Accepted Manuscript

High molecular weight plant heteropolysaccharides stimulate fibroblasts but

inhibit keratinocytes

Munira Shahbudin, Dahlia Shahbuddin, Anthony J. Bullock, Halijah Ibrahim,

Stephen Rimmer, Sheila MacNeil

PII: S0008-6215(13)00134-1

DOI: http://dx.doi.org/10.1016/j.carres.2013.04.006

Reference: CAR 6449

To appear in: Carbohydrate Research

Received Date: 12 February 2013

Revised Date: 4 April 2013

Accepted Date: 4 April 2013

Please cite this article as: Shahbudin, M., Shahbuddin, D., Bullock, A.J., Ibrahim, H., Rimmer, S., MacNeil, S.,

High molecular weight plant heteropolysaccharides stimulate fibroblasts but inhibit keratinocytes, Carbohydrate

Research (2013), doi: http://dx.doi.org/10.1016/j.carres.2013.04.006

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1

High molecular weight plant heteropolysaccharides stimulate fibroblasts but inhibit

keratinocytes.

Munira Shahbudin, Dahlia Shahbuddin1, Anthony J Bullock, Halijah Ibrahim2,

Stephen Rimmer3, Sheila MacNeil.

Department of Materials Science & Engineering, Kroto Research Institute, University

of Sheffield, North Campus, Broad Lane, Sheffield, S3 7HQ

1 School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang

2 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala

Lumpur

3 The Polymer and Biomaterials Laboratories, Department of Chemistry, University of

Sheffield, Sheffield S3 7HF

Corresponding author: s.macneil@shef.ac.uk, ; Telephone: +44 (0) 114 222 5995, Fax:

+44 (0) 114 222 5945

2

Abstract

Konjac glucomannan (KGM) is a natural polysaccharide of (1-4)-Dglucomannopyranosyl

backbone of D-mannose and D-glucose derived from the tuber of Amorphophallus konjac C.

Koch. KGM has been reported to have a wide range of activities including wound healing. In

this study we examined KGM extracts prepared from five plant species, (Amorphophallus

konjac Koch, A. oncophyllus, A. prainii, A. paeoniifolius and A. elegans) for their effects on

cultured human keratinocytes and fibroblasts. Extracts from A. konjac Koch, A. oncophyllus

and A. prainii (but not from A. paeoniifolius or A. elegans) stimulated fibroblast proliferation

both in the absence and presence of serum. However, these materials inhibited keratinocyte

proliferation. The fibroblast stimulatory activity was associated with high molecular weight

fractions of KGM and was lost following ethanol extraction or enzyme digestion with -

mannanase. It was also reduced by the addition of concanavalin A but not mannose

suggesting that these heteropolysaccharides are acting on lectins but not via receptors specific

to mannose. The most dramatic effect of KGM was seen in its ability to support fibroblasts

for 3 weeks under conditions of deliberate media starvation. This effect did not extend to

supporting keratinocytes under conditions of media starvation but KGM did significantly

help support adipose derived stem cells under media starvation conditions.

3

2. Introduction

Complex polysaccharides such as polymers of glucose (glucans), mannose (mannans), xylose

(hemicelluloses), fructose (levans) or other mixtures of sugars are reported to have

immunostimulatory and wound healing properties [1-3]. Glucomannan (GM) and glucans are

major structural components of the heavily glycosylated structures of plant and bacterial cell

walls and are recognised by a range of mammalian cell surface receptors such as the

mannose receptor (MR), toll like receptors 2, 4 (TLR2, TLR4) and mannose binding lectin

(MBL) [4]. Cells possess lectins that recognize specific carbohydrates which are also

involved in many biological functions such as immune responses, cellular recognition,

migration and metabolism [5]. The specificity of lectins for different monosaccharides or

glycans such as fucose, mannose, glucose, N-acetylglucosamine and heparin has been a

major research area for many years [6]. In particular GM active components such as Aloe

Vera have been reported to have wound healing following their interaction with MRs [2].

The aim of this study was to examine the effects of KGM derived from five species of this

plant, looking critically at their effects on metabolic activity and proliferation of human

dermal fibroblasts and epidermal keratinocytes. We looked at the relationship between KGM

extracts of different molecular weights and their effect on fibroblast proliferation.

A number of previous studies have fractionated KGM into different molecular weights

seeking to relate structure to activity. In order to obtain KGM fractions with different

molecular weights, various approaches have been investigated such as radiation, ultrasonic,

and enzymatic hydrolysis. In this study, we used ethanol extraction, ultrafiltration and

glucomannanase enzyme digestion, using recombinant enzymes from a saprophytic soil

bacterium Celvibro japonicus that exhibit specific activity against glucomannan [7].

4

To explore the mechanism of interaction of KGM with cells, we used the plant lectin

concanavalin A (from the common bean Canavalia ensiformis) as a competitor lectin and D

mannose to block MRs and we investigated their effects on the biological activities of KGM

on skin cells. We also in this study explored the ability of KGM to support cells (fibroblasts,

keratinocytes and adipose derived mesenchymal stem cells (ADMSC)) in culture under

conditions of media starvation for 3 weeks - following reports that a mannose rich lectin from

hyacinth bean (Dolichos lab lab) helped to preserve human cord blood progenitor cells in

suspension culture for up to 1 month without media changes [8] and stem cells up to 2 weeks

in culture [9].

