The aim of the present study was to purify the crude pectin extract obtained from gabiroba pulp, elucidate its chemical structure, and evaluate the antitumor potential of crude and purified pectin in a human glioblastoma model.
Trang 1Contents lists available atScienceDirect Carbohydrate Polymers journal homepage:www.elsevier.com/locate/carbpol
Cytotoxic effect of crude and purified pectins from Campomanesia
xanthocarpa Berg on human glioblastoma cells
Sarah da Costa Amarala,1, Shayla Fernanda Barbieria,1, Andrea Caroline Ruthesd,e,
Juliana Müller Barka, Sheila Maria Brochado Winnischofera,b,c, Joana Léa Meira Silveiraa,b,⁎
aPostgraduate Program in Biochemistry Sciences, Sector of Biological Sciences, Federal University of Paraná, Curitiba, PR, 81531-990, Brazil
bDepartment of Biochemistry and Molecular Biology, Federal University of Paraná, CEP 81.531-980, Curitiba-PR, Brazil
cPostgraduate Program in Cellular and Molecular Biology, Federal University of Paraná, CEP 81.531-980, Curitiba-PR, Brazil
dDivision of Glycoscience, Royal Institute of Technology - KTH, Sweden
eDepartment of Entomology and Nematology, University of Florida, Gulf Coast Research and Education Center (GCREC-UF), Wimauma, USA
A R T I C L E I N F O
Keywords:
Gabiroba
Pectin
NMR analysis
Glioblastoma cells
Cytotoxicity
ROS
A B S T R A C T
A new source of pectin with a cytotoxic effect on glioblastoma cells is presented A homogeneous GWP-FP-S fraction (Mw of 29,170 g mol−1) was obtained by fractionating the crude pectin extract (GW) from
Campomanesia xanthocarpa pulp According to the monosaccharide composition, the GWP-FP-S was composed of
galacturonic acid (58.8%), arabinose (28.5%), galactose (11.3%) and rhamnose (1.1%), comprising 57.7% of homogalacturonans (HG) and 42.0% of type I rhamnogalacturonans (RG-I) These structures were characterized
by chromatographic and spectroscopic methods; GW and GWP-FP-S fractions were evaluated by MTT and crystal violet assays for their cytotoxic effects Both fractions induced cytotoxicity (15.55–37.65%) with concomitant increase in the cellular ROS levels in human glioblastoma cells at 25–400 μg mL−1, after 48 h of treatment,
whereas no cytotoxicity was observed for normal NIH 3T3 cells This is the first report of in vitro bioactivity and
the first investigation of the antitumor potential of gabiroba pectins
1 Introduction
Glioblastoma is one of the most frequent and most lethal malignant
primary brain tumors and is associated with poor prognosis (Bailey
et al., 2015) Current therapy includes surgical resection, followed by
radiotherapy and/or concomitant adjuvant temozolomide (TMZ)
che-motherapy (Arbab et al., 2017) Despite modern advance in therapies,
over 90% of patients experience tumor recurrence, and the average
period of survival for patients diagnosed with glioblastoma is only
about 14 months (Bailey et al., 2015;Tanaka, Louis, Curry, Batchelor, &
Dietrich, 2013) Therefore, the search for additional therapeutic
stra-tegies remains a high priority
Recently, polysaccharides (such as pectins) have been exploited for
their anticancer potential, due to their broad spectrum of therapeutic
properties and their low toxicity to healthy cells (Munarin et al., 2015;
Noreen et al., 2017) Although studies have shown that pectins are
effective against a range of tumor models, including prostate cancer
(Prado et al., 2017), breast cancer (Cobs-Rosas, Concha-Olmos,
Weinstein-Oppenheimer, & Zúñiga-Hansena, 2015) and melanoma
(Vayssade et al., 2010), most studies have focused on a colon cancer model (Zhang, Xu, & Zhang, 2015) To our knowledge, no report has been made on the antitumor potential of pectins against glioblastoma cells
Pectins are anionic polysaccharides composed of (1, 4)-linked-D-galacturonic acid residues and a variety of neutral monosaccharides, such as rhamnose, galactose, and arabinose They can be classified into three main types, according to common features: Homogalacturonans (HG), type I Rhamnogalaturonans (RG-I), and type II Rhamnogalaturonans (RG-II) (Caffal & Mohnen, 2009; Chan, Choo, Young, & Loh, 2017;Yapo, 2011)
These polysaccharides are the main component of the peels and pulps of several fruits, although most biological studies are performed with commercial pectins that have been extracted from citrus peel and apple pomace (Noreen et al., 2017;Zhang et al., 2015) As a result, a large number of native fruits around the world continue to go under-used, despite the abundance of the species Brazil is believed to harbor the earth's richest flora, including several native fruits from the Myr-taceae family (Donado-Pestana et al., 2018)
