Extracts from Pulsatilla patens target cancer-related signaling pathways in HeLa cells

The purpose of this study was to determine if a methanolic extract of the Pulsatilla patens (L.) Mill. can inhibit the progression of cancer through the modulation of cancer-related metabolic signaling pathways. We analyzed a panel of 13 inducible luciferase reporter gene vectors which expression is driven by enhancer elements that bind to specific transcription factors for the evaluation of the activity of cancer signaling pathways. The root extract of P. patens exhibited strong inhibition of several signaling pathways in HeLa cells, a cervical cancer cell line, and was found to be the most potent in inhibiting the activation of Stat3, Smad, AP-1, NF-κB, MYC, Ets, Wnt and Hdghog, at a concentration of 40 µg/mL. The methanolic extracts of P. patens enhanced apoptotic death, deregulated cellular proliferation, differentiation, and progression towards the neoplastic phenotype by altering key signaling molecules required for cell cycle progression. This is the first study to report the influence of Pulsatilla species on cancer signaling pathways. Further, our detailed phytochemical analysis of the methanolic extracts of the P. patens allowed to deduce that compounds, which strongly suppressed the growth and proliferation of HeLa cancer cells were mainly triterpenoid saponins accompanied by phenolic acids.

The cancer-related signaling pathways in HeLa cells. The activity of cancer-related signaling pathways was assessed using a panel of luciferase reporter gene vectors, in which luciferase expression is driven by the binding of transcription factors to multiple copies of synthetic enhancers within each vector. The methanolic extracts from the roots of P. patens were more powerful than the compounds from leaf extract of this species inhibiting the activation of 11 pathways analysed, with the exception only two, FoxO and miR-21. The active compounds of methanolic extracts from the roots of P. patens were found to be the most potent in inhibiting the activation of Stat3, Smad, AP-1, NF-κB, MYC, Ets, Wnt and Hdghog signaling (Fig. 1). E2F and Notch signaling were inhibited stronger by compounds of root extract than leaf extract, but similarly inhibited as by an active antitumor compound used in the research, resveratrol analog. Similarly, the activation of the apoptotic mediators pathways FoxO, miR-21 and k-Ras were not observed with either compounds from the methanolic extracts of P. patens (Supplementary Table S1).
The methanolic extracts from the roots of P. patens were stronger in potency than active antitumor compound resveratrol analog in each of their concentrations for inhibition of signaling mediated by Stat3, Smad, AP-1, NF-κB, E2F, MYC, Ets, Wnt and Hdghog, but were similar in potency as resveratrol analog for the inhibition of signaling mediated by Notch and k-Ras. However, the resveratrol analog was more effective than active compounds of P. patens for FoxO and miR-21. It was found that methanolic extracts from the roots of P. patens did not inhibit luciferase expression driven by the minimal thymidine kinase promoter (pTK-control), indicating it as not significant for general cytotoxicity or luciferase enzyme inhibition.
Since active compounds of methanolic extract from the root of P. patens were more forceful than from leaves of this species with respect to inhibiting the activation of pathways, it was determined which concentration of this extract was also a more effective modulator of cancer-related signaling pathways. Active compounds of methanolic extracts from roots of P. patens with a concentration of 40 µg/mL were stronger than these compounds from other concentrations (30 µg/mL and 15 µg/mL) (Supplementary Table S1). Active compounds from roots of P. patens with a concentration of 40 µg/mL were more potent inhibiting the activation of 12 signaling pathways used, with the exception of only two, FoxO and miR-21. The active compounds from the roots of P. patens with a concentration of 40 µg/mL were found to be the most effective in inhibiting the activation of 11 signaling www.nature.com/scientificreports/ pathways used, such as Stat3, Smad, AP-1, NF-κB, MYC, Ets, Wnt, Hdghog, E2F, Notch and k-Ras (Fig. 2). None of the extracts with the concentrations of 40 µg/mL, 30 µg/mL and 15 µg/mL inhibited the apoptotic mediators FoxO, miR-21 and inhibited the minimal thymidine kinase promoter (pTK control) in the luciferase expression, indicating a weak general cytotoxicity. This is the first report of active compounds of methanolic extracts from roots of P. patens from a natural source influencing the cancer-related signaling pathways in HeLa cells, and also the first report of different 13 signaling pathways as transcription factors in cancer processes from the genus Pulsatilla patens. Obtained data suggests that the action of P. patens methanolic extract is based on the induction of apoptosis and on the inhibition of mitosis by disturbing the HeLa cell cycle (Fig. 3). The small-molecule kinase inhibitors, which, through selectivity and specificity, inhibit the activation of specific proteins, also act on the receptors responsible for oncogenesis. Based on the studied cancer-related signaling pathways in HeLa cells we propose that the methanolic extracts of P. patens enhance apoptotic death, deregulated cellular proliferation, differentiation, and progression towards the neoplastic phenotype by altering key signaling molecules required for cell cycle progression. Thereby, this mechanism prevents excessive, harmful proliferation of the HeLa cells and inhibits them.