5

3. Materials and Methods

3.1 Materials

Materials were obtained from the following manufacturers: all KGM samples were provided

by the Institute of Biological Sciences, Faculty of Science, University of Malaya, except for

A. konjac Koch which was obtained without any purification (99% GM content) from Health

Plus Ltd. London U.K.; -mannanase from C.japonicus (EC 3.2.1.78 enzyme activity 5000

U.mg-1) (Megazyme Ltd. Ireland); ethanol (Fisher, U.K.); phosphate-buffered saline (PBS)

tablets (Oxoid, Unipath, Hampshire, U.K.); Dulbeccos modified Eagles medium (DMEM)

(ICN Flow, Thame, Oxfordshire, U.K.); glutamine, penicillin and streptomycin (Gibco

Europe, Life Technologies, Paisley, U.K.) fetal calf serum (FCS) (Advanced Protein Products,

Brairley Hill, West Midlands, U.K.); trypsin, (Difco Laboratories, Detroit, MI, U.S.A.);

isopropanol, (BDH Laboratory Supplies, Lutterworth, Leicestershire, U.K.); 3-[4,5-

dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide-thiazolyl blue (MTT), cholera toxin,

epidermal growth factor (EGF), adenine, insulin, sodium chloride, transferrin,

triiodothryonine, ethylenediamine tetraacetic acid (EDTA) and Trypan blue (Sigma, Poole,

Dorset, U.K.). 4,6-Diamindino-2-phenylidole (DAPI) (Sigma, U.K.), 3.7% of formaldehyde,

SYTO9 (Molecular Probes, U.S.), Propidium Iodide (PI) (Invitrogen, U.K.).

3.2 Determination of glucomannan content in different species of KGM

Selected raw corms of Amorphophallus sp (A. oncophyllus, A. prainii, A. paoenifolius and A.

elegans) were washed with water and scrubbed to remove surface dirt and their skins and

small roots were cut off. They were then sliced, and their sprouts were removed. The slices

were heated in an oven at 60C for 3 days to remove all moisture. The dried slices were

6

ground to a fine powder (

7

obtained using GPC. Confirmation was obtained that the expected KGM fractions remained

biologically active after boiling.

3.6 Analysis of KGM (A. konjac Koch) molecular weights using Gel Permeation

Chromatography (GPC).

Average molecular weights were determined by gel permeation chromatography (GPC,

Agilent Technologies, U.S.A) consisting of Knauer, Smartline Pump 100 (Knauer, Germany),

a Rheodyne 7725i injector loop of 200l and a column HMW Aqueous TSK 4 Viscotech

(950 mm) (Malvern, U.K) at 1 mL.min-1 flow rate on an aqueous GPC, coupled with RI

detector HP1047A (Hewlett Packard, U.S.A). 2 mg of enzymatically hydrolysed and ethanol

extracted KGM was dissolved in 2 mL of 0.1M NaNO3/NaH2PO4 buffer and filtered through

0.4sed and ethanol extracted KGM was dissolved in 2 mL of 0.1into the column. Results

were analysed with Cirrus of 20Multidetector software, Version 3 (Varian, Inc. USA).

3.7 Cell culture

Human keratinocytes and fibroblasts were isolated from skin removed during abdominoplasty

or breast reduction elective surgeries in the Department of Plastic Surgery, Northern General

Hospital, Sheffield with fully informed patient consent for the use of skin for experimental

research. All tissue was banked and used on an anonymous basis under the Human Tissue

Authority Research Tissue Bank Licence number 12179. Human dermal adipose collected at

the same time from these samples was used to isolate mesenchymal stem cells (ADMSC).

Primary keratinocytes were extracted from skin following incubation with 10ml of 1mg.ml-1

Difco Trypsin in PBS overnight at 4oC. 5mL of FCS was added to neutralize the trypsin

followed by separation of epidermis from the dermis. The underside of the epidermis and top

of the dermis were gently scraped into 10% Greens medium (consisting of DMEM high

glucose and Hams F12 medium in a 3:1 ratio supplemented with 10% FCS, 10 ng.mL-1

8

recombinant human epidermal growth factor, 0.4 g.mL-1 hydrocortisone, 0.1 nM cholera

toxin, 1.8 x 10-4 M adenine, 5 mg.mL-1 insulin, 5 g.mL-1 apo-transferrin, 2 x 10-7 M 3,3,5-

tri-idothyronine, 2 x 10-3 M glutamine, 0.625 g.mL-1 amphotericin B, 100 I.U.mL-1

penicillin and 100 g.mL-1 streptomycin) to retrieve keratinocytes. The resulting cell

suspension were transferred into a 25mL universal and centrifuged at 180g and the resulting

pellet was resuspended in Greens media at 37oC and transferred to a T75 flask that was

previously seeded with 5 x 106 i3T3 acting as a feeder layer, i3T3 fibroblasts were cultured in