https://doi.org/10.1016/j.carbpol.2019.115140
Received 16 April 2019; Received in revised form 26 July 2019; Accepted 27 July 2019
⁎Corresponding author at: Department of Biochemistry and Molecular Biology, P.B.19046, Federal University of Paraná, CEP 81.531-980, Curitiba-PR, Brazil
E-mail address:jlms12@ufpr.br(J.L.M Silveira)
1These authors equally contributed to this work
Available online 02 August 2019
0144-8617/ © 2019 Elsevier Ltd All rights reserved
T
Trang 2Biological properties have been reported of polysaccharides
ex-tracted from Myrtaceae family fruits A pectic arabinogalactan from
edible jambo (Syzygium jambos) fruits showed immunomodulatory
properties (Tamiello, Nascimento, Iacomini, & Cordeiro, 2018), while
water-extracted polysaccharides from guava (Psidium guajava and
Psi-dium littorale) fruits exhibited anti-glycated and α-glucosidase
in-hibitory activity (Yan, Lee, Kong, & Zhang, 2013;Zhang et al., 2016)
This work investigated a Brazilian Myrtaceae family species,
Campomanesia xanthocarpa Berg, popularly known as gabiroba (Barroso
& Barroso, 1978) This species is used in folk medicine to treat such
pathologies as inflammatory, renal, and digestive diseases; obesity; and
hypercholesterolemia (Alice et al., 1995) In addition, scientific studies
have confirmed that the leaf and fruit extracts of C xanthocarpa
de-monstrate a broad spectrum of therapeutic effects, including:
anti-oxidant properties (Pereira et al., 2012), antibacterial effects
(Czaikoski, Mesomo, Krüger, Queiroga, & Corazza, 2015),
anti-ulcerogenic effects (Markman, Bacchi, & Kato, 2004), antidiabetic
ef-fects (Vinagre et al., 2010), reduction of cholesterol levels (Viecili et al.,
2014) and obesity (Biavatti et al., 2004) However, most previous
stu-dies focused only on low molar mass compounds, and the
aforemen-tioned health benefits were attributed to the presence of vitamins
(mainly ascorbic acid), flavonoids, volatile oils, carotenoids, and
phe-nolic compounds
In our previous works, polysaccharides extracted from gabiroba
pulp were analyzed (Barbieri et al., 2017), and the presence of pectins
was demonstrated (Barbieri et al., 2019) However, the bioactivities of
pectins extracted from gabiroba fruits, as well as their antitumor
po-tential, have remained unexplored
Thus, the aim of the present study was to purify the crude pectin
extract obtained from gabiroba pulp, elucidate its chemical structure,
and evaluate the antitumor potential of crude and purified pectin in a
human glioblastoma model
2 Materials and methods
2.1 Isolation of pectic polysaccharides
Gabiroba fruits were collected and prepared according toBarbieri
et al (2017) Briefly, crude pectic fraction from gabiroba pulp was
obtained by hot water extraction (GW) as reported in our previous work
(Barbieri et al., 2019) In order to obtain a homogeneous fraction, GW
was submitted to the fractionation process (Fig 1) During this process,
the resulted freezing-thawing precipitated fraction (GWP) was treated
with Fehling solution (Jones & Stoodley, 1965; Ruthes, Smiderle, &
Iacomini, 2015) obtaining the precipitated fraction (GWP-FP), which
was deionized with cation exchange resin and dialyzed with Cellulose 6–8 kDa cut off membrane (Spectrum Labs™), where the precipitation of some of the material could be observed inside the membrane Thus, the sample was removed from the membrane and centrifuged (3000 rpm,
20 min, 4 °C), resulting in an insoluble fraction (GWP-FP-P) and a so-luble fraction (GWP-FP-S) The soso-luble fraction GWP-FP-S was freeze-dried, structurally characterized, and analyzed for the biological ac-tivity tests (Fig 1)
2.2 Monosaccharide composition
Uronic acid content of the GWP-FP-S fraction was quantified by the
colorimetric m-hydroxybiphenyl method, using galacturonic acid as the
standard (Blumenkrantz & Asboe-Hansen, 1973) The identity of the uronic acid was determined by anion exchange chromatography, with pulse amperometric detection (HPAEC-PAD) Samples were hydrolyzed with 2 mol L−1TFA for 8 h at 100 °C, dried, and washed with methanol (x 3) to remove the acid Hydrolyzed samples (1 mg mL−1) were in-jected into a Thermo Scientific Dionex ICS-5000 chromatograph (Thermo Fisher Scientific, USA) with CarboPac PA20 column (3 × 150 mm), using a gradient of 0.5 mol L−1NaOH and 1 mol L−1
NaOAc as eluent (Nagel, Sirisakulwat, Carle, & Neidhart, 2014) in an N2
atmosphere in a flow of 0.2 ml min−1at 24 °C The analysis was carried out in triplicate, and the data were collected and analyzed using the Chromeleon TM 7.2 Chromatography Data System software
