Accepted Manuscript

Title: Galactomannan from Schizolobium amazonicum seed

and its sulfated derivatives impair metabolism in HepG2 cells

Author: Monique Meyenberg Cunha de Padua Silvia Maria

Suter Correia Cadena Carmen Lucia de Oliveira Petkowicz
Glaucia Regina Martinez Guilhermina Rodrigues Noleto

PII: S0141-8130(16)31624-5
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.03.090
Reference: BIOMAC 7256

To appear in: International Journal of Biological Macromolecules

Received date: 14-9-2016

Revised date: 15-3-2017
Accepted date: 16-3-2017

Please cite this article as: M.M. Cunha de Padua, S.M. Suter Correia Cadena,
C.L. de Oliveira Petkowicz, G.R. Martinez, G. Rodrigues Noleto, Galactomannan
from Schizolobium amazonicum seed and its sulfated derivatives impair metabolism
in HepG2 cells, International Journal of Biological Macromolecules (2017),
http://dx.doi.org/10.1016/j.ijbiomac.2017.03.090

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 1  Galactomannan from Schizolobium amazonicum seed and its sulfated derivatives

2  impair metabolism in HepG2 cells

3  Monique Meyenberg Cunha de Paduaa; Silvia Maria Suter Correia Cadenaa; Carmen

4  Lucia de Oliveira Petkowicza; Glaucia Regina Martineza; Guilhermina Rodrigues

t
5  Noletoa*.

ip
a
6  Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná,

cr
7  Curitiba, Paraná, Brasil.

8  *Corresponding author: guinoleto@yahoo.com.br

us
9 

an
10  ABSTRACT

11  This study evaluated the effects of native galactomannan from S. amazonicum seeds and
M
12  its sulfated forms on certain metabolic parameters of HepG2 cells. Aqueous extraction

13  from S. amazonicum seeds furnished galactomannan with 3.2:1 Man:Gal ratio (SAGM)
d

14  and molar mass of 4.34 × 105 g/mol. The SAGM fraction was subjected to sulfation
te

15  using chlorosulfonic acid to obtain SAGMS1 and SAGMS2 with DS of 0.4 and 0.6,

16  respectively. Cytotoxicity of SAGM, SAGMS1, and SAGMS2 was evaluated in human
p

17  hepatocellular carcinoma cells (HepG2). After 72 h, SAGM decreased the viability of
ce

18  HepG2 cells by 50% at 250 µg/mL, while SAGMS1 reduced it by 30% at the same

19  concentration. SAGM, SAGMS1, and SAGMS2 promoted a reduction in oxygen

Ac

20  consumption and an increase in lactate production in non-permeabilized HepG2 cells

21  after 72 h of treatment. These results suggest that SAGM, SAGMS1, and SAGMS2

22  could be recognized by HepG2 cells and might trigger alterations that impair its

23  survival. These effects could be implicated in the modification of the oxidative

24  phosphorylation process in HepG2 cells and activation of the glycolytic pathway.

25  Keywords: galactomannans; sulfation; HepG2 cells.

1 
 
Page 1 of 43
26  1 Introduction

27  Hepatocellular carcinoma (HCC) is extremely aggressive and the third most

28  common cause of death from cancer worldwide [1]. Treatments such as chemotherapy

29  and radiotherapy are rarely effective and cause several side effects. Thus, searches for

t
30  new possibilities of treatments with fewer side effects have been undertaken [2, 3].

ip
31  Polysaccharides have been widely studied aiming at their application in

cr
32  medicine [4]. In recent years, polysaccharides from plants have emerged as an important

33  class of bioactive products [4, 5] mainly because they are water soluble, biocompatible,

us
34  and non-toxic [6]. Among plant polysaccharides, great importance has been given to

an
35  storage galactomannans since they can be obtained from seeds by aqueous extraction in

36  a pure form and in high yields [7, 8].

M
37  Studies have shown that galactomannans from seeds exhibit many biological

38  effects, including antitumoral [9-12], immunomodulatory [13, 14], and leishmanicidal
d

39  [10, 13] activities. In addition, derivatives obtained from chemical modification, such as
te

40  sulfated galactomannans, have been evaluated in different cell lines [9, 15, 16]. Their

41  cytotoxic effects include a decrease in cell proliferation, cell-cycle arrest, and induction
p

42  of cell death by apoptosis.

ce

43  In general, it has been suggested that effects of polysaccharides depend on their

44  chemical compositions and molecular conformations [5]. Thus, the correlation between
Ac

45  the chemical structure and biological activity of polysaccharides has been investigated

46  [5]. These studies show that the most important factors for their activity are molar mass

47  [17], molecular conformation [18] which occurs in highly ordered structures such as

48  triple helices [19, 20], the distribution of glycoside units along the main chain [21], the

49  degree of substitution of the polysaccharide [22], or the presence of different

2 
 
Page 2 of 43
50  substituents such as sulfate [23-25]. It has been shown that the presence of sulfate could

51  increase the cytotoxic potential of a polysaccharide [26].