DMEM supplemented with 10% new born calf serum, glutamine (0.25 mg.mL-1), 0.625

g.mL-1 amphotericin B, 100 I.U.mL-1 penicillin and 100 g.mL-1 streptomycin, before being

growth arrested by a dose gamma irradiation of 60 Gy using a 137Cs source. The cells were

incubated at 37oC, in a 5% CO2 in a humidified atmosphere. The medium was changed every

2-3 days, and keratinocytes were passaged at 70-80% confluency. Only passages 1-2 were

used for experiments. Primary fibroblasts were isolated from skin by mincing the dermal

region of the skin into small pieces, followed by digestion with 0.05% collagenase A in 10%

DMEM (DMEM supplemented with 10% v/v fetal calf serum, glutamine (0.25 mg.mL-1),

0.625 g.mL-1 amphotericin B, 100 I.U.mL-1, penicillin, and 100 g.mL-1 streptomycin)

overnight at 37oC with 5% CO2. The cell suspension was then centrifuged at 400g and

resuspended in 10% DMEM at 37oC. These cells were then seeded into T25 flasks and

incubated at 37oC with 5% CO2. The medium was changed every 2 days and the cells were

passaged as needed, fibroblasts between passage 4 and 9 were used in the experiments.

Human subcutaneous fat was selected as the source of ADMSCs. Tissue was obtained from

discarded skin from elective breast reduction or abdominoplasty surgery after fully informed

consent from Sheffield Teaching Hospitals trust and handled on an anonymous basis under a

research tissue bank licence (number 08/H1308/39) under the Human Tissue Authority.

9

Samples were sectioned with a scalpel in Petri dishes, with 10mL of phosphate-buffered

saline (PBS) and 10 mL penicillin (100 units.ml-1) and streptomycin (100 g.ml-1) (Gibco

Invitrogen, Paisley, UK). Samples were mechanically minced with a scalpel, and the pieces

were collected in 50 mL tubes. Tissue was washed with 15-20 mL PBS before centrifugation

at 330g for 5 minutes. The pelleted tissue was transferred to a new 50ml tube. Hanks

solution containing 0.1% w/v collagenase A (Roche Diagnostics GmbH, Mannheim,

Germany), 0.1% bovine serum albumin (BSA) (Sigma-Aldrich, Dorset, UK) 0.625 g.mL-1

amphotericin B, 100 I.U.mL-1 penicillin, and 100 g.mL-1 streptomycin was added to the

tissue and incubated at 37C for 30 min with periodical shaking to aid chemical

disaggregation. Digested tissues were centrifuged at 330g for 5 minutes. The floating

fractions consisting of adipocytes were discarded and the pellets representing the stromal

vascular fraction (SVF) were resuspended in 10% DMEM. Cells were centrifuged at 330g for

5 minutes, and the pellets re-suspended in 10% DMEM before seeding into one T25

flask. Cells were maintained at 37C and 5% CO2.

After 24 hours, non-adherent cells were discarded by removing the culture medium, and

washing with PBS. Regular visual inspections were undertaken to observe cell morphology

and exclude infection. During the culture period, growth medium was changed three times a

week. After one week, ADMSCs reached 80% - 90% confluence following which cells were

subcultured using 5 mL Trypsin/EDTA (Sigma-Aldrich, Dorset, UK) per T25. 1 x 105 cells

were seeded in each T75 flask, depending on the requirements. Cells between passages 4 and

7 were used in experiments

3.8 The effect of KGM and fractionated KGM on human fibroblasts.

2x104 fibroblasts or 5x104 keratinocyte co-cultured with i3T3 2x104 in 1 mL of 10 % FCS

containing cell medium were seeded into12 well plates respectively and incubated at 37oC,

10

5% CO2 for 24 h before adding 10 mg KGM powder or KGM of different molecular weight

fractions obtained by ethanol extraction, ultrafiltration, or enzyme hydrolysis. In all cases the

cells were then cultured for 1, 3, or 5 days, and measurement of cell viability was undertaken

using an MTT assay[12]. Samples were washed gently with PBS and 1.0 mL of MTT

solution was added per well. The plates were incubated with 0.5 mg.ml-1 MTT in PBS for 40

minutes at 37oC, 5% CO2 in a humidified atmosphere. The MTT solution was subsequently

removed and 600 l of acidified isopropanol (0.125 L 1M HCl in 100 mL of isopropanol)

was used to elute the formazan product from the cells. 200 l of the isopropanol was then

transferred into a 96 well plate and the optical density was read in a Dynex Technologies

MRXII microplate reader attached to a PC running Revelation 2.0 software at 540 nm and

referenced at 630 nm.

3.9 Use of Picogreen to assess the effect of KGM on cell proliferation

Quantification of total cellular DNA by Picogreen was conducted using the method of Ahn

et. al., 1996 [13]. All media was removed, and cells were washed twice with PBS. Then

200L of 10% digestion buffer was added and cells were frozen at -80oC and then thawed in

a dry incubator for three cycles to break the cell membranes and extract the DNA. The cells

were then scraped off and centrifuged at ~1700 g to collect the DNA from the supernatant.

