Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells
- Claudia Urueña†1,
- Claudia Cifuentes†1,
- Diana Castañeda1,
- Amparo Arango1,
- Punit Kaur2, 3,
- Alexzander Asea2, 3 and
- Susana Fiorentino1Email author
© Urueña et al; licensee BioMed Central Ltd. 2008
Received: 29 May 2008
Accepted: 18 November 2008
Published: 18 November 2008
There is ethnopharmacological evidence that Petiveria alliacea can have antitumor activity; however, the mechanism of its cytotoxic activity is not well understood. We assessed multiple in vitro biological activities of an ethyl acetate soluble plant fraction over several tumor cell lines.
Tumor cell lines were evaluated using the following tests: trypan blue exclusion test, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], flow cytometry, cytoskeleton organization analysis, cell cycle, mitochondria membrane depolarization, clonogenicity test, DNA fragmentation test and differential protein expression by HPLC-Chip/MS analysis. F4 fraction characterization was made by HPLC-MS.
Petiveria alliacea fraction characterized by de-replication was found to alter actin cytoskeleton organization, induce G2 cell cycle arrest and cause apoptotic cell death in a mitochondria independent way. In addition, we found down regulation of cytoskeleton, chaperone, signal transduction proteins, and proteins involved in metabolic pathways. Finally up regulation of proteins involved in translation and intracellular degradation was also observed.
The results of this study indicate that Petiveria alliacea exerts multiple biological activities in vitro consistent with cytotoxicity. Further studies in animal models are needed but Petiveria alliacea appears to be a good candidate to be used as an antitumor agent.
Inherent or acquired resistance can occur simultaneously to multiple drugs in the majority of tumor cells [1–4]. Almost 40% of cancer patients with resectable and 80% with unresectable disease have a reduced response to chemotherapy and radiotherapy. Several mechanisms have been associated with this resistance  and in order to overcome it, search for new antitumor agents must target different cell components within the tumor cell. In fact, single antitumoral compounds may be ineffective because of their unique molecular target. Therefore, presence of multiple compounds in well characterized plant extract with synergic activities, may tackle this difficulty since agonist or additive functions may emerge.
Petiveria alliacea L. (Phytolaccaceae) is a perennial shrub indigenous to the Amazon Rainforest, although it can grow in areas as Tropical and Central America, Caribbean and Southeastern United States. In folk medicine, Petiveria alliacea, is used to treat a wide variety of disorders. Root in decoction, powder or leaves infusion are used as antispasmodic, antirheumatic (topical use), anti-inflammatory [6, 7], antinociceptive , hypoglycemiant and abortifacient [9, 10]. Also there are reports describing the plant with sudorific, anti-venereal, diuretic, sedative, antihelminthic, emmenagogue, anesthetic and depurative [6, 9] properties. In some South American countries, alcohol and water infusions have been used in patients with leukemia and breast cancer having good efficacy and reasonable toxicity at higher doses than commonly used by folk medicine [11–13].
Compounds isolated and reported for Petiveria alliacea includes flavonoids as astilbin, myricitrin, engeletin, triterpenes as barbinervic acid, α-friedelinol, steroids as daucosterol, lipids as lignoceric acid, nonadecanoic acid, oleic acid, compounds as allantoin, coumarin, [14–16], and several sulfur-containing amino acids in the roots; as well as S-benzylcysteine sulfoxides, and S-(2-hydroxyethyl) cysteine sulfoxides [17, 18]. It is likely that benzylcysteine sulfoxides serve as precursors to thiosulfinates as S-(2-hydroxyethyl)-phenylmethanethiosulfinate and sulfines as thiobenzaldehyde S-oxide. Isolation and identification of three glutamyl dipeptides from roots of this plant have also been reported . Dibenzyl trisulphide (DTS), a lipophilic compound found in the plant and identified as one of the immunomodulatory compounds , exhibiting anti-proliferative and cytotoxic activity were the cytoskeleton is implicated .
Several reports describe phytochemical characterization of Petiveria alliacea's ethanol and aqueous extracts, [11, 14, 16, 17, 22], and ethnopharmacological evidence describing possible antitumor activity . This learning has not been immersed into common medical practice because lack of reliable experimental data. The present study examines cytotoxic activity in vitro of a partially purified Petiveria alliacea fraction over several tumor cell lines. Results warrant to continue toxicological and pharmacological testing that could lead to a role in tumor treatment.
For decades, pharmacognostic and ethnobotanical studies have focused in the search of single plant drug isolation, assuming that one drug is responsible for all plant biological activity. However, western medicine and even ayurveda, considers the possibility of synergy between different components in phytomedicine. Furthermore, there are clear examples where a single isolated compound is unable to reproduce the plant extract activity .
Current technical development in "omics" technology has permitted development of gene expression signatures for plant specific fractions. The latter technical advance allows validation of traditional plant uses, but unfortunately due to the high costs turns to be a technology quite inaccessible for developing countries. The present study, in addition to partial characterization of the plant fraction, we evaluate "protein expression signature" over melanoma tumor cells .