52  The presence of particular receptors in tumor cells may be used as a therapeutic

53  strategy [27]. On the surface of hepatocytes and HepG2 cells, asialoglycoprotein

t
54  (ASGPR) has been identified, which is able to recognize molecules containing glucose,

ip
55  galactose, and N-acetylglucosamine [28, 29] and allows the uptake of polysaccharides

cr
56  or glycoprotein via endocytosis. It has been suggested that ASGPR could be a desirable

57  target to deliver compounds into these cells aiming at chemotherapeutic effects [29]. In

us
58  this study, we evaluate the effects of galactomannan from S. amazonicum seeds and its

an
59  native and sulfated forms on certain metabolic parameters of HepG2 cells, considering

60  that this polymer could be internalized by these cells and would trigger alterations that
M
61  impair its survival.

62  Our results show for the first time that native galactomannan from S.
d

63  amazonicum and its sulfated forms are cytotoxic to HepG2 cells and impair oxidative
te

64  phosphorylation, likely activating the glycolytic pathway in response to this effect.

65 
p

66  2 Material and Methods

ce

67  2.1 Material

68  HEPES, rotenone, carbonyl cyanide p-trifluoromethoxyphenylhydrazone

Ac

69  (FCCP), oligomycin, 3-methyl-[4-5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium

70  bromide (MTT), trypsin-EDTA, gentamicin, dimethyl sulfoxide (DMSO),

71  trifluoroacetic acid (TFA), sodium borodeuteride (NaBH4), and dialysis tubing were

72  purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Deuterium oxide

73  (D2O) was supplied by Cambridge Isotope Laboratories (Andover, MA, USA).

3 
 
Page 3 of 43
74  Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were

75  supplied by Cultilab (Brazil). All other chemicals used were of analytical grade.

76 

77  2.2 Extraction of galactomannan from S. amazonicum seeds

t
78  Galactomannan extraction was performed according to the procedure described

ip
79  by Petkowicz et al. [30]. The seeds of S. amazonicum were boiled in distilled water for

cr
80  30 min and remained immersed in water for 48 h. After this period, the endosperm was

81  separated from the seed coat and embryo, oven-dried, and then subjected to grinding.

us
82  The ground endosperm underwent aqueous extraction for 16 h at 25 °C. The fraction

an
83  originated, called SAGM (Schizolobium amazonicum Galactomannan), was centrifuged

84  and the supernatant was precipitated with ethanol three times and dried in a vacuum
M
85  oven (25 °C).

86 
d

87  2.3 Monosaccharide composition

te

88  The polysaccharide was hydrolyzed with 2 M TFA for 5 h at 100 °C [31].

89  Monosaccharides were reduced with NaBH4 and acetylated with Ac2O-pyridine (1:1,
p

90  v/v, 16 h, at 25 °C) [32, 33]. The resulting alditol acetates were analyzed by gas-liquid
ce

91  chromatography (GLC) using a 5890 A II HP gas chromatograph at 220 °C (flame

92  ionization detector and injector temperature of 250 °C) with a DB-210 capillary column
Ac

93  (0.25 mm i.d × 30 m), film thickness of 0.25 µm, and N2 (2.0 mL/min) as the carrier

94  gas.

95 

96  2.4 High-performance size-exclusion chromatography

97  The galactomannans were analyzed by high-performance size-exclusion

98  chromatography (HPSEC) using a Waters unit coupled to a refractive index (RI) and a

4 
 
Page 4 of 43
 99  Wyatt Technology Dawn F multiangle laser light scattering (MALLS) detector. Four

100  Waters Ultrahydrogel columns (2,000, 500, 250, and 120) were connected in series and

101  coupled to the multi-detection instrument. A solution of 0.1 M NaNO2 with 0.02%

102  NaN3 was used as eluent at a flow of 0.6 mL/min. Before the analysis, solutions of

t
103  polysaccharides (1 mg/mL) were filtered through a 0.22 µm nitrocellulose membrane.

ip
104  All the analyses were carried out at 25 °C.

cr
105  The refractive index increment of the solvent-solute solution with respect to a

106  change in solute concentration (dn/dc) was determined using a Waters 2410 differential

us
107  refractometer. The average molar mass (Mw) was calculated from light scattering data

an
108  using the ASTRA program.

109 
M
110  2.5 Nuclear magnetic resonance spectroscopy
13
111  The C NMR spectrum was recorded at 70 °C with a Bruker AC-300
d

112  spectrometer at 75 MHz after dissolving the fractions with D2O using acetone as
te

113  internal standard.