100 L of the supernatant was added to 100 L of Picogreen (1:200) and mixed well. 100 L

of this solution was then transferred to a fluorescence plate reader (Biotex Instruments, Inc.,

USA) and samples excited at 340 nm with an emission wavelength at 488nm. A quantitative

estimation of the cell number was obtained by calibrating this reading against a known

number of cells using this method.

3.10 Cell viability assessed using Live/Dead assay

11

5x104 keratinocytes (co-cultured with 2x104 i3T3 in 1 mL of 10% Green) and 2x104

fibroblasts were cultured for 24 hr in 12 well plate respectively then KGM (1, 5, or 10 mg)

was added to the cultures. After 3 days, the medium was removed and the cells were washed

with PBS twice. 1 mL of SYTO9 (1 g.mL-1) and PI (1 g.mL-1) were added to each sample

and incubated for 1 hour in an incubator at 37C. The two-colour fluorescence assay was

observed using an Axon ImageXpress fluorescence microscope (Axoncorp, USA). The

excitation wavelengths were 480 nm for PI and 545 nm for SYTO9. The emission

wavelengths were 500 and 610 nm respectively.

3.11 Investigation of the effect of D-mannose and concanavalin A on the interaction of

KGM with fibroblasts

2x104 fibroblasts in 1 mL of 10% DMEM were seeded into 12 well plates and incubated at

37oC, 5% CO2 for 24h before adding an amount of D-mannose (1, 10, and 20 mg.mL-1) for

60 min. After removal of the medium containing D-mannose, 10 mg of KGM was added with

1 mL of fresh medium to each well. Cell viability was then measured after 1, 3, and 5 days

using an MTT assay.

In other experiments, 2x104 fibroblasts in 1 mL of 10% DMEM were seeded into 12 well

plates and incubated at 37oC, 5% CO2 for 24h then Con A (10, 50, and 100 g.mL-1) was

added for 30 min. After removal of the medium containing Con A, 10 mg of KGM was added

with 1 mL of fresh medium to each well. Cell viability was then measured after 1, 3 and 5

days using an MTT assay.

12

3.12 Blocking of MR on fibroblasts and keratinocytes by D-mannose

2x104 fibroblasts in 1 mL of 10% DMEM and 5x104 keratinocytes (co-cultured with 2x104

i3T3 in 1 mL of 10% Greens) were seeded into 12 well plates respectively and incubated at

37oC, 5% CO2 for 76hours before adding an amount of D-mannose (1, 10 and 20 mg.mL-1)

for 60 min. After removal of the medium containing D-mannose, the cells were then washed

gently twice with PBS. 1 mL of 0.1% Triton-X 100 in PBS was added for 30min at 4oC. The

cells were washed again with PBS. Then 1 mL of Con A-FITC (10 g.mL-1) and of DAPI (1

g.mL-1) in PBS was added to each culture and incubated for 60 min at 37oC. The unbound

Con A - FITC medium was washed twice with PBS and the cells were imaged using an Axon

ImageXpress fluorescence microscope. The excitation and emission wavelengths for DAPI

and Con AFITC were 340 and 488 nm and 545 and 610 nm respectively.

3.13 The effects of KGM (A. konjac Koch) on supporting keratinocyte, fibroblast and

ADMSC metabolic activity in unchanged media for twenty days.

2x104 fibroblasts and 2x104 ADMSC in 1 mL of 10% DMEM were seeded separately in 12

well plates and incubated at 37oC, 5% CO2 for 24h before adding 15 mg of KGM powder to

half of the wells. Then all cells were maintained for 20 days without changing the media.

For keratinocytes, 2x104 i3T3 with 5x104 keratinocytes in 1 mL of 10% Greens were

cultured in 12 well plates and incubated at 37oC, 5% CO2 for 48h before adding 1 mg of

KGM powder to half of the wells. Then all cells were maintained for 20 days without

changing the medium. Cell viabilities for the control and the KGM treated cells were

assessed after 1, 5, 10, and 20 days using an MTT assay.

3.14 Statistical Analysis

13

Quantitative data (e.g. MTT optical density readings or DNA values were analysed using

Minitab (MiniTab Inc. USA) and Microsoft Excel (Microsoft Corporation) to obtain means,

standard deviation (SD) and standard error (SE) from n= number of independent experiments

performed in triplicate. Students t-test was performed to determine whether the observed

differences between means were statistically significant. Where appropriate, results from

statistical analysis are indicated in the corresponding figure or table: ns (not significant;

p0.05), * (significant; p

14

Results

4.1 The effects of KGM from different species of Amorphophallus on fibroblast

proliferation-relationship to glucomannan content

The percentage of GM found in each plant extract and the Glu:Man ratio of Amorphophallus

KGM prepared from five different species are shown in Table 1. Non-modified KGM, A.

konjac Koch had the highest GM content with a 2:1 Glu:Man ratio and 97% of glucomannan

content followed by A. oncophyllus, A. paeoniifolius, A. prainii and A. elegans. Three of

these species, A. konjac Koch, A. oncophyllus and A. elegans have a higher mannose content

compared to glucose.