The neutral monosaccharides were evaluated through total acid hydrolysis, with 2 mol L−1TFA for 8 h at 100 °C The hydrolysates were converted to alditol acetates through treatment with NaBH4(Wolfrom
& Thompson, 1963a), followed by acetylation with acetic anhydride (Ac2O)-pyridine (1:1, v/v, 1 mL) at 100 °C for 30 min (Wolfrom & Thompson, 1963b) The alditol acetates were extracted with CHCl3and were analyzed in a Thermo Scientific Trace GC Ultra gas chromato-graph, with a mixture of He, N2,and compressed air as the carrier gas at
1 ml min−1 The chromatograph also used a DB-225-MS column (0.32 mm internal diameter x 30 m x film thickness 0.25 μm) pro-grammed from 100 °C to 230 °C at a heating rate of 60 °C min−1 The alditol acetates were identified by their profile, and retention times were compared with standards
2.3 High performance size exclusion chromatography coupled to multidetectors (HPSEC-MALLS-RI)
The homogeneity and average molar mass (Mw) of soluble poly-saccharides were evaluated by high performance size exclusion chro-matography (HPSEC), coupled with multi-angle laser light scattering (MALLS) (DSP-F, Wyatt Technology, Santa Barbara, CA, USA) and re-fractive index (RI) detectors (Waters 2410, Milford, MA, USA) (HPSEC-MALLS-RI) The log plot Mwversus elution time was calculated from the Rayleigh-Debye-Gans equation (Wyatt, 1993;Zimm, 1948)
The chromatography was carried out on a Waters system containing four gel permeation columns packed with Ultrahydrogel® 2000, 500,
250, and 120, connected in series, with exclusion limits of 7 × 106,
4 × 105, 8 × 104, and 5 × 103g mol−1, respectively The flow rate used was 0.6 ml min−1, with 0.1 mol L−1sodium nitrite as the mobile phase and 0.2 g L−1sodium azide as a preservative, at a temperature of
25 °C The data was collected and processed by Wyatt Technology ASTRA software, version 4.70.07
2.4 Protein quantification
Protein content of the GWP-FP-S fraction was determined using the Bradford method (Bradford, 1976) A calibration curve of bovine serum albumin was built as a standard, and the results were expressed in g protein/100 g of sample
Fig 1 Scheme of fractionation of water-extracted polysaccharides from
ga-biroba pulp (Campomanesia xanthocarpa) *GW and GWP-TEP was previously
characterized (Barbieri et al., 2019)
Trang 32.5 Nuclear magnetic resonance (NMR) spectroscopy
Mono-dimensional (13C- and 1H-) and bi-dimensional (1H/13C
HSQC) NMR spectra were acquired at 70 °C on a Bruker AVANCE III
400 NMR spectrometer and equipped with a 5-mm multinuclear inverse
detection probe with z-gradient, operating at 9.5 T and observing1H at
400.13 MHz and13C at 100.61 MHz The samples were acquired in D2O
with chemical shifts expressed as δ (ppm), using the resonances of
CH3egroups of acetone (1H at δ 2.22;13C at δ 30.20) as internal
re-ferences The data was collected and processed on Topspin 3.2 (Bruker
BioSpin Corporation, Billerica, MA, USA)
2.6 Cell culture and treatment with pectic fractions
U251-MG and T98 G human glioblastoma cell lines and normal
murine fibroblast NIH 3T3 cell line were kindly provided by Dr Mari
Cleide Sogayar, Cell and Molecular Therapy Center (NUCEL/NETCEM),
Faculty of Medicine, University of São Paulo (FMUSP) Cells were
cul-tured in a DMEM high-glucose medium (Sigma-Aldrich), supplemented
with 10% fetal bovine serum (FBS, Gibco) and 50 μg mL−1gentamicin
(Sigma-Aldrich), and maintained at 37 °C in a 5% CO2atmosphere
For the following experiments, GW and GWP-FP-S fractions
ob-tained from gabiroba pulp were separately dissolved in ultrapure water
(5 mg mL− 1) and stored at −20 °C until utilized
2.7 Cytotoxicity assay
First, 4 × 103cells/well were seeded in 96-well culture plates for
24 h of incubation Then, cells were treated either with vehicle control
(Ultrapure water – 18.2 MΩ cm−1, pH 6.9) or with different
con-centrations (10–400 μg mL−1) of GW and GWP-FP-S fractions
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) method involves metabolically active cells reducing
tetra-zolium salt to formazan (Berridge, Herst, & Tan, 2005; Mosmann,
1983) For the MTT assay (Mosmann, 1983), cells were treated either
with vehicle control or with 10, 25, 50, 100, 200, 400 μg mL−1of GW
and GWP-FP-S fractions Then, the culture medium was removed after
48 h of treatment and replaced with fresh medium containing
0.5 mg mL−1MTT (Sigma- Aldrich); this was protected from light for
3 h at 37 °C in a 5% CO2atmosphere The medium was removed, and
formazan production on viable cells was solubilized in dimethyl
sulf-oxide (DMSO) Absorbance was measured by spectrophotometry