The active compounds of methanolic extracts of P. patens. The results showed that P. patens is a rich source of polyphenolic constituents, mainly saponins and hydroxycinnamic acids. Interestingly, P. patens showed a different profile of triterpenoid saponins in comparison to other species from the Ranunculaceae family.
The calibration curves of reference compounds were linear in the range of 0.02-1 mg/mL and 0.002-0.5 mg/ mL, for Pulsatilla saponin D and ferulic acid, respectively. The linearity was very good with the correlation coefficient (R 2 ) values higher than 0.999, as shown in Table 2. The results obtained in the intraday and interday precision measurements of both reference standards were below 1%, indicating the high stability of the method. Table 1. Cytotoxicity activities of P. patens methanolic extracts from roots towards a panel of mammalian cell lines. a IC 50 µg/mL is the concentration that affords 50% inhibition of cell growth. b-h Human cell lines of epidermal carcinoma, breast carcinoma, ovarian carcinoma, skin melanoma, cervical carcinoma, kidney epithelial cells, and kidney fibroblast respectively, *positive control drug. www.nature.com/scientificreports/ The recovery was in the range from 87.5% (RSD 0.89%) for Pulsatilla saponin D to 97.04% (RSD 0.49%) for ferulic acid. The analyzed compounds vary remarkably with the part of the plant. The total content of phenolic acids was higher in leaves while roots were found to be an abundant source of triterpenoid saponins, representing the dominant class of constituents. The content of saponins was in the range from 0.11 to 2.14 mg/g dw in roots and from 0.14 to 0.32 mg/g dw in the leaves (Table 3; Supplementary Fig. S1). Triterpenoid saponins and phenolic acids present in P. patens extracts were characterized and tentatively identified for the first time. Their MS data were listed in Table 4. Most of the triterpenoid saponins found in the studied P. patens extracts shared common features. The characteristic solvent adduct ions [M + HCOO] − or deprotonated ions [M-H] − were usually observed. The composition of sugar residues was deducted based on the fragmentation pathway observed in MS/MS spectra. The peaks with high abundance intensities were formed from the typical for negative ionization mode, simultaneous loss of 470 Da, which was attributed to the cleavage of the ester bond and the elimination of the entire sugar chain, assigned as Glc-Glc-Rha, from C-28 position 18 . Whereas the successive losses of sugar moieties, such as 162 Da (-Glc), 146 Da (-Rha) or 132 Da (-Ara) correspond to the sequence and composition of sugar fragments linked to C-3 16 . According to the literature data, the majority of saponins with a molecular weight higher than 1000 Da found in Pulsatilla taxa were determined to be bidesmosidic saponins substituted at both C-3 and C-28 positions with oligosaccharides 19 .
P. patens contained two sapogenin skeletons, determined as lupane (Lup-type) and oleane-type (Olean-type) (Fig. 4), which produced characteristic fragment ions at m/z = 471 19 . These fragment ions may be related to both 23-hydroxybetulinic acid (anemosapogenin) and hederagenin structure what may explain the existence of isomers in the analyzed samples. According to Jin et al. 18 these two types of aglycones can be distinguished by their different retention times resulting from smaller Clog P-value of Lup-type, and consequent earlier elution of Olean-type saponins on the reversed-phase column. Additionally, several peaks shared a similar fragmentation pathway but were assigned to other than hederagenin, olenane-type oligoglycosides, which primarily yielded an abundant ion at m/z 487 suggesting the presence of bayogenin structure, reported previously by 20 in another species P. patnes var. multifida.