Figure 1 shows the effect of preparations of commercially available A.konjac Koch and

laboratory prepared preparations of A. oncophyllus, A. paeoniifolius, A. prainii and A. elegans

on fibroblast proliferation. The effects varied depending on the species but were clearly

concentration dependent. From Figure 1, A. konjac Koch, A. oncophyllus and A.

paeoniifolius, with 50% or more percentage of GM stimulated fibroblast proliferation by days

3 and 5 while A. prainii and A. elegans inhibited fibroblast proliferation. 10 mg.mL-1 A.

konjac Koch had the highest stimulation on fibroblast proliferation at day 5 compared to

similar concentrations of A. oncophyllus and A. paeoniifolius. In contrast, A. prainii and A.

elegans inhibited fibroblast proliferation at all concentrations by days 3 and 5 compared to

control cells.

4.2 The effect of KGM from different species on fibroblasts is dependent on serum.

We next examined whether the mitogenic effect of KGM on fibroblast proliferation required

the presence of FCS. This was examined by varying serum concentration from 0 to 10% in

15

cell culture medium and adding 10 mg.mL-1 of KGM. Cell proliferation was measured

indirectly using an MTT assay (which measures metabolic activity) after 5 days. As expected

cell proliferation was extremely low in 0% FCS but addition of KGM (A. konjac Koch) and A.

oncophyllus stimulated fibroblast proliferation by a factor of 5 fold. In 2% FCS, fibroblast

proliferation was increased to 17 fold and 15 fold and addition of 10% FCS increased

proliferation to 35 and 30 fold by addition of KGM (A. konjac Koch) and A. oncophyllus

respectively when compared to control with 0% FCS .

Figure 2A shows the effect of the KGM extract in varying serum concentrations of 0, 2, and

10% in cell culture medium. In 0% FCS, KGM A. konjac Koch and A. oncophyllus

significantly increased proliferation when compared to controls. The other 3 KGM extracts

did not increase proliferation. Similarly fibroblast proliferation was also increased when cells

were cultured in both 2% and 10% FCS with KGM A. konjac Koch and A. oncophyllus while

there was no significant response to KGM prepared from A. paeonifolius and A. prainii. For

KGM prepared from A.elegans this had no significant effect on fibroblasts when cultured in

2% FCS but significantly inhibited these cells when cultured in 10%FCS.

4.3 The effect of KGM on the proliferation of fibroblasts

Fibroblast proliferation was then examined using a PicoGreen assay for total cellular DNA.

This confirmed that (in parallel to an increase in metabolic activity measured with MTT) cell

number for cells cultured with 10% FCS increased as KGM (A. konjac Koch) concentration

increased (Figure 2B). A significant increase was seen with 5 and 10 mg.mL-1 KGM. After 5

days, the cell number increased more than two fold compared to control cells.

16

4.4 The effect of KGM on keratinocytes

KGM (from A. konjac Koch) inhibited keratinocyte metabolic activity as assessed by MTT

and this was clearly evident after 3 days. Figure 3 shows that there was a significant drop in

viability after 3 days with 5 and 10 mg.mL-1 KGM compared to controls. After 5 days of

incubation the decrease in cell viability with 10 mg.mL-1 KGM was more marked, evidenced

with the presence of a larger population of dead cells compared to both control and the lower

doses of KGM (Figure 3B). (Live/Dead staining for fibroblasts shown that KGM did not

reduce viability as shown in the graphical abstract.)

4.5 KGM supports fibroblast and ADMSC but not keratinocyte viability in unchanged

media for up to 20 days.

KGM (from A. konjac Koch) was then examined for its ability to support skin cells and also

ADMSC in unchanged media for up to 20 days. Normally one would change the media of

rapidly proliferating or metabolically active cells every 3 days or so. ADMSC were included

in this comparative study of the ability of KGM to support metabolic activity of cells because

of prior literature suggesting this and to see if the effects were similar in a range of cells or

not.

The addition of 15 mg.mL-1 native KGM enabled fibroblasts to maintain a high level of

viability throughout 20 days of culture (Figure 4A). During this period these cells were

deliberately starved of new media. As can be seen the control cells showed much reduced

metabolic activity during this period-after 20 days in culture the metabolic activity of cells

with KGM was 5 fold greater than control cells.

17

Figure 4B was conducted by allowing fibroblasts to achieve confluence prior to addition of

15mg.mL-1 KGM for a further period of 11days of culture. As for Figure 4A, fibroblasts in

the presence of KGM showed a significantly greater (two fold) metabolic activity throughout

this period compared to control cells, which maintained constant metabolic activity from day

9 to 20.

Figure 4C shows that in contrast KGM did not help sustain metabolic activity of

keratinocytes cultured in the same media for 3 weeks but Figure 4D shows that it did

significantly support ADMSC in culture without media changes for 3 weeks.

Subsequent studies now focussed on trying to understand the mechanism of KGMs

interaction with fibroblasts and keratinocytes.

4.6 The effect of different MW extracts of KGM (A. konjac Koch) on fibroblast

proliferation.