(Epoch, BioTek, USA) at 545 nm, and results were expressed as a
per-centage of control (vehicle), assigned as 100% of metabolically active
cells
Crystal violet is an alkaline dye with high affinity to nucleic acids
extensively used to determine the number of cells through DNA staining
of adhered and fixed cells (Kueng, Silber, & Eppenberger, 1989)
For the crystal violet assay (Kueng et al., 1989), cells were treated
either with vehicle control or with 25, 100, 400 μg mL−1of GW and
GWP-FP-S fractions After 48 h of treatment the culture medium was
removed and washed with PBS Cells were then fixed in methanol for
10 min, stained with crystal violet for 3 min, and then washed with PBS
Afterwards, the stain was diluted in sodium citrate and quantified in a
spectrophotometer (Epoch, BioTek, USA) at 545 nm Absorbance was
proportional to cell number Results were calculated by comparing
absorbance of treated-to-untreated cells (with control as the vehicle
used to dissolve the fractions – Ultrapure water), assigning control
absorbance as 100% of adhered cells
Cell images were obtained using an inverted microscope (Axiovert
40, Carl Zeiss)
2.8 Detection of intracellular reactive oxygen species (ROS) levels
The intracellular levels of ROS were measured using DCFH-DA (2′,
7′-Dichlorofluorescin diacetate, Sigma-Aldrich) a non-fluorescent cell
permeable probe, which oxidizes intracellularly to the highly fluor-escent DCF (2′,7′-dichlorofluorescein) (Kalyanaraman et al., 2012) U251-MG and T98 G glioblastoma cells (4 × 103 cells/well) were seeded on black 96-well plates for 24 h of incubation, and treated as previously described for the cytotoxicity assay Subsequently, the cul-ture medium was removed, cells were washed with PBS, and then in-cubated with 5 mM of DCFH-DA at 37 °C for 30 min in the dark Fluorescence was measured on a spectrofluorometer (Infinite M200, Tecan Trading AG, Switzerland) with a 485 nm excitation filter and a
520 nm emission Hydrogen peroxide (H2O2,400 μM) was used as a positive control 30 min before the measurement Values were normal-ized using crystal violet assay standardization and the intracellular fluorescence levels of control cells (vehicle used to dissolve the extract -milliQ water) were considered as 1
2.9 Statistical analysis
The statistical analyses are expressed as mean ± SD of at least three independent experiments in quadruplicate Analyses were per-formed on GraphPadPrism software version 5.01 for Windows (GraphPad Software, San Diego, CA, USA), using a one-way analysis of
variance (ANOVA) followed by Tukey’s A difference of p < 0.05 was
considered statistically significant
3 Results and discussion
3.1 Structural characterization of purified fraction extracted from gabiroba pulp
GW fraction (5.06% yield), recently characterized byBarbieri et al (2019), was submitted to fractionation by freeze-thawing and the Fehling’s solution treatment, giving rise to a GWP-FP fraction (1.63% yield) This fraction was purified to give the fraction of interest, GWP-FP-S (0.64% yield) (Fig 1)
Fig 2A presents the HPSEC-RI elution profiles of the GWP-FP and GWP-FP-S fractions The GWP-FP fraction presented three peaks that eluted at retention times between 42 and 60 min, showing a hetero-geneous profile, such as the GW fraction analyzed by Barbieri et al (2019); meanwhile, the GWP-FP-S fraction presented a homogeneous profile, with a single elution peak at 51 min, demonstrating that the polysaccharide fractionation process was efficient Assuming that achieve the homogeneity is the primordial step for studies of poly-saccharide structure, pharmacology, and its structuactivity re-lationships (Shi, 2016), the GWP-FP-S was suitable for the investigation
of its cytotoxic potential
HPSEC-MALLS-RI chromatogram and molar mass distribution of the GWP-FP-S fraction were performed (Fig 2B) Through the light scat-tering signal (90°), a major peak can be observed between 35 and
45 min (with an invisible signal to the RI detector), corresponding to its very low concentration A minor peak between 45 and 55 min detected
by light scattering (90°) was demonstrated as a major peak by the RI detector, showing a high concentration corresponding to the GWP-FP-S fraction The average molar mass (Mw) for GWP-FP-S was determined to
be 29,170 g mol−1(dn/dc 0.113).
GWP-FP-S was predominantly composed of galacturonic acid (GalA, 58.8%), followed by arabinose (Ara, 28.5%), galactose (Gal, 11.3%), and rhamnose (Rha, 1.1%), suggesting the presence of pectins (Table 1)