The detailed spectrometric data revealed that peaks 22, 25 and 28 shared the same basic skeleton of bayogenin identified based on the characteristic fragment ion at m/z 487, which further generated a product ion at m/z 471 as a result of the neutral elimination of H 2 O 19 . The difference between analyzed compounds originated from residues at an α-sugar chain.    www.nature.com/scientificreports/ and Ara, respectively. The identical ions found in both analyzed compounds confirmed the existence of the same sugar chain at C-3. Saponin 27 and 29 were expected to be isomers with different aglycone structure belonging to lupane or hederegenin. The saponin 27 eluted earlier at 29.407 min was characterized as Lup-type and tentatively identified as Pulsatilloside D 18,21 , whereas saponin 29 eluted at 30.282 min as Leonloside D (= leiyemudanoside) 14,18 . The compound 30 characterized with an adduct ion [M + HCOO]at m/z 1119, gave the precursor ion at m/z 1073, which was 162 Da less than that of peak 29, corresponding to the lack of one Glc group in the β-sugar chain of compound 30. Comparing with the fragmentation of compound 29 and appearance of similar product ions at m/z 603 we could deduce that C-28 residues correspond to Glc-Glc-Rha, whereas ions observed at m/z 471, resulted from a loss of one Ara moiety from C-3. Thus saponin 30 eluted at 31.265 min was identified as 3-Oarabinopyranosyl-23-hydroxybetulinic acid 28-O-rhamnopyranosyl(1-4)-glucopyranosyl(1-6)-glucopyranosyl ester. Consequently, its isomer 35 eluted later at 33.889 min was assigned as 3-O-arabinopyranosyl-hederagenin Another pair of isomers 38 and 40, which produced identical precursor ions at m/z 617 have been found. Further fragmentation yielded ion at m/z 471 indicating the neutral elimination of Rha moiety from both saponins. In the MS/MS spectra of compounds 38 and 40 the characteristic ion at m/z 247, formed from the ring cleavage reaction has been observed. Unfortunately, in this case, data derived from negative-ion mode was insufficient to assume the location of Rha fragment neither in C-3 nor C-28 position. Based on the differences in elution behavior of analyzed compounds, saponin 38 was assigned as Lup whereas 40 as Olean-type and they have proposed to be 23-hydroxybetulinic acid O-rhamnopyranoside and hederagenin O-rhamnopyranoside, respectively 18 .
Interestingly, four isomeric compounds 33, 37, 41 and 42 showed the same molecular formula of C 41 H 66 O 13 and afforded an adduct ion [M + HCOO]at m/z 811, which fragmented in slightly different ways. For saponins 41 and 42 the most prominent precursor ion was observed at m/z 765 and further fragmentation of [M-H]formed product ions at m/z 603 and 471, which correspond to one Glc (-162 Da) in either C-3 or C-28 and one Ara (− 132 Da) molecule in C-3 position. While for the second pair of isomers 33 and 37 an abundant product ion at m/z 603, correlating to neutral loss of Glu, was observed in MS/MS spectrum. The differences in fragmentation behavior of analyzed isomers and their fragment ions abundances might originate not only from aglycone structure but also from the variation in C-3 or C-28 composition. The MS data obtained from negative-ion mode was insufficient to clearly determine the localization of glucose moiety. The elution behavior of analyzed compounds and literature data, indicate the slightly more polar saponins 33 and 37 eluted at 33.510 and 35.472 min, respectively to be Lup rather than Olean-type saponins, and tentatively identified as two isomers of O-arabinopyranosyl-glucopyranosyl 23-hydroxybetulinic acid ester, while 41 and 42 isomers were proposed Table 3. Content of saponins and hydroxycynnamic acid type compounds in P. patens extracts. Araarabinose; Glc -glucose; Rha -rhamnose; DW -dry weight.

Hydroxycynnamic acid type compounds
Dicaffeic acid (5)  www.nature.com/scientificreports/ to be hederagenin esters with various glucose location (Fig. 4). According to Jin et al. 18  Compound 32 characterized with precursor ion at m/z 1219 and two notable fragment ions at m/z 749 and 603, showed a decrease of 146 Da compared to that of saponin 30, indicating the presence of one Rha moiety (146 Da) in ẞ-sugar chain. The sequence of sugar moieties linked to C-3 position was determined based on literature data 23 . The characteristic product ion at m/z 749 might indicate on Anemoside B4 structure also found in P. chinensis and P. koreana [21][22][23] . However, in the absence of sufficient data, the compound 32 might be as well tentatively identified as Olean-type saponin, namely Hederacoside C 18 .
Another group of secondary metabolites identified in samples of P. patens were phenolic acids. In the category of hydroxycinnamic acid the most abundant were caffeic and ferulic acids derivatives, which occurrence was in accordance with data reported for P. pratensis (L.) Mill by 24 .