Ethanol, ultrafiltration and enzymatic treatment were used to fractionate the KGM extracts

and the relationship between molecular weight and biological activity of the extracts on

fibroblast proliferation were examined. The results in Figure 5 show highly significant

stimulation with the non-modified KGM compared to extracts obtained by (b) ultrafiltration,

(c) ethanol and (d) -mannanase treated KGM. Ultrafiltration with a 30,000 g.mol-1 cut off

filter provided a high MW component (HMW) which retained some stimulatory activity on

fibroblast proliferation after 3 and 5 days when compared to non-modified KGM (compared

to controls in the absence of any KGM). Extraction of LMW KGM using ultrafiltration

produced an extract which did not significantly affect the rate of proliferation when compared

to control cells after 5 days as shown in Figure 5B.

18

Treatment with ethanol (see Figure 5C) reduced KGM biological activity after 3 and 5 days

by approximately 30% compared to non-modified KGM. The subsequent extraction of an

HMW KGM component using ethanol showed retention of some stimulatory activity, which

was evident after 3 and 5 days. LMW KGM components after ethanol extraction had no

significant effect on fibroblast proliferation after 3 days, but was shown to inhibit fibroblast

proliferation by -25% after 5 days.

Figure 5D summarises the effect of a range of concentration of -mannanase hydrolysed

KGM at room temperature and 4oC. There was no difference in the biological activity of 0.1

U.mL-1 -mannanase hydrolysed KGM at room temperature, or at 4oC for 10 minutes with no

loss of stimulatory effect on fibroblast proliferation. Heat inactivation used to inactivate -

mannanase also did not affect KGM stimulation of fibroblast proliferation. However, a

complete loss of all ability to stimulate fibroblast proliferation was seen with KGM

hydrolysed at 10 and 100 U.mL-1 of this enzyme.

Figure 6 shows the relationship between the molecular weight of the KGM extracts and the

effect of these extracts on fibroblast and keratinocyte proliferation assessed after 5 days. The

effect of KGM on both fibroblasts and keratinocytes was clearly dependent on the molecular

weight of the fractionated material it was evident that the native KGM which contained a

high molecular weight component of around 1x106 g.mol-1 stimulated fibroblast proliferation

(by + 240%) but was inhibitory to keratinocytes proliferation (-60%). With HMW

components of approximately 5x105 g.mol-1 there was some stimulatory activity on

fibroblast proliferation (+ 144%) and still -50%.inhibition of keratinocyte proliferation.

Once the molecular weight of the fractions dropped beneath 100,000 g.mol-1 there was no

evidence of any stimulatory activity on fibroblasts. With LMW components of less than

1x103 g.mol-1 then inhibition of both fibroblasts and keratinocytes was seen .

19

4.7 Blocking of MR on skin cells by D-mannose

The manner of KGM interaction with fibroblasts and keratinocytes was next examined by

attempting to block the MR on these cells using D-mannose and assessing the effect of this

on KGM activity. Figure 7 shows the addition of increasing concentrations of D-mannose

from 1 to 20 mg.mL-1. It is well-known that Con A binds to mannose residues on

glycosylated cell surface proteins and other reports show that this lectin also binds to

glucomannan residues [14]. In figure 7 we show that Con A bound to cell surface glycan

features on the fibroblasts used in this study but this binding was then blocked by adding

mannose, which competitively blocked the binding of Con A to the fibroblasts at

concentrations > 10 mg.ml-1 . The technique also provides a semi-quantitative assessment of

the MR density and for keratinocytes, figure 7 shows that it was necessary to go up to 40

mg.mL-1 of D-mannose to block the attachment of Con A. Thus, these data suggest that one

of the differences between these two cell types is that keratinocytes have a higher density of

MR (a larger fraction of the added mannose binds to MR on keratinocytes so a higher

concentration is required to block the binding of Con A) and we hypothesise that this feature

may relate to the different behaviour of the cells in the presence of KGM.

The effect of MR blocking by D-mannose on fibroblast proliferation was then assessed with

the MTT assay (Figure 8A). The blocking of MR did not significantly affect the rate of

proliferation nor did it adversely affect native KGM stimulation of fibroblast proliferation.

We then assessed the effect of Con A on fibroblast proliferation and on the biological activity

of KGM (Figure 8C-D). Adding Con A at 10-100 g.mL-1 did not affect fibroblast

proliferation but Con A at 50-100 g.mL-1 significantly inhibited the stimulatory effect of

KGM on fibroblast proliferation - the biological activity of KGM on fibroblasts was reduced

by more than 50% by 10 g.mL-1 Con A. Thus, the MR do not appear to be involved in the

20

action of KGM on fibroblasts but providing another lectin (Con A) does reduce the action

KGM, presumably because binding of KGM to Con A prevents its binding to another

alternative lectin on the cell surface.