Petiveria alliacea fraction preparation
Plant material was collected in Viota, Cundinamarca, Colombia, and identified by Antonio Luis Mejia (botanical consultant) as Petiveria alliacea Linne. Plant material was compared with the Herbario Nacional Colombiano sample, registry number 333406 of August 12 de 1991. Dry ground leaves and stems (300 g) from Petiveria alliacea were extracted under reflux (60°C) with 1.5 liter of 96% ethanol for 3 h. The ethanol extract was filtered and evaporated until half its volume. An equal volume of water was added and heated (65°C) for 20 minutes to allow flocculation. The precipitate was eliminated by filtration and the liquid part subjected to liquid-liquid extraction with ethyl acetate (EtOAc) seven times. All the EtOAc fractions were combined and taken to dryness at 40°C under vacuum conditions. The dry extract was submitted to column chromatography on RP-C18 column (30 × 4 cm), and mobile phase methanol: water (MeOH:H2O). For ratio (1:1), 600 ml were eluted, yielding F-1 to F3 fractions. F-4 fraction eluted within the first 150 ml of ratio (7:3), and F-5 to F11 fractions eluted from the last 450 ml of ratio (7:3) and (9:1). F-1 to F11 fractions were assayed at concentrations ranging from 125 to 1.9 μg/ml but only fraction named F4 exhibited high cytotoxicity causing relevant changes in tumor cell lines morphology, reason why the biological testing was carryout on F4 fraction.
Cell lines and growth conditions
Mel-Rel was established as a melanoma cell line from tumors developed in REL transgenic mice (gift from Dr. Armell Prevost, Cohin Hospital, Paris, France). A375 are human melanoma cells, courtesy of the Instituto de Investigaciones de la Universidad del Rosario (Bogotá, Colombia) and K562 a human erythroleukemia cell line from ATCC. Cells were placed in RPMI-1640 supplemented medium (10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.01 M Hepes) and incubated under humidified environment at 37°C and 5% CO2. Adherent cells at 75% of confluence were detached (trypsin/EDTA), washed (PBS) and suspended in complete medium. Human peripheral blood mononuclear cells (PBMC) from healthy volunteers were separated by density gradient centrifugation (Ficoll-Hypaque, Amersham, Biosciences) and the human fibroblasts from gingival tissue of healthy volunteers. PBMC and human fibroblasts were suspended in RPMI-1640 supplemented medium (10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.01 M Hepes) and incubated under humidified environment at 37°C and 5% CO2.
In vitro cytotoxicity (IC50) and normal cell assays
All tumor cell lines were incubated and treated with F4 fraction (125 to 1.9 μg/ml), ethanol (0.2%), as negative control and vincristine (0.1 to 0.0015 μg/ml) as positive control, during 48 h at 37°C. Adherent cells trypsinized, and washed with saline phosphate buffer (PBS). Human PBMC and fibroblasts were seeded (2 × 105 cells/well) on 96-well plates and incubated with or without phytohemagglutinin (PHA, GibcoBRL) for 12 h. Afterwards, PBMC and fibroblasts were treated with F4 fraction (125 to 1.9 μg/ml), ethanol (0.2%) and vincristine, for 60 h and 24 h, respectively. After treatment cells were centrifuged, F4 fraction removed and lastly cells were carefully washed 3 times (PBS) before adding the MTT. Next 12 μl of MTT 12 mM [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Molecular Probes, Eugene, Oregon, USA) in PBS was added to each well and incubated for 4 h at 37°C. Formazan crystals were dissolved with SDS-HCl 0.01 M. MTT results were read at 540 nm in a Multiskan MCC/340 (LabSystems). In addition cell viability was assessed with a trypan blue dye exclusion test. The IC50 (50% inhibition of cell growth) value was calculated using Probit analysis (MINITAB® Release 14.1. Minitab Inc. 2003 Statistical Software).
Cell cycle analysis
Mel Rel, A375 and K562 tumor cells lines, starved for 72 h (to induce arrest in G1 phase), seeded in 12-well plate (4 × 105 cells/well) were treated with concentrations of F4 fraction at 12, 18, 24 and 48 h under humidified environment at 37°C and 5% CO2. After treatment, cells were washed and fixed with ethanol (70%, ice-cold) during 18 h. After fixing, cells were suspended in PBS 1X, 100 U/ml RNase, 50 μg/ml of propidium iodide (Sigma, St. Louis, MO) and incubated at room temperature for 30 min. Cell DNA content was measured by flow cytometry using a FACScalibur, (Becton Dickinson, Fullerton, CA). For cytometric data 50,000 cellular events were collected per sample and analyzed with Cell Quest software (Becton Dickinson). Cell cycle distribution percentages are calculated by Modfit LT software. FACScalibur calibration is performed with the DNA QC Particle Kit (Becton Dickinson). Treatments were performed in triplicate, and results express as mean ± SEM.
Cytoskeleton organization analysis
A375 human cells (5 × 104 cells/ml) plated on glass coverslides (13 mm diameter), precoated with collagen (Sigma, St. Louis, MO) were allowed to adhere for 16 h. Afterwards, treated with F4 fraction for 24 h and incubated under humidified environment, at 37°C and 5% CO2. Treated cells were washed (PBS) and fixed (2% paraformaldehyde in PBS) for 30 min at 4°C. Fixed cells were wash twice with 1% PBS-BSA, incubated with cold acetone for 1 min, washed (1% PBS-BSA) and incubated with phalloidin conjugated to Oregon-green (Molecular Probes, Eugene, Oregon, USA), diluted in 1% PBS-BSA (1/40) for 30 min. Slides were mounted with prolong antifade kit (Molecular Probes, Eugene, Oregon, USA) and analyzed under fluorescence microscope (Olympus, Japan).
DNA fragmentation analysis
A375 human cells were treated and incubated as described on cytoskeleton organization procedures except for last step were cells are stained with 300 nM of DAPI (Sigma, St. Louis, MO) for 5 min. Slides were mounted with prolong anti-fade kit (Molecular Probes, Eugene, Oregon, USA) and cells analyzed under fluorescence microscope (Olympus, Japan).