Compound 20 presented the precursor ion [M-H]at m/z 193 and yielded the fragment ions at m/z 178 and 134, attributed to the cleavage of CH 3 group and characteristic for phenolic acids loss of carbon dioxide from the carboxylic acid group 25 . By comparison with the UV-vis spectra, retention time and fragments generated  www.nature.com/scientificreports/ during CID of the reference standard, compound 20 was unambiguously identified as ferulic acid, while peak 19 was its isomer. The remaining ferulic acid derivatives were tentatively assigned as ferulic acid glucoside (isomers 13 and  www.nature.com/scientificreports/ (tartaric acid) as well as m/z 135, corresponding to caffeic acid decarboxylation. Because of lack of product ion at m/z 341 (relevant for caftaric acid glycoside) in both MS/MS spectra and based on the work 29 , the compounds 23 and 26 could be classified as isomers of chicoric acid (= di-caffeoyltartaric acid). Similarly, compound 7 characterized by precursor ion at m/z 311 and a molecular formula of C 13 H 12 O 9 , was tentatively identified as caftaric acid. The tree notable product ions observed in MS/MS spectrum at m/z 179, 149 and 135 were in accordance to data reported by 29,30 for other plant materials.
Among hydroxycinnamic acids, trace amounts of coumaric acid primarily in roots and its derivatives (in leaves) were also found. Compound 6 yielded precursor ion at m/z 163 and a molecular formula of C 9 H 8 O 3 . The most prominent product ions were at m/z 135 (-28 Da) and 119 (− 44 Da). Regarding presented data, peak 6 was presumed to be coumaric acid. Similarly, for compound 31 the fragmentation of [M-H]at m/z 487 produced two notable product ions at m/z 325 and at m/z 163. Based on this data compound 31 was tentatively identified as coumaric acid derivative.
The ESI-MS signals for hydroxybenzoic acid derivatives were also identified in analyzed samples, mainly at m/z 167, 181 and 137, corresponding to valinic (10), hydroxymelilotic (3) and hydroxybenzoic acids isomers (4 and 18), respectively. Compared with literature data reported by 30 and spectrum obtained from Human Metabolome Database the fragmentation ions at m/z 152 and 108 confirmed the valinic acid structure. In terms of compounds 4 and 18 further fragmentation by CID, revealed the characteristic loss of CO 2 from its precursor ion, generating the diagnostic ion at m/z 93. Due to the identical fragmentation pattern, we deduced compounds 4 and 18 to be isomers of hydroxybenzoic acid. Compound 2 characterized by precursor ion at m/z 315 and fragment ions at m/z 153, 109 was tentatively identified as dihydroxybenzoic acid hexoside, by comparison with spectral data reported for other plant samples by 27,31 .
Compound 17 generated product ions at m/z 463 and 301 from precursor ion [M-H]at m/z 608. It's characteristic UV-vis spectra (λ max 257, 357 nm) as well as detected in MS/MS spectrum diagnostic fragment ions at m/z 179 and 151 supported the existence of a quercetin aglycone. So the chemical structure of compound 17 was tentatively identified as quercetin-3-O-hexose-deoxyhexose 32 .
According to 33 the anemonin structure was also reported in the roots of P. patens. The peak 9 was characterized with a molecular formula of C 10 H 8 O 4 and fragment ions at m/z 191, 147 and 101.
Unfortunately, some of the constituents could not be identified 12, 43 and 18 (sesquiterpene) because of the insufficient data.

Discussion
Uncontrolled cell division, escaping the external and internal control of the cell cycle, leads to unlimited proliferation (multiplication) of cancer cells and neoplastic processes (neoplasia, carcinogenesis) 34 . This is often due to mutations in DNA regions encoding genes important for cell cycle control mechanisms. A panel of 13 vectors (transcription factors) induced by the luciferase reporter gene was used in our research. These vectors enable efficient expression of the protein encoded by the luciferase reporter gene in the system under investigation. Their expression was carried out by enhancer elements (inducers) (such as, IL6, TGF-β, PMA, Wnt 3a) that bind to specific transcription factors. The vectors and inducers included in this study and the studies of other authors 17,35 are used to assess the activity of most of the cancer-related signaling pathways. Similarly [(E)-3,5,4′trihydroxystilbene], a parent compound of the analog used in this study, which is present in a wide range of plants, such as Arachis hypogaea L. (Fabaceae) or Vitis vinifera L. (Vitaceae), is used as a positive control 35 . It is the type of natural phenol, and a phytoalexin produced by several plants in response to injury or when the plant is under attack by pathogenic bacteria and fungi. Resveratrol is the most well-known stilbene, which is postulated as a potential modulator of signal transduction pathways for cancer and the carcinogen response 35 .