114 
p

115  2.6 Preparation of sulfated derivatives

ce

116  Galactomannan was sulfated according to the method described by O’Neill [34]

117  with slight modifications, as follows. SAGM (2.0 g) was solubilized in formamide (100
Ac

118  mL) and pyridine (100 mL) by vigorous stirring for 24 h, followed by dropwise addition

119  of chlorosulfonic acid (25 mL) over 1 h at 0 °C, the mixture being maintained at 4 °C

120  for 12 h. Ice-water was added, followed by 10% (w/v) aqueous NaHCO3 until

121  effervescence ceased. The solution was then dialyzed against water to remove pyridine,

122  salts, and potential degradation products and then freeze-dried, providing the sodium

123  salt of Schizolobium amazonicum Galactomannan Sulfated 1 (SAGMS1). Sulfated

5 
 
Page 5 of 43
124  galactomannan was re-sulfated as mentioned before in order to increase the degree of

125  sulfation, which originated S. amazonicum Galactomannan Sulfated 2 (SAGMS2). The

126  degree of sulfation (DS) of sulfated derivatives was determined by hydrolysis with 1 M

127  HCl for 5 h at 100 °C, the resulting BaSO4 being measured turbidimetrically. After 15

t
128  min at room temperature, absorbance was determined at 360 nm. The sulfate content

ip
129  was determined in relation to a standard curve of sodium sulfate (20 to 200 g/mL)

cr
130  [35].

The DS was calculated using the formula according to [36]:

us
131 

132  DS = 162 x S / 3,200-102 x S

an
133  Where:

134  162 represents 1 mol of hexoses units;

M
135  3,200 is the atomic weight of sulfur (32) x 100

136  102 represents 1 mol of the ester substituent (SO3Na)-1

d

137  S represents the sulfur content given in percentage

te

138 

139  2.7 Infrared spectroscopy (FTIR) analysis

p

140  The chemical structure of the derivatives was characterized using Fourier
ce

141  Transform Infrared Spectroscopy (BOMEM MB-100 - Hartman & Braun, Canada). The

142  sulfated samples were ground with potassium bromide at a ratio of 1:50 and pressed
Ac

143  into a thin pellet, which was used for FTIR analysis.

144 

145  2.8 Galactomannan solution

146  The galactomannan solutions were prepared in ultrapure water at a concentration

147  of 3 mg/mL. The solution was sterilized by filtration through 0.22 µm Millipore®

148  membrane, frozen, and diluted in culture medium for the biological experiments.

6 
 
Page 6 of 43
149  2.9 Cell culture

150  HepG2 cells were acquired from the Rio de Janeiro Cell Bank, Brazil. The cells

151  were maintained in DMEM High-Glucose medium supplemented with 10% (v/v) fetal

152  bovine serum (FBS), 50 µg/mL gentamycin at 37 °C, and 5% CO2. Cells were cultured

t
153  only in medium or in medium plus galactomannan at different concentrations (50, 100,

ip
154  and 250 µg/mL) in a 96-well microculture plate (104 cells/well), 60 mm plate (5 × 105

cr
155  cells/well), and 12-well microculture-plate (105 cells/well) and cell growth was

156  monitored with an Olympus® inverted microscope.

us
157 

an
158  2.10 Cytotoxicity of galactomannans

159  Cell viability was determined by the MTT method [37], in which viable and
M
160  metabolically active cells reduce the tetrazolium salt to form formazan crystals soluble

161  in DMSO absorbing a wavelength of 550 nm. HepG2 cells were trypsinized and seeded
d

162  (104 cells/well) in 96-well plates. After 24 h of adhesion, the culture medium was
te

163  removed and solutions of native and sulfated galactomannans at concentrations of 50,

164  100, and 250 μg/mL diluted in culture medium were added, followed by incubation at
p

165  37 °C in 5% CO2 atmosphere. After incubation for 24, 48, and 72 h, the medium was
ce

166  removed and replaced with HBSS plus MTT solution for a final concentration of 500

167  µg/mL. This preparation was then incubated for 3 h at 37 °C in a 5% CO2 atmosphere,
Ac

168  after which the supernatant was removed and DMSO added to dissolve the formazan

169  crystals. The absorbance of the samples was measured in a microplate reader.

170 

171  2.11 Determination of oxygen consumption

172  Oxygen consumption was monitored by high-resolution respirometry

173  (Oxygraph-2k, Oroboros® Instruments, Innsbruck, Austria) [38]. Measurements were

7 
 
Page 7 of 43
174  made in two chambers at 37 °C under gentle agitation. HepG2 cells were plated at a

175  density of 5  105 cells per 60 mm diameter plate and allowed to adhere for 24 h.

176  Subsequently, the cells were treated with concentrations of 100 and 250 μg/mL of

177  SAGM, SAGMS1, and SAGMS2 for 72 h. The adherent cells were released with

t
178  trypsin-EDTA solution, re-suspended in DMEM, and transferred to Oroboros®

ip
179  chambers. Oxygen consumption was rated in different states of respiration defined as

cr
180  follows: Basal, oxygen consumption in the absence of inhibitors and uncouplers; leak

respiration in the presence of olygomicin (2 µg/mL); and uncoupled, oxygen

us
181 

182  consumption in the presence of the uncoupler FCCP (0.5 µM). The oxygen flow in

an
183  these states was corrected by subtracting non-mitochondrial respiration, which was

184  obtained after addition of rotenone (0.5 µM) and antimycin (3 µg/mL). The results are
M
185  expressed as the oxygen flow per cell ((pmol/(s × 5 × 105 cells)) as mean ± SD.