K562 human cells (2.5 × 105 cells/well) plated (96-well plate) were treated with F4 fraction at 31.2, 15.6 and 7.8 μg/ml, or 200 μg/ml etoposide, or 0.1 μg/ml vincristine or 0.2% ethanol (in PBS) and incubated for 24 h under humidified environment at 37°C and 5% CO2. After treatment cells were re-plated onto 0.5% agar dishes (60-mm, 20,000 cells/dish), incubated for 14 days (37°C and 5% CO2) and stained with violet crystal (0.4% in ethanol). Cell colonies with more than 50 cells were counted. Treatments were performed in triplicate, and results expressed as mean ± SEM.
Evaluation of Mitochondrial Membrane potential (MMP)
Mitochondria membrane potential (MMP) was measured on human K562 cells by flow cytometry, using JC-1, a lipophilic cationic probe (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolcarbocyanine iodide), (Sigma, St. Louis, MO). JC-1 (10 μg/ml in PBS) is added to 3 × 105 cells/ml and incubated for 10 min at 37°C. Data analysis was processed by Cell Quest software (Becton Dickinson). All treatments were performed in triplicate, and results expressed as mean ± SEM.
Characterization and identification of proteins
A375 cells treated with F4 fraction (31.2 μg/ml for 24 h) lysed in lysis buffer, supplemented with phosphatase and proteinase inhibitors. Protein samples were de-salted in 10 K microcon, diluted with 100 ml of ammonium bicarbonate buffer (100 mM). Cysteine residues were reduced with DTT (10 mM) by incubation at 65°C for 45 min. After cooling to room temperature, sulfhydryls were alkylated with iodoacetamide (55 mM) for 30 min at room temperature in a dark environment. The reduced and alkylated sample was diluted (1:1) with water. Trypsin (Promega, Madison, WI) was added at a 1:50 enzyme:substrate ratio, and incubated overnight at 37°C. Tryptic peptides were completely dried in a SpeedVac and reconstituted with 10 ml of 0.1% TFA.
A 1 ml sample of peptides was injected onto an LC/MS system consisting of an 1100 Series liquid chromatograph, HPLC-Chip Cube MS interface, and 1100 Series LC/MSD Trap XCT Ultra ion trap mass spectrometer (all Agilent Technologies). The system is equipped with an HPLC-Chip (Agilent Technologies) that incorporated a 40-nl enrichment column and a 43-mm × 75-mm analytical column packed with Zorbax 300SB-C18 5-mm particles. Peptides were loaded onto the enrichment column with 97% solvent A (water with 0.1% formic acid). They were then eluted with a gradient from 3% B (acetonitrile with 0.1% formic acid) to 45% B in 25 min, followed by a steep gradient to 90% B in 5 min at a flow rate of 0.3 ml/min. The total runtime, including column reconditioning, was 35 min. The column effluent was directly coupled to an LC/MSD Trap XCT Ultra ion trap mass spectrometer (Agilent Technologies) via a HPLC-Chip Cube nanospray source operated at ~1900 volts in ultra-ultra mode. The gain control (ICC) was set to 500000 with a maximum accumulation time of 150 milliseconds. CID was triggered on the six most abundant, not singly charged peptide ions in the m/z range of 450–1500. Precursors were set in an exclusion list for 1 min after two MS/MS spectra.
CID data was searched against the SwissProt all species database, using the Agilent Spectrum Mill Server software (Rev A.03.03.) installed on a HP Intel® Xeon (TM) dual processor server. Peak lists were created with the Spectrum Mill Data Extractor program with the following attributed: scans with the same precursor ± 1.4 m/z were merged within a time frame of ± 15 s. Precursor ions needed to have a minimum signal to noise value of 25. Charges up to a maximum of 7 were assigned to the precursor ion, and the 12C peak was determined by the Data Extractor. The SwissProt database was searched for tryptic peptides with a mass tolerance of ± 2.5 Da for the precursor ions and a tolerance of ± 0.7 Da for the fragment ions. Two missed cleavages were allowed. A Spectrum Mill auto-validation was first performed in the protein details, followed by peptide mode using default values [Minimum scores, minimum scored peak intensity (SPI), forward minus reversed score threshold, and rank 1 minus rank 2 score threshold]. All protein hits found in a distinct database search by Spectrum Mill were non-redundant. Analysis of the increase or decrease in proteins was performed by comparing each sample with the control. Those values above and below 0.250 from the control value were considered up- or down-regulated.
HPLC chromatogram was recorded on a Waters HPLC Alliance 2690 (Waters, Milford, MA) chromatograph with PDA detector (Waters 2690), and RP-C18 column (5 μm, 2.1 × 150 mm, Waters), at 0.3 ml/min with acetonitrile-water (4:6). MALDI-TOF spectra was recorded in a mass spectrometer (Bruker Reflex III), equipped with a 337 nm N2 laser and HCCA matrix.
The mean fluorescent intensity was used to compare flow cytometry data of controls and samples and expressed as the mean ± SEM. The unpaired Student's t-test was used (p < 0.05) to measure differences between treatments and controls. IC50 was estimated using Minitab 14 Statistical Software Probit analysis [(MINITAB® Release 14.1. Minitab Inc. 2003 Statistical Software).
F4 fraction Characterization
Petiveria alliacea F4 fraction induces morphological changes on tumor cell lines without affecting normal human cells
Comparative IC50 values of F4 fraction and vincristine over tumor cell lines and normal human cells.