21

DISCUSSION

The aim of the study was to evaluate the biological effects of KGM on skin cells. KGM is a

linear polysaccharide that composed of (1-4)--D-Glc and (1-4)--D-Man and reportedly to

have presence of few short side chains which may contain galactose residues and exhibit

some degree of acetylation which depends on the plant species [15, 16]. We used KGM of 5

different species and found that 3 out of the 5 KGM samples (which each had 50% or more of

glucomannan content) significantly stimulated fibroblast and paradoxically inhibited

keratinocyte viability. Specifically A. konjac Koch, A. oncophyllus and A. paeoniifolius were

stimulatory while A. prainii and A. elegans was inhibitory to the proliferation of fibroblasts.

We found that the KGM with the higher mannose to glucose ratios were biologically active in

stimulating proliferation of fibroblasts. The stimulatory effect of KGM could be seen in the

absence of FCS but was most evident in culture media containing foetal calf serum (a rich

source of platelet mitogens). The combined effects of the two were roughly additive. This

may be explained by a report showing that the expression of MR increases (about two fold)

by the presence of 10% FCS when compared to 1% FCS [17].

On balance our results suggest that plant extracts with a high proportion of glucomannan are

capable of stimulating fibroblast metabolic activity and proliferation. In contrast, keratinocyte

viability was reduced with KGM (Figure 3). In the right panel of Figure 3, keratinocyte

viability was the same for cultures with 5 and 10 mg.mL-1 however, Live/Dead staining

showed a larger population of homogenous dead cells in 10 mg.mL-1 compared to 5 mg.mL-1

KGM. It was not clear how the KGM reduced the cell viability but not killing the cell but

this could suggest the decrease in keratinocyte viability before death phase.

22

The stimulatory and inhibitory effects on cell proliferation were clearly related to the

molecular weight of the KGM extracts. KGM extracts having high molecular weight

fractions (>100,000) stimulated fibroblast and inhibited keratinocyte proliferation. Our

results were coherent with the relationship of Aloe vera molecular weight fractions and

higher mannose content on the stimulation of murine T-cell proliferation [18]. We suggest

that structure-activity relationship of KGM was found to be similar to (1-3)--glucan, where

molecular weight >550,000 showed the highest immunopotentiating activity, while fractions

(from the same source) with molecular weights of 5000-10000 showed no activity

irrespective of the chemical structure [19-22]. However, anti-tumour activity was found in

(1-3)--glucan with degree of branching (DB)

23

competitor to cell bound MR [29]. Con A is a plant lectin that binds to glycosylated surfaces

on cell membranes and mediates numerous interactions with a range of carbohydrate

configurations [30-32]. The blocking of MR in fibroblasts by mannose at 1-20 mg did not

affect fibroblast proliferation cultured with and without KGM. In contrast, the addition of

Con A to fibroblast cultures with KGM significantly inhibited KGM stimulation on

proliferation suggesting the involvement of other lectins (not MR) are responsible for these

effects. The molecular mode of action remains unclear but it is likely to relate to KGM

interacting with membrane bound lectins as suggested by Con A blocking KGMs actions.

Our results do not allow us to identify with any confidence the exact mechanism of how high

molecular weight KGM extracts interact with the cell surface of fibroblasts or keratinocytes

in the case of fibroblasts the evidence does not support them interacting with mannose

receptors, whereas in the case of keratinocytes the data suggests that KGM may stimulate

mannose receptors and our results indicate that these are at higher density on these cells

compare to fibroblasts.

Irrespective of the mechanism by which KGM interacts with these skin cells, the results on

fibroblasts were sufficiently dramatic that they merit further exploration in a 3D wound

healing model and this is on-going. There are some important points to note about the

mechanism of the KGM action on fibroblasts. The stimulatory activity we saw was much

greater than that seen in the presence of 10% foetal calf serum. Thus, KGM further

stimulated metabolic activity and proliferation beyond that seen in the presence of a

saturating concentration of platelet mitogens (as would be found in 10% foetal calf serum).

There are many conditions when one would wish to support cells in less than ideal conditions,

such as transporting cells across countries and to deliver these cells into patients without

damaging cell viability. The basis that mannose rich lectins can help to preserve human cord

24

blood progenitor cells in suspension culture for up to one month without media change [8]

and stem cells up to 2 weeks in culture [9] by specifically reacting with Flt3+ (a tyrosine

kinase receptor central to regulation of stem cells) by preventing their proliferation and

differentiation [33] were the main motivation behind the study on KGMs ability to support

skin cells and ADMSC in nutrient deprived condition. Besides, in very recently alginate

encapsulation had been use for the short term storage of stem cells for use in cell therapy [34].

In this study, we shown that the presence of KGM enabled fibroblasts and ADMSC (but not

keratinocytes), to maintain a high level of metabolic activity in starved media conditions. In

the absence of KGM the metabolic activity of all three cell types clearly decreased. This may

be due to a lack of glutamine (which is normally added fresh to media prior to addition to

cells) and / or to a lack of cell mitogens. A KGM dose of 15 mg.mL-1 was chosen for

prolonged culture of fibroblasts and ADMSC since 10 mg.mL-1 stimulated proliferation for

up to 5 days, and that it was thought that the addition of 15 mg.ml-1 would be sufficient to

sustain cell viability in 20 days of unchanged medium. As for keratinocytes, we had

previously shown that 5-10 mg.mL-1 KGM inhibited proliferation, and resulted in cell death,

which the lower concentration of 1 mg.mL-1 did not. For this reason we chose 1 mg.mL-1

KGM to mimic mannose rich lectin biological effect on stem cells to suspend keratinocyte

proliferation and differentiation that would be useful for cell preservation in long term culture.