186 
d

187  2.12 Lactate and pyruvate production

te

188    The supernatant from HepG2 cells treated to test oxygen consumption (item

189  2.11) with galactomannan solutions after 72 h of incubation (SAGM, SAGMS1, and
p

190  SAGMS2) were used for the determination of lactate and pyruvate according to the
ce

191  method described by Gutmann & Wahlefeld [39] and Czoc & Lamprecht [40],

192  respectively. Lactate levels were determined in a final volume of 300 µL in reaction
Ac

193  medium containing 0.1 M glycine, 0.4 M hydrazine buffer pH 9.5, 1.5 mM NAD+, and

194  lactate dehydrogenase (3U). The system was maintained at 37 °C for 90 min and the

195  amount of NADH formed was measured at 340 nm in a microplate reader (Epoch®). For

196  the determination of pyruvate, the final volume of 300 µL was also in a reaction mixture

197  containing 0.1 M Tris-HCl pH 7.4, 0.15 mM NADH, and lactate dehydrogenase (0.1

198  U). The amount of NADH oxidized was measured spectrophotometrically at 340 nm

8 
 
Page 8 of 43
199  after 20 min incubation at 37 °C. The amount of lactate and pyruvate was calculated

200  from the molar extinction coefficient of NADH (6,220 M-1 cm-1).

201 

202  2.13 Statistical analysis

t
203  Results were expressed as mean ± SD and statistical analysis was performed

ip
204  using ANOVA to determine the significant differences among the groups, followed by

cr
205  Student’s t-test with p < 0.05 considered significant.

206 

us
207  3 Results

an
208 

209  3.1 Native and chemically sulfated galactomannans from S. amazonicum seeds
M
210  Native galactomannan from S. amazonicum seeds (SAGM) was obtained by

211  aqueous extraction at 25 °C and monosaccharide analysis showed a 3.2:1 Man:Gal ratio.
d

212  This value is in agreement with previous reports for galactomannans obtained from the
te

213  same species under similar conditions [30, 41, 42].

214  The galactomannan from S. amazonicum seeds (SAGM) was analyzed by high-
p

215  performance size-exclusion chromatography (HPSEC) and showed a unimodal molar

ce

216  mass distribution (Fig. 1a). The molar mass determined by light scattering was 4.34 ×

217  105 g/mol (Table 1). The ratio of average molar mass and numeric mass, Mw/Mn, also
Ac

218  known as polydispersity index, was low at 1.123 ± 0.012, which indicates low

219  polydispersity of the sample.

220  The galactomannans from S. amazonicum seeds have been previously

13
221  characterized [7, 8, 30, 41, 42]. The structure of SAGM was confirmed by C NMR

222  (Fig. 2a) and is in agreement with previous reports. The spectrum showed a

223  galactomannan composed of a backbone of D-manopyranose units linked by β (1 → 4)

9 
 
Page 9 of 43
224  glycosidic bonds with lateral branches of D-galactopyranoses linked by α (1 → 6)

225  glycosidic bonds (Table 2 and Fig. 2a) [7, 8, 41, 43].

226  The sulfated galactomannan (SAGMS1) was prepared from SAGM using

227  chlorosulfonic acid in formamide-pyridine. SAGMS1 had a sulfate content of 6.2%

t
228  corresponding to a degree of sulfation (DS) of 0.4. The molar mass of the sulfated

ip
229  galactomannan was Mw of 3.41 x 105 g/mol. In an attempt to obtain a sample with a

cr
230  higher DS, the SAGMS1 sample was subjected to a new sulfation process resulting in

231  the SAGMS2 fraction showing a DS of 0.6, sulfate content of 8.2%, and molar mass of

us
232  2.43 × 105 g/mol (Table 1). The sulfated galactomannans were analyzed by HPSEC-

an
233  MALLS. After sulfation, a bimodal elution profile was observed, suggesting the

234  sulfation process had promoted degradation (Fig. 1b,c).

13
M
235  The C NMR spectra of SAGMS1 showed a reduction in the intensity of the

236  signals related to C-6 of galactose and unsubstituted mannose units, suggesting that
d

237  sulfation occurred in this position (Fig. 2b). An additional intense signal at δ 67.5 could
te

238  correspond to the C-6 signal of the units that were sulfated. SAGMS2 NMR spectra

239  showed signals related to C-6 of galactose and unsubstituted mannose units became
p

240  virtually non-existent, suggesting complete sulfation of the polymer in these positions
ce

241  (Fig. 2c) and the DS determined for the sample (0.6) confirms this hypothesis.

242  Infrared spectroscopy was also used to analyze the biopolymers. SAGMS1 and
Ac

243  SAGMS2 showed an absorption band at 1,250 cm-1 resulting from asymmetrical

244  stretching of S=O [15, 16], in the vibrational mode, absent from the spectrum of the

245  native galactomannan (SAGM) (Fig. 3a, b, and c).

