/ml) for 24 h. Suspensions of 2 × 106 MSTO-211H cells x mouse (n = 6) were subcutaneously injected in PBS 1x/Matrigel (BD Biosciences San Jose, CA, USA) into 6-weeks-old female CD1 mice (Charles River, Milan). Tumor volume was calculated by using the formula: V 1/2 x length x width2 (by electronic caliper).
Formalin-fixed and paraffin-embedded 5 μm sections from mice tumor sections were stained with haematoxylin and eosin or stained with anti-ki67 antibody (ab15580, Abcam). Seven fields chosen randomly from each sample were scored.
A standardized extract from dropwort inhibits cell proliferation and impairs migration and invasion of MPM cells
Dropwort impairs MPM ALDH-bright positive cells viability and affects in vivo tumor growth
Dropwort extract sensitizes MSTO-211H cell to cisplatin or Pemetrexed-induced cell killing
Dropwort affects multiple pathways in mesothelioma cells
Dropwort impairs YAP and TAZ activity in MSTO-211H cells
Although the anti-tumoral activity of Fil. v. extract is likely maximized by its intrinsic complexity, to find out which components might be responsible for Fil. v. effects, we treated MSTO-211H cells with either Fil. v. total extract or single synthetic components of the extract, such as Salicylic, Chlorogenic, Gallic acid and Quercetin (Table 1 and Additional file 3: Figure S3a). Among them, we found that Gallic acid and Quercetin affected cells viability, while Salicylic and Chlonogenic acid did not impair MSTO-211H vitality (Additional file 3: Figure S3a). Consequently, we assessed whether Gallic acid and Quercetin inhibited YAP and TAZ protein levels. Unlike Gallic acid, Quercetin inhibited YAP and TAZ protein levels (Additional file 3: Figure S3b) in MSTO-211H cells. Moreover, we found that Quercetin and Fil. v. impaired YAP and TAZ protein levels in MPP-89, H-28 and H-2052 MPM cell lines (Additional file 3: Figure S3c).
Post-translational modifications of YAP and TAZ are mainly related to their cytoplasmic distribution or their degradation mediated by the proteasome pathway . Thus, we firstly tested whether Fil.v. extract induced a different nucleo-cytoplasmic distribution of YAP and TAZ proteins. We observed that upon Fil.v. extract treatment the reduction of YAP and TAZ occurred mainly in the nucleus (Fig. 6d and Additional file 4: Figure S4a), but concomitantly we did not find any re-localization of YAP and TAZ from the nucleus to the cytoplasm (Fig. 6d and Additional file 4: Figure S4a). Strikingly, we observed that Fil.v. extract induced an increased ubiquitination of both YAP and TAZ proteins (Fig. 6e, f), thus suggesting that the treatment with the plant extract impaired YAP/TAZ oncogenic activities by promoting their degradation.
Inactivation of YAP/TAZ/TEAD axis favors dropwort extract anticancer activity
We previously reported that MPM cells with elevated ALDH activity endow with tumor initiating features, thus contributing strongly to in vitro and in vivo chemoresistance of MPM . Fil.v. treatment affects ALDH-bright MPM cells, thus it might hold the potential to tackle actively MPM subcellular populations highly chemoresistant. At least, at the preclinical stage, Fil.v. synergizes with both cisplatin and Pemetrexed and reduces the volume of MPM xenografted tumors as efficienttly as Pemetrexed. As a whole, these findings support the possibility that Fil.v. might complement and reinforce either cisplatin- or Pemetrexed-induced anticancer effects on MPM cells. Furthermore, Fil.v. exhibits the ability to tackle MPM tumor initiating cells, thereby having at the preclinical stage a profound cell killing effect on highly resistant cell subpopulations of MPM.
MPM is still a poorly treated tumor. Either novel therapeutic approaches or the repurposing of existing drugs might contribute to tackle this unmet clinical need. Despite the apparent biochemical complexity of plant extracts that are derived from Filipendula vulgaris, for the specific formulation of Fil.v. extract used here, an anticancer activity could be documented in several cell culture and animal models of MPM. It would be important now to identify the complete mechanism via which the extract achieves anti-tumoral properties in order to candidate it to become a potent drug for the MPM treatment. Our previous published natural compound derived from leaf extracts of artichoke is actually in clinical trial (NCT 02076672) for the treatment of subjects with asbestosis  . This leads the way to the potential clinical application of Filipendula vulgaris either alone or in combination with leaf extracts of artichoke.
E.K. was supported from a fellowship from Aboca SPA. Sabrina Strano and Giovanni Blandino are corresponding and co-corresponding author respectively.
Consent for pubblication
GB and SS conceived the study, coordinated the experiments and revised the manuscript. CP performed in vitro experiments, phosphoarray assay and participated in coordinating the experiments and drafting the manuscript. MV, LC performed in vitro metabolomic studies and statistical analysis. FM performed the in vivo tumorigenic assays. EK performed in vitro biological functional assays. MF and FLS performed migration and biochemical assays. AS performed statistical analysis. GG performed and analyzed immunoistochemical assay. AM and JL produced standardized Filipendula vulgaris extract. MS and PM participated to design the study and revised the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The authorization to use animals were obtained by the Italian Health Authority. The Care and Husbandry of animals are in accordance with European Directives no. 86/609 and with the Italian Regulatory system (D.L. vo no. 26/14). All methods were carried out in accordance with relevant guidelines and regulations and all experimental protocols were approved by the Italian Ministry of Health. All efforts were made to minimize animal suffering and to reduce the number of animals used.
The authors declare that they have no competing interests.
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