The modulation of cancer-related signaling pathways and determining the mechanism of action of transcription factors on the inhibition of the neoplastic process is an important topic in recent years. STAT3 (Signal Transducers and Activators of Transcription) is a protein, involved in the transmission and the activation of gene transduces from the STAT protein family. In the Stat3 signaling pathways, cytokine IL-6 used as an inducer in our research leads to its activation (Janus kinase JAK) (Fig. 3). Stat3 is phosphorylated by the receptor 36 and translocated to the nucleus, where it promotes transcription of target genes, including prooncogenetic ones 37 . The inhibition of STAT3 in HeLa cell signaling caused by methanolic extract of P. patens is associated with the inhibition of growth and inhibition of cell proliferation and their apoptosis (Fig. 3).
Smad is a signaling pathway activated by TGF-β and taking part in the regulation of target gene expression (Fig. 3). In our study, in Smad/ TGF-β signaling pathway, the ligand TGF-β was bound and connected with two pairs of type I and type II receptors, which are transmembrane Ser/Thr kinases. In these receptor complexes, the type II receptor phosphorylated and activated the type I receptor kinase, which then phosphorylated and activated Smad. The Smad/TGF-β signal transduction pathway is important in signaling and regulation of cells functions during the whole life of the organism 38 .
The MYC-is an induced nuclear protein antigen, encoded by the MINA gene. MYC is a transcription factor involved in proliferation, growth, and cellular development, and direct regulation of target genes. MYC is controlling the complex networks of microRNAs and apoptosis mediators 39 . In this study, MYC was activated by PMA (protein kinase activator), which is phorbol 12-myristate-13-acetate (Fig. 3). As reported previously by several researchers, MYC can be activated by the direct interaction with the acetyltransferases p300 and CBP 39 . In our modulation of cancer-related signaling pathways, many transcription factors were activated by PMA (protein kinase activator). These are NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), AP-1 (Activator Protein 1), Ets, Hedgehog, and Notch (Fig. 3). NF-κB can also be activated by the enzyme IκB kinase and then translocated into the nucleus. The Notch can be activated by specific Serine/threonine (Ser/ Thr) protein kinase and E2F by-Cyclins, cyclin-dependent kinases CDKs connected with the cyclin-dependent www.nature.com/scientificreports/ kinase inhibitors (p15, p16). This article reports the use of the methanolic extract of the P. patens which inhibits the progression of cancer through the modulation of cancer-related metabolic signaling pathways. Numerous studies support a pro-oncogenic function for Notch signaling too. It was reported that the modulation of the Notch pathway is important in controlling the development of cancer cells and has bidirectional complicated interaction with multiple other pathways that include candidate therapeutic targets 40 . Same for the Ets family (Erythroblast Transformation Specific) which are one of the largest families of transcription factors to be associated with cancer, such as through gene fusion 41 . Their deregulation following changes in expression and transcriptional activity plays a critical role in carcinogenesis (neoplasm, tumor formation), apoptosis (programmed cell death), and neoplastic angiogenesis by the ability to form blood vessels within a neoplastic tumor enables further growth tumor mass. All ETS family members are identified through a highly conserved DNA binding domain, the ETS domain, which is a winged helix-turn-helix structure that binds to DNA sites with a central GGA (A/T) DNA sequence 38 .
The E2F family comprises of basic helix-loop-helix transcription factors that supervise the expression of genes associated with the regulation of the cell cycle 42 . The transcriptional targets of E2F include cyclins, cyclindependent kinases, checkpoints regulators, DNA repair, and replication proteins (Fig. 3). The cancer-related proliferative roles of E2F family members represent a recent evolutionary adaptation 42 . The evolutionarily conserved Hedgehog (Hh) signaling pathway (synonyms: Hedgehog-Patched (Hh-Ptch), Hedgehog-Patched-Smoothened (Hh-Ptch-Smo)) is responsible for signal conveyance from the cell membrane into the nucleus (Fig. 3). The Hh signaling pathway plays a significant role in normal embryonic development and the maintenance of stem cells important for tissue repairs, such as mammary, skin, neural, lung stem cells, and some epithelial cells of internal organs 43 .