246 

247  3.2 Effects of galactomannans on HepG2 cell viability

248  The effects of native (SAGM) and chemically sulfated galactomannans

249  (SAGMS1 and SAGMS2) of S. amazonicum seeds on the viability of HepG2 cells are

10 
 
Page 10 of 43
250  shown in Figure 4. SAGM at 250 µg/mL was cytotoxic after 48 h of incubation (~30%)
251  (Fig. 4a), decreasing the cell viability by 50% after 72 h of treatment. However, for
252  sulfated derivatives, only SAGMS1 significantly reduced cell viability, reaching 30% at
253  250 μg/mL for 72 h (Fig. 4b). In the same conditions, the polymers were not cytotoxic
254  to HepG2 cells when incubated for 24 h (data not shown). In respect to difference
among the biopolymers, only SAGM and SAGMS1 at the concentrations 50 and 250

t
255 

ip
256  μg/mL lowered viability with statistically significance after 72 h of treatment.
257  Furthermore, SAGM reduced the cell viability around 30% and 50% at 48 h and 72 h,

cr
258  respectively, indicating a dose dependent profile. Thus, we decided to use 250 μg/mL
259  for 72 h for the next tests, because 50% of cytotoxicity gives the possibility to identify

us
260  several alterations in the cells”.
261 

an
262  3.3 Effect of galactomannans on respiration of HepG2 cells

263  Respiration of HepG2 cells was evaluated in basal, leak, uncoupled, and
M
264  inhibited states of respiration as illustrated in Figure 5, where the solid line represents

265  the oxygen concentration (µM) and the dashed line represents the oxygen flow
d

266  ((pmol/(s × 5 × 105 cells)). The basal state in the absence of inhibitors or uncouplers
te

267  was significantly decreased by addition of oligomycin. The oxygen uptake in the

268  presence of oligomycin results from the re-entry of protons into the mitochondrial
p

269  matrix, characterizing the leak state. The addition of a classical uncoupler, FCCP,
ce

270  promotes a significant increase in oxygen consumption, characterizing the uncoupled

Ac

271  state. Finally, mitochondrial respiration was completely inhibited by the addition of

272  rotenone and antimycin (inhibited state) [44-46]. The values of oxygen flow

273  corresponding to the inhibited state were subtracted from the other states of respiration

274  so that only the oxygen uptake resulting from mitochondrial respiration was represented

275  in each state.

276  The results of respiration assays were analyzed by the DataLab4® software and

277  are shown in Figure 6. All polysaccharides inhibited the basal state (Fig. 6a) of HepG2

11 
 
Page 11 of 43
278  cells after 72 h of incubation. SAGM at 100 and 250 µg/mL inhibited respiration by

279  62% and 85%, respectively. Both sulfated galactomannans, SAGMS1 and SAGMS2,

280  also inhibited respiration. However, SAGMS1 at concentration of 250 µg/mL inhibited

281  by 82% mitochondrial respiration while SAGMS2 at the same concentration decreased

t
282  it by 60%. Galactomannans decreased respiration in the leak state by ~40% as observed

ip
283  in Figure 6b, except for SAGMS2 (100 µg/mL). Galactomannans also promoted a

cr
284  significant inhibition, around 90%, of the uncoupled state at all concentrations evaluated

285  (Fig. 6c). As observed for basal respiration, SAGMS2 was less effective and reduced

us
286  the respiration rate by 50% and 85% at 100 and 250 µg/mL, respectively.

an
287  These results suggest that SAGM and SAGMS1 significantly impair cellular

288  respiration and this effect could be associated with the loss of cell viability.
M
289  Interestingly, SAGMS2 inhibited respiration, but it did not affect cell viability (Fig. 4),

290  which could indicate that additional effects, besides alterations in respiration, are
d

291  involved in the maintenance of cell viability.

te

292 

293  3.4 Effect of galactomannans on lactate and pyruvate levels

p

294  The final metabolites of the glycolytic pathway are pyruvate in normal cells and
ce

295  lactate in most tumor and proliferative cells [47]. Respiration inhibition by SAGM,

296  SAGMS1, and SAGMS2 may promote a decrease in ATP production by oxidative
Ac

297  phosphorylation, which could result in the activation of the glycolytic pathway.

298  Initially, to evaluate this possibility, the levels of pyruvate and lactate in the supernatant

299  of HepG2 cells were measured after 72 h of incubation with the polysaccharides.

300  SAGM increased lactate levels by ~25% at 250 µg/mL, as observed for SAGMS1 and

301  SAGMS2 at the same concentration (Fig. 7a). SAGM and SAGMS1 decreased pyruvate

12 
 
Page 12 of 43
302  levels by ~13% for SAGM (100 µg/mL) and ~20% for SAGMS1 (250 µg/mL).