PBMC no PHA
PBMC with PHA
F4 fraction (IC50 μg/ml)
35.2 ± 1.35*
32 ± 1.41
36.3 ± 1.64.
121 ± 2.6 *
151 ± 8.3 *
440 ± 15 *
Vincristine (IC50 nM)
132 ± 10*
61 ± 4*
124.5 ± 15*
247 ± 22*
197 ± 20*
85.5 ± 24*
F4 fraction induces apoptosis in a mitochondria independent way
Effect of F4 fraction on tumor cell cycle distribution
F4 fraction reduces tumor cells clonogenic survival
Proteomic characterization of F4 fraction activity over tumor cells
Proteins down regulated by F4 Fraction
Down regulated proteins
Dhx9 (DEAH) P-9, RNA Helicasa A (RHA)/DEAH (Asp-Glu-Ala-His)
Eukariotic translation elongation factor 1 gamma
Eukariotic translation elongation factor-2 (EF-2).
Eukaryotic initiation factor 4A (eIF-4A)
Heparin-binding protein HBp15 solo un articulo
Heterogeneous nuclear ribonucleoprotein H1
Heterogeneous nuclear ribonucleoprotein U isoform a (scaffold attachment factor-A)
Interleukin enhancer binding factor 3, 90 Kda
Nucleosome assembly protein 1-like 1
Poly(rC)-binding protein 2 isoform b variant PCBP2
Small nuclear ribonucleoprotein Sm D1
Tryptophanyl-tRNA synthetase (IFP53)
Tu-transcription elongation factor. ET-1 o EF-Tu
DEAD (Asp-Glu-Ala-His) box polypeptide 21
DEAD (Asp-Glu-Ala-His) box polypeptide 9
Ribosomal protein L4
Ribosomal protein L5
Ribosomal protein L7a
Ribosomal protein L8
Ribosomal protein L9
Ribosomal protein L10
Ribosomal protein L10a
Ribosomal protein L11
Ribosomal protein L12
Ribosomal protein L13
Ribosomal protein L13a
Ribosomal protein L14
Ribosomal protein L18
Ribosomal protein L18a
Ribosomal protein L19
Ribosomal protein L23
Ribosomal protein L26
Ribosomal protein L27
Ribosomal protein L27a
Ribosomal protein L28
Ribosomal protein L31
Ribosomal protein L32
Ribosomal protein L36
Ribosomal protein L37a
Ribosomal protein S2
Ribosomal protein S4
Ribosomal protein S5
Ribosomal protein S6
Ribosomal protein S8
Ribosomal protein S11
Ribosomal protein S12
Ribosomal protein S13
Ribosomal protein S15a
Ribosomal protein S18
Ribosomal protein S19
Ribosomal protein S24
Ribosomal protein S25
Ribosomal protein S27
Ribosomal protein P0 variant
CSE1 chromosome segregation 1-like protein
ErbB3 (HER3) binding protein 1
Protein Kinase, DNA-activated, catalytic polypeptide isoform 1
26S proteasome subunit p45
Flap structure-specific endonuclease 1
Fumarate hydratase, isoform CRA_b
Histone cluster 1, H1d
Hydroxysteroid (17-beta) dehydrogenase 10 isoform 1
Mitochondrial acetoacetyl-CoA thiolase
Mitochondrial trifunctional protein, alpha subunit precursor
Prohibitin 2 (Phb2)
Solute carrier family 25 (SLC25A5 protein)
Actin, gamma 1 propeptide
Adenylyl cyclase-associated protein
Chaperonin containing TCP-1 (subunit 6 A and 7)
Dynein light chain 1
F-actin capping protein alpha 1
Lamin A/C, isoform CRA_c
Miller-Dieker lissencephaly protein
Myosin heavy polypeptide 9
Myosin, light polypeptide 6B, alkali, smooth muscle and non-muscle, isoform CRA_c
t-complex polypeptide 1 (TCP-1)
Tubulin alpha 6
RAB5C, member RAS oncogene family isoform b
Guanine nucleotide binding protein (G-protein)
IQ Motif containing GTPase activating protein 1
RAN member RAS oncogene familly
Tumor rejection antigen (gp96) or Heat schock protein 90 Kda beta
Heat shock protein 90 kDa alpha (HSP90)
ACLY variant protein
ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit precursor
Dihydropyrimidinase-like 2 variant
Enolase 1, variant
Fatty acid synthase
Glucose phosphate isomerase
Lactate dehydrogenase A
Phosphoglicerate kinase (PGK)
Prostaglandin E synthase 3 (cytosolic)
Pyruvate Kinase, muscle isoform CRA_c
Melanoma-associated antigen 4 (MAGE 4 antigen)
Calcium binding proteins
Annexine A2 isoform 1
Proteins up regulated by F4 Fraction
Up regulated Proteins
Alanyl t-RNA synthetase variant
CDA02 (Eukaryotic translation initiation factor 2A)
Eukaryotic translation initiation factor 2, subunit 1 alpha, 35 kDa
Eukaryotic translation initiation factor 3 subunit A, KIAA0139
GA17 protein (eukaryotic translation initiation factor 3, subunit M)
GCN1 general control of amino-acid synthesis 1-like 1, KIAA0219
Leucyl-tRNA synthetase, cytoplasmic, KIAA1352
Methionine adenosyltransferase II, alpha
Mitochondrial isoleucine tRNA synthetase
Prt1 homolog, Eukaryotic translation initiation factor 3 subunit B
Synaptotagmin binding RNA interacting protein, SYNCRIP
DNA-binding protein A (Cold shock domain-containing protein A)
Heterogeneous nuclear ribonucleoprotein M isoform a
Small nuclear ribonucleoprotein polypeptide F
Small nuclear ribonucleoprotein Sm D1
Ribosomal protein S15
Ribosomal protein S9
Ribosomal Protein S3A
Proteasa de Cisteina del Retículo (ER60)
Proteasome 26S ATPase subunit 1 variant
Proteasome 26S ATPase subunit 2