Activator protein 1 (AP-1) is a transcription factor responsible for the regulation of gene expression in reaction to various stimulants, including viral and bacterial infections, growth factors, cytokines and stress. AP-1 controls proliferation, differentiation and apoptosis 44 . A variety of extracellular matrix and genotoxic agents were shown to induce AP-1 activity, which supports the involvement of AP-1 in programmed cell death.
The FoxO transcription factors oversee the protection from oxidative stress by regulating antioxidants and controlling the protein quality. If the expression or activation of FoxO becomes dysregulated it may lead to a variety of age-related disorders affecting CNS (central nervous system) bones, muscles, or leading to cancer 45 . Moreover, the FoxO regulates the cell cycle, the proliferation of cells and apoptosis.
Protein Wnt-3a is encoded by the WNT3A gene 46 which belongs to the WNT gene family. The WNT includes a series of genes that encode secreted signaling proteins (Fig. 3) which are involved in adipogenesis, oncogenesis, and the regulation of embryogenesis. WNT3 and WNT3A genes are two human paralogues of mouse protooncogene Wnt3, which induces carcinogenesis through activation of the β-catenin-TCF signaling pathway 46 .
Reporter genes were introduced by transfection into cells grown in cell culture ( Table 5). The inhibition of the progression of human cancer cells by many cancer-related signaling pathways were studied on the species Pulsatilla patens (L.) Mill. The Pulsatilla species is a good candidate as a natural product for use in the treatment of cancer. This is indicated by the research of other authors about P. koreana Nakai 16 or P. chinensis (Bunge) Regel 47 . In the study with P. koreana it was found that SB365, Pulsatilla saponin D isolated from the root of this species, strongly suppressed the growth and proliferation of colon cancer cells and induced their apoptosis. Additionally, SB365 showed antiangiogenic activity and significantly inhibited tumor growth 16 . In anticancer mechanism, SB365 effectively suppressed the AKT/mTOR pathway both in vitro and in vivo. Moreover, deoxypodophyllotoxin (DPT) from the extract of P. koreana was found as a cytotoxic and antiangiogenic component combined with antitumor activity. The DPT from the extract of Pulsatillae Radix which exhibited antiangiogenic activity and good antitumor activity against mice bearing Lewis lung carcinoma (LLC) is an active constituent of SB31 48 . The biologically active compounds isolated from the root extract of P. koreana have been found to show www.nature.com/scientificreports/ antibacterial, antiparasitic and anti-inflammatory 49 activities too. Other species P. patens and P. vulgaris from our previous research showed antibacterial and antifungal activities 50 . P. chinensis has been found to show antitumor activities too. Pulsatilla saponin A from this species significantly inhibited the growth of human hepatocellular carcinoma SMCC-7721 cells and pancreatic BXPC3 and SW1990 cancer cells 47 . Similar inhibitory activities were observed when the compound was tested in mouse xenograft tumor models using human hepatocellular carcinoma Bel-7402 and pancreatic cancer SW1990 cells. Pulsatilla saponin A may exert its antitumor effect by inducing DNA damage and causing G2 arrest and apoptosis in cancer cells 47 . The triterpene glycosides (Pulsatilla saponins A and B) and the triterpene acid isolated from P. chinensis shows high cytotoxic activity against the malignant cells of the lung cancer 21 and are used for antibacterial, antiparasitic, antiprotozoal, antifungal and molluscicidic 51 activities. Our study about the active compounds of methanolic extracts of P. patens is very important too because these are the first studies of Pulsatilla patens species which its antitumor properties in the luciferase reporter test performed.
There are various classes of plant secondary metabolites influencing cancer-related signaling pathways. Triterpenoid saponins, naphthoquinones, chalcones, sesquiterpene lactones, curcuminoids, flavonoids and isothiocyanates are selective for cancer cells because they are much more active within cancer cells than in normal cells (Supplementary Table S2). Transformed cells are less able to handle the oxidative stress induced by these compounds and therefore redox-sensitive targets are more greatly impacted. These classes of compounds are great for targeting cancer-relevant signaling pathways in cancer cells and play a major role in reactive oxygen species (ROS) generation 52 , the depletion of the antioxidant glutathione (GSH), target redox reactions, and redox-sensitive cysteine 53 .