303  SAGMS2 did not alter pyruvate levels in comparison to the control (Fig. 7b).

304 

305  4. Discussion

t
306  The chemical modification of polysaccharides can alter their biological

ip
307  properties [5]. Chemical sulfation of galactomannans may be performed using different

cr
308  methodologies. In this study, galactomannan from S. amazonicum was sulfated

309  according to O’Neill [34]. The molar mass for both sulfated galactomannans SAGMS1

us
310  (3.41 × 105 g/mol) and SAGMS2 (2.43 × 105 g/mol) decreased when compared to the

an
311  native polymer. As demonstrated by other authors, sulfation and degradation occur

312  simultaneously in the derivatization process [48]. The decrease in molar mass was also
M
313  observed during the sulfation of galactomannan from guar gum and from D.

314  gardneriana [16, 49] under the same conditions of the present study. In addition,
d

315  sulfation could modify the parameters of chain conformation, which may also affect
te

316  biological properties [49].

317  Yang et al. [50] evaluated the sulfation of polysaccharide from Chinese lacquer
p

318  tree (Rhus vernicifera) by sulfur trioxide-pyridine (SO3 Py) as a reagent and DMSO,
ce

319  DMF, and FA (formamide) as solvents at different temperatures. The authors showed

320  that polymers with lower molar mass exhibited high DS and suggested that the
Ac

321  degradation of the polysaccharide in the sulfation reaction could be a hydrolytic

322  degradation depending on the nucleophilicity of the solvents that were used.

323  The preferred sulfating for the more reactive 6-CH2 groups has been previously

324  described in galactomannans from Mimosa scabrella, Dimorphandra gardneriana,

325  Senna macranthera, and guar gum [15, 16, 48, 49, 51]. The absence of signals of
13
326  exocyclic C from galactose and unsubstituted mannose in the SAGMS2 C NMR

13 
 
Page 13 of 43
327  spectra suggests complete sulfation of the polymer in the O-6 position. Similar results

328  were found by Wang et al. [49] for guar galactomannan. Those authors also observed

329  the disappearance of C-6, suggesting the C-6 OH group was completely sulfated.

330  Concerning SAGMS1, the appearance of a new peak at δ 67.5, which corresponds to the

t
331  signal of substituted C - 6, suggests sulfation of O-6. According to the literature, the

ip
332  carbon attached to a sulfate group suffers a shift by 6-8 ppm to a lower field [16, 49].

cr
333  The appearance of this new signal after sulfation was also observed for a sulfated

334  galactomannan from guar [49]. Doctor and Esho [52] compared different native and

us
335  sulfated polysaccharides and concluded that sulfation occurred preferentially at the most

an
336  accessible primary free hydroxyl groups, according to the results of the present study in

337  primary carbons of galactose and unsubstituted mannose units.

M
338  Infrared spectroscopy was also used to analyze the biopolymers. The broad and

339  intense band at centered at ~3450 cm−1 is the result of stretching of hydroxyl groups
d

340  while the band at 2950 cm-1, it is caused by C–H stretching of CH2. The intensity of this
te

341  band decreased with the sulfation probably due to effects of the sulfation at C – 6 of

342  galactose and unsubstituted mannose units. The band at 1650 cm-1 is due to the
p

343  associated water or –OH deformation vibration [53] and the differences between native
ce

344  and its sulfated forms could be due to stronger associations of water with sulfate groups.

345  SAGMS1 and SAGMS2 showed an absorption band at 1250 cm−1 resulting from
Ac

346  asymmetrical stretching of S=O [15, 16], in the vibrational mode, absent from the

347  spectrum of the native galactomannan (SAGM).

348  It has been shown that chemical sulfation of polysaccharides may be associated

349  with their antitumor activity [26]. However, the relation between DS and antitumor

350  activity is not conclusive. Galactomannan from Leucaena leucacephala and its

351  chemically sulfated derivative, both at 5 µg/mL, decreased HepG2 cells viability by

14 
 
Page 14 of 43
352  ~30% and 50%, respectively [12]. Chen et al. [54] observed that polysaccharides

353  containing Gal, Man, Rha, and Ara isolated from Gynostemma pentaphyllum subjected

354  to chemical sulfation showed a better cytotoxic effect (46%) on HepG2 cells than native

355  polymer, both at the highest concentration evaluated (1 mg/mL for 48 h). After

t
356  sulfation, the polymer with the highest DS (1.34) attained ~30% in molar mass over the

ip
357  non-sulfated form. Liu et al. [55] analyzed four sulfated derivatives of a β-D-glucan

cr
358  with different DS (0.62 to 2.02) with non-degradation. The polysaccharide with DS of

359  1.80 was the most effective, reducing HepG2 cell viability by ~25% at 250 μg/mL. A

us
360  highly branched (1→4)-α-D-glucan lost its activity against H-22 tumor cells after

an
361  sulfation [56].

362  In the present study, the highest cytotoxic effect was not correlated with the
M
363  increase in the DS. This could be related to the chemical modification to which the

364  polymer has been subjected, not being recognized by membrane receptors in HepG2
d

365  cells, maybe ASGPR receptors which interact with glucose, galactose, and N-
te

366  acetylglucosamine units [57].