Proteasome 26S non-ATPase subunit 11 variant
Proteasome 26S non-ATPase subunit 2 variant
Putative ubiquitin-conjugating enzyme E2 D3-like protein
SUMO1 activating enzyme subunit 1
Tripeptidyl peptidase II
Ubiquitin-Activating enzime E1
Amino acid transporter E16
ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 isoform 1
Coatomer protein complex subunit alpha isoform 1 (Cop I)
Karyopherin beta 1, Importin subunit beta-1
SEC13-like 1 (S. cerevisiae), isoform CRA_b
Signal recognition particle 72 kDa
Solute carrier family 25 (mitochondrial carrier, Aralar), member 12
Actin related protein 2/3 complex subunit 2
ARP3 actin-related protein 3 homolog
Chaperonin containing TCP1-subunit 2 beta
Chaperonin containing TCP1-subunit 3 gamma
Destrin, isoform a
Dynactin 1 isoform 1
Dynamin 1-like, isoform CRA_c
Filamin A, FLJ00343
MYO1C variant protein (myosin-I beta)
T-complex protein 1 subunit epsilon, KIAA0098
Alpha isoform of regulatory subunit A, protein phosphatase 2
Minichromosome maintenance complex component 6
Poly (ADP-ribose) polymerase family, member 1
Regulator of chromosome condensation 1, isoform CRA_c
Septin 9, KIAA0991
GTP-binding protein PTD004 isoform 1
Phosphofructokinase, platelet, isoform CRA_a
Protein kinase C inhibitor protein 1, YWHAZ
Oxygen regulated protein precursor
TNF receptor-associated protein 1 variant
5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase
Acyl-CoA synthetase long-chain family 3
Aldehyde dehydrogenase 18 family, member A1
Alkylglycerone phosphate synthase, isoform CRA_b
Carbamoylphosphate synthetase 2/aspartate transcarbamylase/dihydroorotase
Enoyl Coenzyme A hydratase
Glucosamine–fructose-6-phosphate aminotransferase (GFAT 1)
HMT1 hnRNP methyltransferase-like 2 isoform 1
Human rab GDI
Hydroxyacyl-Coenzyme A dehydrogenase
Inosine monophosphate dehydrogenase 2, hCG2002013
Ornithine aminotransferase precursor
Phosphoribosyl pyrophosphate synthetase 2, PRPS2
Efficiency in synthesis of cytoskeleton proteins is required for tumor colony formation, partly explaining why treatment with F4 cells cannot form colonies in soft agar. In addition, these results explain why morphology of treated cells is also abhorrent under light microscope. Also treatment of tumor cell lines with F4 fraction affected proteins associated with metabolism (e.g., peroxiredoxin 6, glucose phosphate isomerase, ACLY variant protein, phosphoglycerate dehydrogenase, pyruvate kinase, muscle isoform CRA, enolase 1, variant Fatty acid synthase, lactate dehydrogenase A, phosphoglicerate kinase (PGK), ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit precursor, glyceraldehyde-3-phosphate dehydrogenase, glucosidase II, prostaglandin E synthase 3 (cytosolic), dihydropyrimidinase-like 2 variant); some were drastically down-regulated, while others were greatly up-regulated. Chaperone proteins (e.g., Hsp70, Hsp60, tumor rejection antigen (gp96), Hsp90, Hsp90alpha) were also down-regulated after F4 fraction treatment; these proteins are critical for cell survival and protection from stressful stimuli.
Significant attained information from ethnopharmacological reports for our study is the Petiveria alliacea's antitumor and immunomodulatory reported activities. To date at a molecular level, there is a lack of scientific evidence to explain such activities. For example, a methanolic extract was unable to induce cytotoxicity on Hep G2 cells. Nonetheless, no specific reason was given for the lack activity in this case . Several compounds isolated from Petiveria alliacea, such as astilbin and dibenzyl trisulphide have been demonstrated to induce apoptosis or influence cell cycle or affect actin dynamics [20, 26]. The present study demonstrates that Petiveria alliacea's F4 fraction contains substances capable of inducing G2 arrest in a dose and time dependent manner (Fig. 5). The ability of F4 fraction to change cell morphology and induce G2 arrest was further investigated. Previous reports demonstrate that dibenzyl trisulphide (DTS), one of the sulfur compounds found in Petiveria alliacea, might be responsible for this dual activity . DTS has been previously reported to exhibit potent immunomodulatory function, capable of increasing murine thymic weight along with up-regulation of parameters associated with the reticuloendothelial system, a system essential for molecules involved in immunomodulatory functions . Mice exposed to lethal dose of E. coli were protected from death probably because an increase in phagocytic activity [27, 28]. DTS has also been reported having anti-fungal activity in vitro , as well as insecticidal, acaricidal and insect repellent activities in vivo .