In comparison to the crude fraction GW (54.5% Ara; 33.5% GalA; 7.6% Gal) (Barbieri et al., 2019), the purified fraction GWP-FP-S, ex-hibited an increase in GalA and Gal followed by a decrease in Ara content (Table 1) Nevertheless, the content of GalA from the GWP-FP-S fraction was similar to that found by Maxwell et al (2016) for a modified sugar beet pectin (52 5% of GalA), which induced apoptosis
of colon cancer cells
The relative amount of HG and RG-I domains of the pectins was
Trang 4estimated from the monosaccharide composition according toM’sakni
et al (2006), using the equations:
HG (%) = GalA (%) – Rha (%) and,
RG-I (%) = [GalA (%) – HG(%)] + Rha + Ara + Gal
Thus, the GWP-FP-S chain is represented by 42.0% of RG-I and
57.7% of HG These results showed a decrease in the RG-I proportion
and an increase in the HG proportion of GWP-FP-S in relation to the GW
fraction, which presented 65.3% RG-I and 31.9% HG (Barbieri et al.,
2019) Structural differences in the main chain can be attributed to the
process of purification According to the literature, HG-rich chains form
a complex with Cu2+and precipitate after being treated with Fehling’s
solution (Nascimento, Iacomini, & Cordeiro, 2017)
In addition, in order to estimate the extension of neutral side chains
attached to the RG-I (Houben, Jolie, Fraeye, Van Loey, & Hendrickx,
2011), the ratio (Gal + Ara)/Rha was calculated The value obtained
for GWP-FP-S was 37.6 (Table 1) Despite the differences in the
pro-portions of the HG and RG-I domains of GWP-FP-S and GW, it can be
observed that the values obtained to the neutral side chains attached to
the RG-I (37.6 for GWP-FP-S and 38.8 for GW) was similar for both
fractions, demonstrating that the side chain extension was not affected
by the fractionation process adopted
Regarding the protein content, GWP-FP-S showed no significant
amount when evaluated by the Bradford method
The GWP-FP-S fraction was investigated by13C-NMR The13C-NMR
spectrum (Fig 3) showed a typical chemical shift from HG observed at δ
99.7 and 70.3, which correspond to C-1 and C-5, respectively, of
un-esterified α-D-GalAp units The remaining assignments of D-GalAp ring
carbons were seen at δ 78.1 (O-substituted C-4/H-4), δ 68.3 (C-3), and δ
68.0 (C-2) Signals of C-6 from unesterified α-D-GalAp units could be
assigned as δ 171.9 In the literature, the signal of methyl groups linked
to α-D-GalAp units can usually be observed at δ 52.8 ppm (Colodel,
Vriesmann, & Petkowicz, 2018;Nascimento et al., 2017); however, this
signal was not observed for GWP-FP-S A de-esterification may have occurred due to the use of alkaline pH in the Fehling’s treatment De-esterification was also observed byNascimento et al (2017)on sweet
pepper (Capsicum annum) pectin fraction after treatment with Fehling’s
solution
The13C-NMR spectrum also showed signals at δ 107.6, δ 107.2,
106.5, and 106.3 that are attributed to α-L-Araf (C-1) Signals for the → 4)-β-D-Galp-(1→units were observed at δ 104.3 (C-1), δ 72.0 (C-2), δ 73.4 (C-3), δ 77.6 (O-substituted C-4), δ 74.5 (C-5), and δ 60.8 (C-6).
All assignments obtained by13C-NMR were confirmed by the ana-lysis of the 1H/13C heterocorrelated HSQC-NMR spectrum (Table 2, Fig 4) The highest intensity peaks in the spectrum were attributed to
→4)-α-D-GalAp-(1→ units, confirming the presence of HG in the
GWP-FP-S fraction Signals with lower intensity were also observed and were
attributed to unsubstituted →2)-α-L-Rhap-(1→ and substituted →2,4)-α-L-Rhap-(1→ units from the backbone of type I rhamnogalacturonan.
Arabinose and galactose side chains were ascribed to signals of linear
backbone of →5)-α-L-Araf-(1→ units, as well as branched →3,5)-α-L-Araf-(1→ units In addition, the presence of galactans was observed at correlations of →4)-β-D-Galp(1→ units (Colodel et al., 2018; Klosterhoff et al., 2018;Tamiello et al., 2018) With the analysis of
1H/13C heterocorrelated HSQC-NMR, it was possible to propose that the purified GWP-FP-S fraction consists of a pectin formed predominantly
by HG regions and containing branched RG-I inserts with side chains of arabinans, galactans, and possibly arabinogalactans
3.2 Cytotoxic effect of crude and purified pectin fractions extracted from gabiroba pulp
Fig 5shows the cytotoxic effect of GW and GWP-FP-S pectins on glioblastoma cell lines U251-MG and T98 G cells were exposed to dif-ferent concentrations of pectins (25–400 μg mL−1) for 48 h, and cell cytotoxicity was determined by crystal violet assay
Both of these fractions were able to reduce the number of adherent
Fig 2 HPSEC-MALLS-RI elution profile (A) Elution profile by RI of the GWP-FP and GWP-FP-S fractions obtained from the gabiroba pulp (B) GWP-FP-S Fraction.