367  Some studies on the internalization of polysaccharides on different types of cells

p

368  have been reported [28, 29, 58]. Lai et al. [29] evaluated the effect of Cy3 fluorescent
ce

369  dye and galactose derivatives with different ratios on the surfaces of magnetic

370  nanoparticles (MNPs) in HepG2 cells. The authors observed that MNPs interacted with
Ac

371  ASGP-R on the cell surface of liver cancer cells (HepG2) and triggered subsequent

372  internalization via receptor-mediated endocytosis. A kinetic study in vivo with

373  fluorescein-labeled pullulan  suggests the occurrence of the receptor-mediated hepatic

374  uptake of this polymer in rats [28]. Nevertheless, Péterszegi et al. [58] demonstrated the

375  effects of oligos and polysaccharides containing galactose, galacturonic acid, and fucose

15 
 
Page 15 of 43
376  in human fibroblasts. The authors showed that polysaccharides labeled with FITC have

377  two sites of interaction with fibroblasts, i.e., the membrane and nucleus.

378  Regarding cytotoxic effects observed in galactomannan and its sulfated

379  derivatives on HepG2 cells, our study sought to evaluate interferences of galactomannan

t
380  on metabolism of HepG2 cells. After all, the differential metabolism of tumor cells is

ip
381  the subject of study for the development of anticancer drugs [59-61], and heretofore no

cr
382  data have been reported regarding the effects of galactomannans on any parameter of

383  tumor cell metabolism.

us
384  Among the targets of interest in the study of metabolism for anticancer effects,

an
385  great emphasis has been placed on interference in mitochondrial metabolism [62]. It is a

386  fact that, by impairing mitochondrial activity, the provision of energy and certain
M
387  metabolites is suppressed, which can culminate in cell death. At the time of writing,

388  studies evaluating the effect of polysaccharides on the HepG2 respiration were not
d

389  found.
te

390  The effects of SAGM, SAGMS1, and SAGMS2 on respiration of HepG2 cells

391  suggest two possibilities: Galactomannans could interact with receptors on the cell
p

392  surface and become internalized, for example, via endocytosis by ASGPR, so the
ce

393  biopolymers could act directly in the mitochondria; or, after their interaction with cell

394  membrane receptors, trigger alteration of signal pathways that would lead to a decrease
Ac

395  in oxygen consumption. Although we have not investigated the process of

396  galactomannan internalization, their significant interference on cellular respiration

397  points to evidence of this possibility, considering the monosaccharide composition of

398  galactomannans (mannose and galactose) and the presence of ASGPR HepG2 cells [28,

399  29].

16 
 
Page 16 of 43
400  The involvement of mitochondria in the resistance to apoptosis is one of the

401  essential contributions of these organelles to tumor progression [60], which make them

402  a potential candidate for antitumor therapies [63]. Concerning polysaccharides, Wang et

403  al. [64] had shown that sulfated polysaccharide from E. intestinalis promoted loss of

t
404  mitochondrial membrane potential and released cytochrome c in HepG2 cells. The

ip
405  authors suggested the occurrence of cell death by apoptosis. This result is consistent

cr
406  with the results from the present study in relation to the alterations in mitochondrial

407  respiration by the polysaccharides.

us
408  The increase and decrease of lactate and pyruvate levels, respectively, by the

an
409  polymers may be induced by the inhibition of cell respiration and could indicate that

410  HepG2 cells would be shifting from oxidative phosphorylation to aerobic glycolysis as
M
411  a survival mechanism. It can therefore be suggested that the changes in conformation by

412  the sulfation process impaired the recognition of the polymer derivatives by HepG2
d

413  cells. However, additional studies must be performed to clarify this hypothesis, and
te

414  such studies are currently in progress in our laboratory.

415 
p

416  5. Conclusion
ce

417  Two sulfated galactomannan derivatives from S. amazonicum seeds with degrees

418  of sulfation of 0.4 and 0.6 were obtained. NMR analysis suggested that sulfation
Ac

419  occurred at the primary carbons of galactose and unsubstituted mannose units.

420  The cytotoxic potential of the native galactomannan from S. amazonicum seeds

421  (SAGM) was higher than sulfated derivatives. However, all polymers evaluated

422  promoted a significant inhibition in cell respiration, which likely resulted in increased

423  levels of lactate, indicating activation of aerobic glycolysis (Warburg effect). The

424  results of the present research motivate further studies on the antitumor potential of this

17 
 
Page 17 of 43
425  galactomannan since the significant inhibition of cell respiration by the polymers could

426  impair cell metabolism and culminate in tumor cell death. However, additional studies

427  to clarify their mechanisms of action are necessary.