The Authors hereby would like to point out the limitation related to the presented results. The suggested influence of P. patens on apoptosis was based on the studies of cancer-related signaling pathways in HeLa cells, being the main goal of our research, and not on the direct measurements of apoptosis. We acknowledge that the potential proapoptotic activity of P. patens requires additional research focusing on apoptosis with the use of direct measurement of cellular caspases (caspase-3, -7, -8 or -9), DNA fragmentation, and Annexin V based assays using fluorescence microscopy or flow cytometry.
Our phytochemical analysis of the methanolic extracts of the P. patens shows that compounds, which strongly suppressed the growth and proliferation of HeLa cancer cells are mainly saponins. The triterpenoid saponins and phenolic acids were characterized and identified from a methanolic extract of the P. patens roots and leaves. Similar biological properties of saponins of the genus Pulsatilla in the treatment of cancer and other diseases together with P. koreana 54 59 , neuroactive, immunomodulatory, antioxidant, cognition-enhancing and neuroprotective 55 activities. In our research was found that active compounds resulted in inhibition of cell growth/proliferation and induction of apoptosis of HeLa cells-cervical cancer. The saponins isolated from the methanolic extract of P. patens subsp. multifida (G.A. Pritel) Zämelis, the native species from the USA and Asia, have been used for ages in traditional medicine as the agent showing anticancer properties, inhibiting the growth of skin cancer 20 . But no one until now has identified the antitumor properties of P. patens subsp. patens of a native species from Europe using cancer-related signaling pathways in HeLa cells.

Conclusion
Research on the methanolic extracts of P. patens confirms strong inhibition of signaling pathways in HeLa cells, a cervical cancer cell line, which may also be of importance for the chemotherapeutic ability in other types of cancer. A characteristic feature of active compounds from P. patens is their activity at many levels of cell signaling. In this study it was found, that the root extract of P. patens is the most potent in inhibiting the activation of Stat3, Smad, AP-1, NF-κB, MYC, Ets, Wnt, and Hdghog. The pleiotropic nature of this extract is in line with current trends in oncological pharmacology, where it is to replace drugs, in favor of multidirectional therapies. The methanolic extracts of P. patens enhanced apoptotic death of HeLa cells, deregulated cellular proliferation, differentiation, and progression towards the neoplastic phenotype by altering key signaling molecules required for cell cycle progression. Since the proapoptotic activity of P. patens was observed during studies of cancer-related signaling pathways in HeLa cells the future studies should be focused on the detailed analysis of apoptosis using alternative methods, ex. direct measurement of cellular caspases. This is the first study to report the influence of Pulsatilla species on cancer signaling pathways. Effects of the methanolic extracts of P. patens in these signaling pathways identified here should be further evaluated in tumor-bearing mice and in the next step in preclinical studies of cervical cancer patients, treated with active compounds from this species. The results showed that P. patens is a rich source of polyphenolic constituents, mainly saponins, and hydroxycinnamic acids, wherein their triterpenoid saponins showed a different profile in comparison to other species from the Ranunculaceae family. Our research will be helpful to determine the relevance of each cancer-related signaling pathways that may be used to develop novel therapies that combine extracts of P. patens with other agents including STAT, SMAD, AP-1, NF-κB, MYC, Ets, Wnt, and Hdghog blockers, to effectively treat cervical cancer, and other cancers that utilize these pathways. , the temperature was 100 °C and the pressure was 120 bar. Three extracts were prepared from each raw material. Extracts were filtered through paper filters. Methanol was dried using a rotor evaporator. The obtained extracts were subjected to detailed phytochemical analysis.

Preparation of reference compounds solutions.
To provide the quantitative analysis of saponins and hydroxycynnamic acid type compounds two reference substances, pulsatilla saponin D and ferulic acid were used. The pulsatilla saponin D was obtained from TRC-Canada (Toronto, Canada) with 90% purity, while ferulic acid was purchased from Sigma-Aldrich (Madrid, Spain). All standards were precisely weighed and individually dissolved in methanol HPLC-grade (J.T. Baker, Deventer, The Netherlands) to achieve stock solutions of 1 mg/ mL and stored at 4 0 C.