DTS causes reversible microtubule disassembly, which may be due to attenuation of the tyrosyl residues dephosphorylation of the MAP kinases (erk1/erk2) . Along with the fact that MAP kinases are involved in development and apoptotic responses, this event suggests a molecular linkage between these two observations. Mixed-lineage kinase 3 (MLK-3, a kinase of the family controlling MAP kinases activity) inhibition, can cause mitotic arrest by a mechanism involving disruption of microtubule formation and spindle pole assembly . The latter data indicates that Petiveria alliacea F4 fraction might inhibit MLK3.
Presence of apoptotic cells after treatment with F4 fraction clearly suggests that cell cycle arrest induces cell death (Fig. 5A). The F4 fraction from Petiveria alliacea did not cause mitochondrial membrane depolarization, suggesting that cell death is caused by mitochondrial independent mechanisms (Fig. 4A and 4B). Differentiation of cell death mechanisms, such as necrosis or apoptosis, become necessary since an inflammatory response after tissue injury might be different. The induction of an immune response in situ could be the consequence of equilibrium between apoptosis and subsequent necrotic death.
The types of compounds tentatively found in Petiveria alliacea's F4 fraction are sulfur compounds, flavonoids, flavonoid glycosides, coumarin, a monomethylated cyclo hexitol and a fatty acid. The sulfur compounds reported for Petiveria alliacea and probably present in F4 fraction are: thiobenzaldehyde S-oxide, dibenzyl sulfide, S-(2-hydroxiethyl)-phenylmetanethiosulfinate, glutamyl-S-benzyl cysteine and dibenzyltrisulfide (Fig. 1B y 1C). It is likely that these compounds are produced by petiverins (benzyl sulfoxides) degradation during the plant extraction process , and are associated with antitumor activity. Dibenzyl trisulfide, an immunomodulatory compound isolated from Petiveria [20, 21], is likely to be present in our fraction. Therefore, could be one of the compounds responsible for the biological activity present in F4 fraction. Pinitol, a monomethylated cyclohexitol reported in Petiveria alliacea and possibly present in our fraction, has been reported to exhibit anti-inflammatory properties , possibly acting on dendritic cells . Myricitrin, a flavonoid glycoside probably present in F4 fraction, has been reported to have analgesic, anti-inflammatory and antinociceptive properties . Coumarin, another compound possibly found in the F4 fraction is reported to exhibit anti-tumor activity in prostate cancer models , and anti-inflammatory activities . Other compounds possibly present in F4 fraction, includes senfol (1,2 diisothiocyanato ethane), 3,5 diphenyltritiolan, 4 ethyl petiveral, 5-O-methyl leridol and lignoceric acid have no literature reports related to anti-tumoral activity.
Down-regulation of cytoskeleton proteins detected by mass spectrometric analysis is consistent with the cytoskeleton disruption observed by fluorescent microscopy. Moreover, changes in the concentration of proteins involved in translation and transduction processes, as well as those involved in cellular metabolism, could explain the decrease of tumor cells clonogenic ability, as well as the anti-tumor activity of Petiveria alliacea. Currently, we are evaluating the coding genes for these proteins in order to determine if the changes are at the transcriptional level or whether the proteomic results are a consequence of differential management of the existing proteins in the tumor cells. The mechanism by which tumor cells undergo death should be determined. Our results indicate that there is DNA fragmentation; however, it is possible that oxidative stress, metabolic changes, necrosis or senescence are also ways by which tumor cells may undergo death. In fact, necrotic death can provide the necessary danger signals to induce dendritic cells activation, giving anti-tumoral protective immune response ; although other mechanisms can be implied in this antigen transfer [38, 39]. Induction of an effective immune response is unknown, but possibly Petiveria alliacea F4 fraction, can act as Sho-Saiko-to, or Juzen-taiho-to [40, 41], inducing reduction of primary tumors, metastasis, and generating a specific CD8+ CTL responses. Mechanisms implied in the process are unknown. However, it is critical to understand and elucidate the molecular mechanisms before the plant fraction can be used in the design of effective cancer drug therapeutics.
In conclusion, our study demonstrates that Petiveria alliacea's F4 fraction, exhibits multiple anti-tumoral activities against human (K562, A375) and mouse (Mel Rel) tumor cells. F4 fraction exerts G2 cell cycle arrest, induces actin cytoskeleton reorganization, affects cell morphology, causes DNA fragmentation and decreases clonogenicity. Furthermore, our findings indicate that F4 fraction may use multiple molecular targets to exert its antitumor activity.
seventy kilo-Dalton heat shock protein
mixed-lineage kinase 3
peripheral blood mononuclear cells
phosphate buffer saline.
Grant support: The Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnología "Francisco Jose de Caldas" (COLCIENCIAS) Bogotá, Colombia. Grant number 1203-05-14660 (S. Fiorentino). US National Institutes of Health grant RO1CA91889, institutional support from Scott & White Memorial Hospital and Clinic, the Texas A&M Health Science Center College of Medicine, the Central Texas Veterans Health Administration and an Endowment from the Cain Foundation (A. Asea), and we thank Lei Shi, Vadiraja B and Preethi Rao for expert technical assistance.