Molar mass distribution (MwD), light scattering (LS-90°), and refractive index (RI)
Table 1
Monosaccharide composition of crude and purified pectin fractions extracted from gabiroba pulp
Rha
a % of peak area of monosaccharide composition relative to the total peak area, determined by GLC
b Uronic acids, determined using the m-hydroxybiphenyl method (Blumenkrantz & Asboe-Hansen, 1973), and identified by HPAEC-PAD
c HG = GalA – Rha ; RG-I = 2(Rha) + Ara + Gal (M’sakni et al., 2006); tr = trace
dGW fromBarbieri at al (2019)
Trang 5glioblastoma cells (stained by crystal violet assay) (Fig.5) These effects were also evidenced by optical microscopy, which showed that both
GW (Fig 5D) and GWP-FP-S (Fig 5E) treatments promote significant changes in U251-MG cell morphology beyond altering cell number, as compared to a vehicle-treated control group (Fig 5C)
All tested concentrations of both GW and GWP-FP-S fractions were able to reduce the percentage of adherent U251-MG and T98 G cells, reaching a decrease of 18.04–33.74% and 20.05–37.65% of adherent U251-MG cells after GW and GWP-FP-S treatment, respectively, at different concentrations Similar effects were also achieved for T98 G glioblastoma cells, where GW and GWP-FP-S treatments were able to inhibit 15.78–27.32% and 25.48–35.55% of adherent T98 G cells, re-spectively, indicating that the cytotoxic effect of these pectins was not selective, and is independent of cell-line-specific characteristics In view
of this, the next investigations were continued with only one glio-blastoma cell line (U251-MG)
The cytotoxic effects were also confirmed by MTT assay (Fig 6) When U251-MG cells were treated with GW and GWP-FP-S at high concentrations (100–400 μg mL−1), the percentage of metabolically-active U251-MG cells were reduced in an equal manner However, U251-MG cells seemed to respond differently when pectin fractions were used at low concentrations A decrease in metabolic U251-MG cell
Fig 3.13C NMR spectrum of the GWP-FP-S fraction from the pulp of gabiroba fruits, obtained at 70 °C in D2O (chemical shifts are expressed in δ, ppm)
Table 2
1H and13C NMR chemical shifts (δ, ppm) of GWP-FP-S fraction from the pulp of
gabiroba
Glycosil residues Nucleus Chemical shifts, δ (ppm)
→4)-α -D-GalAp-(1→ 13 C 99.7 68.0 68.5 78.1 70.3
→2)-α-L-Rhap-(1→ 13 C 99.1 76.7 – – – 16.5
→2,4)-α-L-Rhap-(1→ 13 C 99.1 – – – – 16.8
→5)-α-L-Araf-(1→ 13 C 107.7 81.4 76.8 82.8 66.8
→3,5)-α-L-Araf-(1→ 13 C 106.6 – – 82.3 66.6
t-α-L-Araf-(1→ 13 C 107.6 79.8 76.7 84.1 61.3
→4)-β-D-Galp-(1→ 13 C 104.3 72.0 73.4 77.6 74.5 60.8
Acetone was used as internal standard (δ2.22/30.2), and the analysis was
carried out at 70 °C
Fig 4.1H/13C HSQC- NMR spectrum of the GWP-FP-S fraction from the pulp of gabiroba fruits, obtained at 70 °C in D2O (chemical shifts are expressed in δ, ppm)
Trang 6activity (19.55%) was observed using 50 μg mL−1of GW (similar to
obtained with high concentrations), while half that amount (25 μg
mL−1) of GWP-FP-S was necessary to achieve the same effect
(inhibi-tion of 20.96% of metabolic human glioblastoma cell activity)
GW and GWP-FP-S treatments at 25 μg mL−1decreased comparable
numbers of adherent cells; nevertheless, U251-MG cells treated with
GW fraction did not alter their cellular metabolic activity, likely in an attempt to maintain survival This differential effect was no longer observed when the treatment reached higher concentrations of either fraction
It is important to note that, from a pharmacological point of view, cytotoxicity at lower concentrations is one of the most valuable po-tentials for clinical application (Cobs-Rosas et al., 2015) In the litera-ture, modified sugar beet pectin reduced the number of viable HT29 colon cancer cells by 20.7% in 48 h at 1000 μg mL−1(Maxwell et al.,
2016) Low-molecular-weight citrus pectin decreased cell viability of AGS (gastric cancer) and SW-480 (colorectal cancer) cells by 24% and 28%, respectively, at a concentration of 5.0 mg mL−1for 24 h (Wang,
Li, Lu, & Ling, 2016) However, until now, only one study has been reported for glioblastoma model, whenFan et al (2012)evaluated the
effect of Panax ginseng (ginseng) pectin on U87 human glioblastoma
cells, and no cytotoxicity was observed at any tested concentrations (10–500 μg mL−1)
Considering that one of the pivotal pathways to activate cell death is
by excessive reactive oxygen species (ROS) levels (Kalyanaraman et al.,
2018), the impact of GW and GWP-FP-S on intracellular ROS levels was measured using DCFH-DA probe As illustrated inFig 7, the ROS levels were significantly increased after treatment with 100–400 μg mL−1of
GW, and 25–400 μg mL−1of GWP-FP-S, in comparison to untreated U251-MG cells, which suggest that cytotoxic effect of pectin fractions could be related to increased intracellular ROS levels
Citrus pectin (CP) and apple pectin (AP) have also been reported to suppress viability in MDA-MB-231, MCF-7 and T47D human breast cancer cells by increasing ROS content, which led to the caspase-de-pendent apoptosis (Salehi, Behboudi, Kavoosi, & Ardestani, 2018) The
Fig 5 Cytotoxic effect of GW and GWP-FP-S pectins Glioblastoma cell lines: (A) U251-MG and (B) T98 G cells were treated with GW or GWP-FP-S at different
concentrations (25–400 μg mL−1) for 48 h The percentage of adherent cells were evaluated by crystal violet assay The values represent the means ± SD of
percentage of cells compared with the control (data represent at least three independent experiments, each in quadruplicate) *p < 0.05; **p < 0.01;
***p < 0.001 Morphology of U251-MG cells: (C) Control group (cells treated with vehicle) (D) Cells treated with GW at 400 μg mL−1 (E) Cells treated with GWP-FP-S at 400 μg mL−1 Magnification 100 ×
Fig 6 Cytotoxic effect of GW and GWP-FP-S pectins U251-MG cells were
treated with GW or GWP-FP-S at different concentrations (10–400 μg mL−1) for
48 h The percentage of metabolically active cells were evaluated by MTT assay
The values represent the means ± SD of percentage of cells compared with the
control (data represent at least three independent experiments, each in
quad-ruplicate) *p < 0.05; **p < 0.01; ***p < 0.001.