428 

t
429  Acknowledgement

ip
430  This study was supported by the Brazilian research funding agencies CNPq

cr
431  (472651/2012-9) and CAPES. G.R.M. was also supported by INCT de Processos Redox

432  em Biomedicina – Redoxoma.

us
433 

434 

435 
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cr
623  mitochondrial pathway, Tumor Biology 35(2) (2013) 1641-1647.
624 

us
625  Figure and table captions

626 

627 

628  an
Table 1 – Degree of sulfation (DS) and molecular weight (Mw) of polysaccharides from

S. amazonicum (SAGM) and its chemically sulfated derivatives (SAGMS1 and

M
629  SAGMS2).

630 
d

631  Table 2 – 13C NMR chemical shifts for galactomannan obtained from the endosperm of
te

632  S. amazonicum
p

633 
ce

634  Fig. 1. Elution profile obtained by high-performance size-exclusion chromatography

635  (HPSEC) of native and chemically sulfated galactomannans from the endosperm of S.
Ac

636  amazonicum. (A) native galactomannan, SAGM; (B) chemically sulfated galactomannan

637  with DS 0.4, SAGMS1; and (C) chemically sulfated galactomannan with DS 0.6,

638  SAGMS2.

639 

640  Fig. 2. 13C NMR spectra of SAGM (A), SAGMS1 (B), and SAGMS2 (C) in D2O at 70

641  °C; numerical values expressed in ppm (δ).

642 

22 
 
Page 22 of 43
643  Fig. 3. Infrared spectra (FTIR) of native and chemically sulfated galactomannans from

644  endosperm of S. amazonicum. (A) Native galactomannan, SAGM; (B) chemically

645  sulfated galactomannan with DS 0.4, SAGMS1; and (C) chemically sulfated

646  galactomannan with DS 0.6, SAGMS2. The samples (2 mg) were homogenized in pre-

t
647  dried KBr (100 mg) and then analyzed as pellets. The arrow points to the location where

ip
648  the sulfation band occurs at 1,250 cm -1.

cr
649 

650  Fig. 4. Evaluation of cytotoxicity of the galactomannans SAGM, SAGM:S1 and

us
651  SAGM:S2 on HepG2 cells. (A) 48 h and (B) 72. Cell viability was determined by MTT

an
652  assay. Culture medium was used as negative control, corresponding to 100%. The values

653  represent the mean ± the standard error of six different experiments. *, ** and ***denote
M
654  values significantly different from the control or between the different concentrations at

655  p < 0.05; p < 0.001 and p < 0.0001, respectively and ns means not significant.
d

656 
te

657  Fig. 5. Representative trace of HepG2 cell respiration in OROBOROS-2K oxygraph

658  chambers. Legends: A) Routine state; B) Leak state; C) Uncoupled state; D) Inhibited
p

659  state; oligo – oligomycin; FCCP – Carbonyl cyanide p-

ce

660  trifluoromethoxyphenylhydrazone; ROT – rotenone; ANT – antimycin A. Solid line:

661  oxygen concentration; Dashed line: oxygen flow.

Ac

662 

663  Fig. 6. Effects of SAGM, SAGMS1, and SAGMS2 on the oxygen uptake of HepG2

664  cells. Cells (5 × 105) after incubation for 72 h with galactomannans were analyzed by

665  Oroboros 2-K oxygraph and the oxygen consumption was determined in the absence of

666  inhibitors or uncouplers (A), in the presence of oligomycin (B), *, and ** denote values

667  significantly different from the control at p < 0.05, p < 0.001, respectively, and in the

23 
 
Page 23 of 43
668  presence of FCCP (C). The values represent the mean ± the standard error of six

669  different experiments. *, **, and ***denote values significantly different from the

670  control or from the different concentrations at p < 0.05, p < 0.001, and p < 0.0001,

671  respectively and ns means not significant. 

t
672 

ip
673  Fig. 7. The lactate and pyruvate levels in the supernatants of HepG2 cells after

cr
674  incubation for 72 h with SAGM, SAGMS1, and SAGMS2. Lactate and pyruvate

675  concentrations were measured as described in the Methods section. (A) lactate levels

us
676  (100% of lactate = 233.8 ± 25.10-9mol/105 cells), (B) pyruvate levels (100% of pyruvate

= 155 ± 4.10-9/105 cells). Results are expressed as the mean ± standard error of six

an
677 

678  different experiments. *, **, and ***denote values significantly different from the
M
679  control or from the different concentrations at p < 0.05, p < 0.001, and p < 0.0001,

680  respectively and ns means not significant.  

d

681  Table 1
te

Polysaccharide DS Mw
p

SAGM 0 4.34 x 105 M

ce

SAGMS1 0.4 3.41 x 105 M

Ac

SAGMS2 0.6 2.43 x 105 M

682 

683  Table 2 -

(ppm) δ

24 
 
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 C1 C2 C3 C4 C5 C6

α-Gal(1→ 98.8 68.5 69.4 69.6 70.0 61.3

→4)-β-Man-(1→ 100.1 71.4 71.5 76.6 77.0 60.6

t
ip
→4,6)-β-Man-(1→ 100.3 71.4 71.5 76.9 73.4 66.5

cr
684 

us
685   

686   

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