- El-Deiry WS: Meeting report: The international conference on tumor progression and therapeutic resistance. Cancer Res. 2005, 65: 4475-84. 10.1158/0008-5472.CAN-05-0620.View ArticlePubMedGoogle Scholar
- Johnstone RW, Ruefli AA, Lowe SW: Apoptosis: a link between cancer genetics and chemotherapy. Cell. 2002, 108: 153-64. 10.1016/S0092-8674(02)00625-6.View ArticlePubMedGoogle Scholar
- Bellamy WT: P-glycoproteins and multidrug resistance. Annu Rev Pharmacol Toxicol. 1996, 36: 161-83. 10.1146/annurev.pa.36.040196.001113.View ArticlePubMedGoogle Scholar
- Gottesman MM, Pastan I: Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem. 1993, 62: 385-427. 10.1146/annurev.bi.62.070193.002125.View ArticlePubMedGoogle Scholar
- Gouaze V, Yu JY, Bleicher RJ, Han TY, Liu YY, Wang H, Gottesman MM, Bitterman A, Giuliano AE, Cabot MC: Overexpression of glucosylceramide synthase and P-glycoprotein in cancer cells selected for resistance to natural product chemotherapy. Mol Cancer Ther. 2004, 3: 633-9.PubMedGoogle Scholar
- Lopes-Martins RA, Pegoraro DH, Woisky R, Penna SC, Sertie JA: The anti-inflammatory and analgesic effects of a crude extract of Petiveria alliacea L. (Phytolaccaceae). Phytomedicine. 2002, 9: 245-8. 10.1078/0944-7113-00118.View ArticlePubMedGoogle Scholar
- Morales C, Gomez-Serranillos MP, Iglesias I, Villar AM, Cáceres A: Preliminary screening of five ethnomedicinal plants of Guatemala. Farmaco. 2001, 56: 523-526. 10.1016/S0014-827X(01)01107-7.View ArticlePubMedGoogle Scholar
- di Stasi LC, Costa M, Mendacolli SL, Kirizawa M, Gomes C, Trolin G: Screening in mice of some medicinal plants used for analgesic purposes in the state of Sao Paulo. J Ethnopharmacol. 1988, 24: 205-11. 10.1016/0378-8741(88)90153-5.View ArticlePubMedGoogle Scholar
- de Lima TC, Morato GS, Takahashi RN: Evaluation of antinociceptive effect of Petiveria alliacea (Guine) in animals. Mem Inst Oswaldo Cruz. 1991, 86 (Suppl 2): 153-8.View ArticlePubMedGoogle Scholar
- De Sousa PJ, JR DA, Afonso AM: Guiné: erva medicinal ou tóxica. Ciênc Cult. 1987, 39: 645-646.Google Scholar
- Bernal HYC, Enrique Jaime: Especies Vegetales promisorias de los países del convenio Andrés Bello, Bogotá: Secretaría ejecutiva del Convenio Andrés Bello. SECAB edn. 1998Google Scholar
- Gupta M: Petiveria alliacea in 270 plantas medicinales iberoamericanas, Presencia ed edn. 1995Google Scholar
- Garcia B: Flora medicinal de colombia, Imprenta nacional ed. Bogotá edn. Bogotá. 1974Google Scholar
- De Sousa JR, Demuner AJ, Pinheiro JA, Breitmaier E, Cassels BK: Dibenzyl trisulphide and trans-N-methyl-4-methoxyproline from Petiveria alliacea. Phytochemistry. 1990, 29: 3653-3655. 10.1016/0031-9422(90)85294-P.View ArticleGoogle Scholar
- Delle-Monache F, Menichini F, Cuca LE: Petiveria alliacea: II. Further Flavonoids and Triterpenes. Gazzeta Chimica Italiana. 1996, 126: 275-278.Google Scholar
- Delle-Monache F, Cuca LE: 6-C-formyl and 6-C hidroxymethyl flavonones from Petiveria alliacea. Phytochemistry. 1992, 31: 2481-2482. 10.1016/0031-9422(92)83304-H.View ArticleGoogle Scholar
- Kubec R, Musah RA: Cysteine sulfoxide derivatives in Petiveria alliacea. Phytochemistry. 2001, 58: 981-5. 10.1016/S0031-9422(01)00304-1.View ArticlePubMedGoogle Scholar
- Kubec R, Kim S, Musah RA: S-Substituted cysteine derivatives and thiosulfinate formation in Petiveria alliacea-part II. Phytochemistry. 2002, 61: 675-80. 10.1016/S0031-9422(02)00328-X.View ArticlePubMedGoogle Scholar
- Kubec R, Musah RA: gamma-Glutamyl dipeptides in Petiveria alliacea. Phytochemistry. 2005, 66: 2494-7. 10.1016/j.phytochem.2005.06.029.View ArticlePubMedGoogle Scholar
- Rosner H, Williams LA, Jung A, Kraus W: Disassembly of microtubules and inhibition of neurite outgrowth, neuroblastoma cell proliferation, and MAP kinase tyrosine dephosphorylation by dibenzyl trisulphide. Biochim Biophys Acta. 2001, 1540: 166-77. 10.1016/S0167-4889(01)00129-X.View ArticlePubMedGoogle Scholar
- Williams LA, Rosner H, Levy HG, Barton EN: A critical review of the therapeutic potential of dibenzyl trisulphide isolated from Petiveria alliacea L (guinea hen weed, anamu). West Indian Med J. 2007, 56: 17-21.PubMedGoogle Scholar
- Benevides PJ, Young MC, Giesbrecht AM, Roque NF, Bolzani VS: Antifungal polysulphides from Petiveria alliacea L. Phytochemistry. 2001, 57: 743-7. 10.1016/S0031-9422(01)00079-6.View ArticlePubMedGoogle Scholar