Trang 7elevated levels of ROS are reported to increase the vulnerability of
cancer cells to oxidative damage of various macromolecules, including
proteins, lipids, and DNA, which in turn results in cell death (Wang
et al., 2019)
Furthermore, it is well established in the literature (Galadari,
Rahman, Pallichankandy, & Thayyullathil, 2017; Panieri & Santoro,
2016) that cancer cells have an inherent elevated ROS level compared
to normal cells This effect suggests that therapeutic strategies that
in-crease ROS generation may push cancer cells beyond the breaking
point, leading to a toxic level, thereby activating ROS-induced cell
death pathways This may explain why GW and GWP-FP-S were only
cytotoxic on glioblastoma cells (Figs 5 and 6) and showed no
cyto-toxicity for NIH 3T3 cells (Fig 8)
Interesting, only the purified pectin (GWP-FP-S) at 25 μg mL−1was
capable to increase significantly (1.3 fold higher than control) the levels
of ROS, corroborating to cytotoxic effects observed by MTT assay
(Fig 6) In addition, the effect observed for 400 μg mL−1of GW (1.71
fold) is achieved in a 4-fold lower concentration of GWP-FP-S (100 μg
mL−1)
These results indicate that, despite differing chemical structural
characteristics between GW and GWP-FP-S, a similar cytotoxic effect
was observed in high concentrations (the cytotoxic effect was not sta-tistically different for treatments using more than 25 μg mL−1) However, is it possible to suggest that the major component responsible for the biological activity of the crude GW is purified GWP-FP-S structure, since after purification process, lower concentrations of this homogeneous fraction were sufficient to exert the cytotoxic effect The crude GW fraction was revealed to be a heterogeneous fraction, with a wide range of molar mass distribution and a degree of methyl-esterification (DM) of 60% It was composed mostly of RG-I portion (65.3%) and presented high extension of the neutral side chains and high amounts of arabinose (54.5%) and galactose (7.6%) (Barbieri
et al., 2019) Evidence from the literature suggests that the antitumor activity may be related to the RG-I domain, possibly by adopting a conformation that maximizes the availability of neutral sugar side chains for cellular interaction (Maxwell et al., 2016;Prado et al., 2017; Vayssade et al., 2010) In addition, the importance of arabinose’s pre-sence in the structure has recently been recognized due to its partial influence on the pectin’s effect on cancer cell lines Maxwell et al
pro-liferation—reaching 7–15% of inhibition—after selective removal of arabinose in modified sugar beet pectin
In contrast, GWP-FP-S structure, represented by 57.7% HG and lower proportions (42.0%) of branched RG-I (containing 28.5% of Ara and 11.3% of Gal) and de-esterified (due to the employed alkaline pH in the Fehling’s treatment) By the HPSEC-MALLS-RI analysis, it showed as
a homogeneous fraction, with low molar mass (29,170 g mol−1) The molar mass of GWP-FP-S was similar to a modified citrus pectin (MCP, 30,000 g mol−1) (Gao et al., 2012) Modified pectins with lower molar mass have been reported as producing more profound antitumor ac-tivity than naturally large pectins (Naqash, Masoodi, Rather, Wani, & Gani, 2017;Zhang et al., 2015) These authors showed the MCP as a galectin-3 (gal3) inhibitor (a carbohydrate recognition domain with the ability to modulate important functions for cell survival migration and metastasis) that represents an attractive target for cancer therapy (Vladoiu, Labrie, & St-Pierre, 2014)
Another structural characteristic of pectins attributed to the antic-ancer effect by inhibiting gal3 function is the presence of
→4)-β-D-Galp-(1→ units on the branched chain in the RG-I (Gunning, Bongaerts,
& Morris, 2009;Zhang et al., 2015) This may explain the difference of biological effect of GW and GWP-FP-S observed in lower concentrations (25 μg mL−1 by MTT and ROS assay), where only GWP-FP-S (which contains 1.5 times more galactose than GW) is capable to induce cy-totoxicity
In order to evaluate the cytotoxic effect of GW and GWP-FP-S fractions on normal cells, NIH 3T3 murine fibroblast cells were exposed
to the same treatment conditions as GW and GWP-FP-S (10–400 μg
mL−1for 48 h), and cytotoxic effect was measured by MTT assay Both pectin fractions showed no cytotoxicity for NIH 3T3 cells after 48 h of treatment at all tested concentrations (Fig 8) By contrast, traditional anticancer agents are generally toxic to normal cells, producing severe side effects (Huettemann & Sakka, 2005;Wang, Huang, Sun, & Pan,
2015) Indeed, pectins have been described as a biocompatible and non-toxic biopolymer (Chan et al., 2017;Munarin et al., 2015)
In addition, 400 μg mL−1 (or 13.7 μmol L-1) of GWP-FP-S, the highest concentration tested on the human glioblastoma model, in-hibited 26.5% of metabolically active glioblastoma cells; in contrast, temozolomide (TMZ), the standard chemotherapy for glioma patients (Bailey et al., 2015), is known to inhibit 24.7% of U251-MG viable cells (also evaluated by MTT assay, on 48 h of treatment) at 100 μmol L−1
(Shen, Hu, & Zheng, 2014) Interestingly, GWP-FP-S concentration is approximately 7 times lower than the necessary TMZ concentration for
a similar effect
The antitumor activities of polysaccharides can be mediated through three main approaches: direct cytotoxicity, immunoenhance-ment, and synergistic effects combined with conventional antitumor drugs (Yang et al., 2013) In this context, GW and GWP-FP-S pectin
Fig 7 Effect of GW and GWP-FP-S pectins on intracellular ROS levels
U251-MG cells were treated with GW or GWP-FP-S at different concentrations
(25–400 μg mL−1) for 48 h The intracellular ROS levels were evaluated
spec-trofluorometrically using a DCFH-DA probe U251-MG cells treated with
hy-drogen peroxide (H2O2) were used as a positive control (400 μM, 30 min) The
values represent the means ± SD of percentage of cells compared with the
control (data represent at least three independent experiments, each in
quad-ruplicate) *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 8 Cytotoxic effect of GW and GWP-FP-S pectins on normal fibroblast cells.