Conditions for Qualitative and Quantitative LC-ESI-QTOF-MS analysis. The LC-ESI-QTOF-MS
analysis was done on Agilent 1200 Infinity HPLC coupled to Agilent 6530BQTOF Accurate-Mass QTOF system equipped with Dual Agilent Jet Stream spray source (ESI) (Agilent Technologies, Santa Clara, CA, USA) and operated with N 2 generator (Parker Hannifin Corporation, Haverhill, MA; generating a nitrogen supply at purities > 99%). For the chromatographic separation Gemini column (3 μm i.d. C18 with TMS end-capping, 110 Å, 100 × 2 mm) supported with a guard column (PhenomenexInc, Torrance, CA, USA) was used. Chromatographic and spectrometric conditions followed our previous report 31 . The MS spectra acquired in the negative mode provided greater molecular ion abundances and better sensitivity compared to the positive-ion mode. Compounds were tentatively identified based on fragmentation patterns and supported by comparison of obtained mass spectra with mass spectra obtained for reference compounds, mass spectra found in the literature and records available in Metlin database (https:// metlin. scrip ps. edu). Quantification was done based on the UV spectra recorded in 210 and 320 nm for saponins and hydroxycynnamic acid type compounds, respectively.
Method validation. Calibration curves, the limit of detection and quantification. The method linearity was checked by evaluation of the dilutions prepared from stock solutions of reference substances. The construction of calibration curves was based on seven for pulsatilla saponin D, and on eight for ferulic acid concentrations, prepared in triplicate. The detected peak areas were correlated with the corresponding concentrations of reference compounds. The limits of detection (LOD) and limits of quantification (LOQ) under the chromatographic conditions were separately calculated as a signal-to-noise ratio (S/N) of 3 and 10, respectively according to The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines.
Precision and repeatability. The relative standard deviation (RSD) was taken as a measure of precision and repeatability. The intra-and interday variance were measured to obtain the precision of the LC-QTOF-MS method using the standard solutions at two different concentration levels. Each concentration was injected in three replicates on the same day and further on three consecutive days for the determination of the intraday and interday precision, respectively. To investigate the repeatability, five solutions at one concentration were extracted and analyzed.
Recovery. To evaluate the accuracy of the method applied, a recovery test was done by spiking the reference solutions to the P. patens sample and measuring the concentrations in triplicate.
Cytotoxicity assays. The assessment of cytotoxicity was made using cell lines obtained from the ATCC (American Type Culture Collection Rockville, MD, USA) in accordance with the methodological procedure used in the National Center for Natural Products Research (University of Mississippi, MS, USA). The cell line panel included five human cancer cell lines-epidermal carcinoma (KB), breast carcinoma (BT-549), ovarian adenocarcinoma (SK-OV-3), skin malignant melanoma (SK-MEL), cervical adenocarcinoma (HeLa) and two noncancerous kidney cell lines -epithelial cells (LLC-PK-1), fibroblast (Vero). Briefly, cells were passaged (25,000 cells/well) into 96-well microplates (tissue culture-treated) and incubated for 24 h. Afterward, different concentrations of the extract were added and incubation continued for 48 h. Finally, after incubation, a modified Neutral Red assay was used to determine the cell viability 64 . The IC 50 values were calculated from dose-response curves. The doxorubicin was used as a reference cytotoxic drug (positive control), whereas DMSO was used as a negative control (vehicle control). www.nature.com/scientificreports/ were transfected with respective plasmids using X-tremeGENE HP transfection reagent (Roche Applied Science, Indianapolis, IN, USA). Luciferase vectors used in this assay are summarized in Table 5. Luciferase reporter constructs were transfected into Hela cells as previously described 35 . After 24 h of transfection, the test agents were added to the transfected cells, followed 30 min later by an inducing agent: IL-6 (50 ng/mL, R&D Systems, Inc., Minneapolis, MN, USA) for Stat3 35 , TGF-beta (5 ng/mL, R&D Systems, Inc., Minneapolis, MN, USA) for Smad 35 , Wnt-3a (500 ng/mL, Peprotech Corporation, Rocky Hill, NJ, USA) for Wnt 35 and PMA (phorbol 12-myristate 13-acetate, 77 ng/mL, Sigma Chemical Company, St. Louis, MO, USA) for AP-1, NF-κB, E2F, MYC, Ets, Notch and Hdghog. No inducer was added for FoxO, miR-21, k-Ras and pTK-control (thymidine kinase promoter). After 4 h or 6 h of induction, the cells were lysed by the addition of the One-Glo luciferase assay system (Promega Corporation, Madison, WI, USA). The light output was detected in a Glomax Multi + detection system with Instinct Software (Promega Corporation, Madison, WI, USA). This luciferase assay determines if the test agent was able to inhibit the activation of cancer-related signaling pathways. In the case of FoxO and mi-R21 enhanced luciferase activity by the test agent was assessed 17,35 .