- Williamson EM: Synergy and other interactions in phytomedicines. Phytomedicine. 2001, 8: 401-9. 10.1078/0944-7113-00060.View ArticlePubMedGoogle Scholar
- Ulrich-Merzenich G, Zeitler H, Jobst D, Panek D, Vetter H, Wagner H: Application of the "-Omic-" technologies in phytomedicine. Phytomedicine. 2007, 14: 70-82. 10.1016/j.phymed.2006.11.011.View ArticlePubMedGoogle Scholar
- Ruffa MJ, Ferraro G, Wagner ML, Calcagno ML, Campos RH, Cavallaro L: Cytotoxic effect of Argentine medicinal plant extracts on human hepatocellular carcinoma cell line. J Ethnopharmacol. 2002, 79: 335-9. 10.1016/S0378-8741(01)00400-7.View ArticlePubMedGoogle Scholar
- Yan R, Xu Q: Astilbin selectively facilitates the apoptosis of interleukin-2-dependent phytohemagglutinin-activated Jurkat cells. Pharmacol Res. 2001, 44: 135-9. 10.1006/phrs.2001.0838.View ArticlePubMedGoogle Scholar
- Delaveau P, Lallouette P, Tessier AM: [Stimulation of the phagocytic activity of R.E.S. by plant extracts (author's transl)]. Planta Med. 1980, 40: 49-54. 10.1055/s-2008-1074941.View ArticlePubMedGoogle Scholar
- Wagner HPA: Immunostimulatory drugs of fungi and higher plants. London. 1985Google Scholar
- Johnson L, Williams LAD, Roberts E: An Insecticidal and Acaricidal Polysulfide metabolite from the Roots of Petiveria alliacea. Pesticide Science. 1997, 50: 228-232. 10.1002/(SICI)1096-9063(199707)50:3<228::AID-PS575>3.0.CO;2-J.View ArticleGoogle Scholar
- Cha H, Dangi S, Machamer CE, Shapiro P: Inhibition of mixed-lineage kinase (MLK) activity during G2-phase disrupts microtubule formation and mitotic progression in HeLa cells. Cell Signal. 2006, 18: 93-104. 10.1016/j.cellsig.2005.03.028.View ArticlePubMedGoogle Scholar
- Kubec R, Kim S, Musah RA: The lachrymatory principle of Petiveria alliacea. Phytochemistry. 2003, 63: 37-40. 10.1016/S0031-9422(02)00759-8.View ArticlePubMedGoogle Scholar
- Singh RK, Pandey BL, Tripathi M, Pandey VB: Anti-inflammatory effect of (+)-pinitol. Fitoterapia. 2001, 72: 168-70. 10.1016/S0367-326X(00)00267-7.View ArticlePubMedGoogle Scholar
- Lee JS, Jung ID, Jeong YI, Lee CM, Shin YK, Lee SY, Suh DS, Yoon MS, Lee KS, Choi YH: D-pinitol inhibits Th1 polarization via the suppression of dendritic cells. Int Immunopharmacol. 2007, 7: 791-804. 10.1016/j.intimp.2007.01.018.View ArticlePubMedGoogle Scholar
- Meotti FC, Fachinetto R, Maffi LC, Missau FC, Pizzolatti MG, Rocha JB, Santos AR: Antinociceptive action of myricitrin: involvement of the K+ and Ca2+ channels. Eur J Pharmacol. 2007, 567: 198-205. 10.1016/j.ejphar.2007.03.039.View ArticlePubMedGoogle Scholar
- Maucher A, Kager M, von Angerer E: Evaluation of the antitumour activity of coumarin in prostate cancer models. J Cancer Res Clin Oncol. 1993, 119: 150-4. 10.1007/BF01229529.View ArticlePubMedGoogle Scholar
- Shimizu M, Shogawa H, Matsuzawa T, Yonezawa S, Hayashi T, Arisawa M, Suzuki S, Yoshizaki M, Morita N, Ferro E: Anti-inflammatory constituents of topically applied crude drugs. IV. Constituents and anti-inflammatory effect of Paraguayan crude drug "alhucema" (Lavandula latifolia Vill.). Chem Pharm Bull (Tokyo). 1990, 38: 2283-4.View ArticleGoogle Scholar
- Wells AD, Malkovsky M: Heat shock proteins, tumor immunogenicity and antigen presentation: an integrated view. Immunol Today. 2000, 21: 129-32. 10.1016/S0167-5699(99)01558-3.View ArticlePubMedGoogle Scholar
- Fiorentino S, Barreto A, Castañeda D, Cifuentes C: Anti-tumor response and heat shock proteins (HSP): a friend or foe relationship. 2007, Netherlands: Springer ednGoogle Scholar
- Asea A: Mechanisms of HSP72 release. J Biosci. 2007, 32: 579-84. 10.1007/s12038-007-0057-5.View ArticlePubMedGoogle Scholar
- Kato M, Liu W, Yi H, Asai N, Hayakawa A, Kozaki K, Takahashi M, Nakashima I: The herbal medicine Sho-saiko-to inhibits growth and metastasis of malignant melanoma primarily developed in ret-transgenic mice. J Invest Dermatol. 1998, 111: 640-4. 10.1046/j.1523-1747.1998.00341.x.View ArticlePubMedGoogle Scholar
- Dai Y, Kato M, Takeda K, Kawamoto Y, Akhand AA, Hossain K, Suzuki H, Nakashima I: T-cell-immunity-based inhibitory effects of orally administered herbal medicine juzen-taiho-to on the growth of primarily developed melanocytic tumors in RET-transgenic mice. J Invest Dermatol. 2001, 117: 694-701. 10.1046/j.0022-202x.2001.01457.x.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/8/60/prepub
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