NIH 3T3 cells were treated with GW or GWP-FP-S at different concentrations
(10–400 μg mL−1) for 48 h, and MTT assay was performed The values
re-present the means ± SD of percentage of metabolically active cells compared
with the control (data represent at least three independent experiments, each in
quadruplicate) *p < 0.05; **p < 0.01; ***p < 0.001.
Trang 8fractions from gabiroba pulp could prove to be effective antitumor
candidates The purification and chemical structural characterization
are important factors in establishing structure-function relationships
According to the literature, the cytotoxic effects of pectins on cancer
cell lines may be related to different factors involving their structure, as
conformation of the molecule, degree of esterification, ratio of HG /
RG-I and side chain of these polysaccharides RG-In this work, by comparing
the data obtained about the chemical structure and response to
cyto-toxicity in glioblastoma cells, it may suggests that the antitumor
ac-tivity of the pectic fractions extracted from gabiroba pulp (GW and
GWP-FP-S) is probably related to the presence of →4)-β-D-Galp-(1→
units on the branched chain in the RG-I (Gunning et al., 2009;Zhang
et al., 2015) The amount of galactose on the GWP-FP-S structure (1.5
times higher than GW fraction) may explain the difference of cytotoxic
effect of in lower concentrations (25 μg mL−1by MTT and ROS assay),
where only GWP-FP-S is capable to induce cytotoxicity
Else more, for the first time, a crude (GW) and purified (GWP-FP-S)
pectins from gabiroba pulp have demonstrated cytotoxicity in
glio-blastoma cell lines In terms of purely cytotoxic purposes, crude pectin
GW presented similar effects, when compared to the purified
GWP-FP-S, with the advantages of higher yield and fewer steps in the extraction
process, which in turn mean lower cost and time of acquisition
Nevertheless, GWP-FP-S requires less concentration to exert this effect
and is more suitable to continue further studies on the potential
anti-tumor effect, since it is a homogeneous purified pectin
4 Conclusion
This paper presents a new source of pectin with a cytotoxic effect on
glioblastoma cells GWP-FP-S pectin with low molar mass
(29,170 g mol−1) was purified from a crude (GW) pectin fraction from
gabiroba pulp and analyzed through monosaccharide composition,
homogeneity, and 1D and 2D nuclear magnetic resonance The results
indicate the presence of HG, RG-I, and long side chains composed by
arabinose and galactose in the RG-I backbone Crude GW and purified
GWP-FP-S pectins selectively exhibited a cytotoxic effect, even in low
concentrations, against human glioblastoma cells (U251-MG and T98 G
cell lines) and a concomitant increase in the cellular ROS levels,
sug-gesting that these pectins could mediate cytotoxicity by altering the
cellular redox status Also, no cytotoxicity was observed in normal
fi-broblast cells (NIH-3T3) The hypothesis that the antitumor effect of
pectins is associated with the Gal and Ara content of side chains on the
RG-I/HG backbone is corroborated by these results
Acknowledgements
The authors gratefully acknowledge the following Brazilian
agen-cies for financial support: the National Council for Scientific and
Technological Development – CNPq, the Coordination for the
Improvement of Higher Education Personnel - CAPES; the Araucaria
Foundation, Nanoglicobiotec and Ministry of Science and Technology/
CNPq, and the Federal University of Parana – Brazil J.L.M.S is a
re-search member of the CNPq Foundation (nº 476950/2013-9; 308296/
2015-0; 309225/2018-3); S.M.B.W is a research member of the CNPq
and Araucaria Foundation (nº 479356/2010-6; 307066/2012-6; 219/
2010-17497); S.C.A is the beneficiary of a post-graduation scholarship
(nº 141692/2018-9) provided by CNPq, and S.F.B is the beneficiary of
a post-doctoral scholarship from Coordination of Superior Level Staff
Improvement - CAPES, nº 88887.335103/2019-00 The authors would
like to thank the NMR Center of UFPR for recording the NMR spectra,
the Brazilian Agricultural Research Corporation/Embrapa Forestry,
Rossana Catie Bueno de Godoy and Maria Cristina Medeiros Mazza for
provide the gabiroba pulp
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