In conclusion we report that plant derived KGM has significant stimulatory effects on

fibroblasts (and ADMSC cells which in this study were studied only under conditions of

media starvation) but not keratinocytes. It now remains to be established how these effects

might influence wound healing, and also the transport of cells.

25

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Table 1. Comparison of the mannose and glucose content of KGM extracted

from five different species of Amorphophallus.

Species Man:Glc ratio GM(%)

A. konjac Koch 2.2:1 97

A. oncophyllus 2.5:1 57.28

A. paeoniifolius 0.8:1 49.82

A. prainii 0.18:1 29.86

A. elegans 235:1 16.78

Figure 1. Comparison of the effect of KGM extracted from five

different species of Amorphophallus on fibroblast metabolic activity.

2x104 fibroblasts were cultured in 1 mL of 10% DMEM in 12 well

plate for 24 hours. Cells were then cultured in medium with 10%

FCS supplemented with various concentrations of KGM for 1, 3, and

5 days (A) A.konjac Koch, (B) A.oncophyllus, (C) A.paeoniifolius,

(D) A.praiini and (E) A.elegans. Cell viability was assessed using

MTT assay. Results shown are means+SD, n=2 expts ,each expt

with 3 replicates ***p

Figure 2 (A). Investigation of the serum dependency of the stimulatory

effect of KGM on fibroblast metabolic activity. 2x104 fibroblasts were

cultured in 1 mL of cell medium containing 0, 2 and 10% FCS respectively

for 5 days with 10 mg.mL-1 extracted from 5 species, A.konjac Koch,

A.oncophyllus, A.paeonifolius, A.prainii and A.elegans. Cell viability was

measured with the MTT assay (n=2 expts each with 3 replicates). (B) The

effect of KGM (A.konjac Koch) on proliferation of fibroblasts. 2x104

fibroblasts were cultured in 1 mL of DMEM with 10% FCS

supplemented with various concentrations of KGM for 1, 3, and 5 days.

DNA content was measured with PicoGreen, and calibrated to give cell

number. The results were compared to control cells without KGM.

Results shown are mean+SD, n=2 expts each with 3 replicates ***p

A B

C D

Figure 3. Effect of KGM (A.konjac Koch) on human keratinocyte metabolic

activity. 2x104 keratinocytes were co-cultured with 2x104 i3T3 for two days

in 1 mL of 10% Greens medium, then treated with KGM for 1, 3, and 5

days. Assessment of keratinocyte proliferation was conducted by MTT

assay. Results shown are mean+SD, n=3expts each with 3 replicates,

***P

Figure 4. Investigation of the ability of KGM (A. konjac Koch) to support

the metabolic activity of (A-B) fibroblasts, (C) keratinocytes and (D)

ADMSC under conditions of media starvation. 2 x 104 fibroblasts, 5 x 104

keratinocytes were co-cultured with 2 x 104 i3T3 and 2 x 104 ADMSC

were seeded in 1 mL of culture medium with 10% FCS respectively for 24

hr. Then the media was removed and fresh medium with (A,B and D) 15

and (C) 1 mg.mL-1 KGM was added and then not changed for 20 days.

Metabolic activity was assessed at 1, 3, 5, 9 and 20 days via MTT assay.

(B) Investigation of ability of KGM to increase fibroblast metabolic

activity, not proliferation. 2 x 104 fibroblasts were seeded in 1 mL of

DMEM media with 10% FCS and left until they reached confluency at day

9. Then, at day 9, 15 mg.mL-1 KGM was added to the media and cell

viability was assessed at days 12, 15 and 20 using the MTT assay. control, - fibroblasts + 15 mg.mL-1 KGM (A. Konjac Koch) except for keratinocytes 1 mg.mL-1 . Results shown are means+SD, A,C and D) n=3

expts and B) n=1 expt ( each with triplicates) ***p

Figure 5. The effect of extracts of KGM (A. konjac Koch) on fibroblast

proliferation. 2 x 104 fibroblasts were seeded in 1 mL of DMEM with 10%

FCS containing 10 mg.mL-1 KGM extracts. Metabolic activity was measured

after 1, 3 and 5 days of incubation using the MTT assay. (A) non-modified

KGM extract, (B) high molecular weight and low molecular weight ultra-

filtration extracts, (C) ethanol extracted KGM further divided into high

molecular weight and low molecular weight extracts and a combined extract

of low molecular weight and high molecular weight KGM. (D) shows the

effects of enzyme treatment with -mannanase at a range of concentrations

and temperatures on KGM activity. Results shown are mean+SD, n=5 expts

( each with triplicates) ***p

Figure 6. The relationship between the distribution of KGM (A.konjac Koch)

molecular weight and the ability to stimulate fibroblast metabolic activity at day

5 as measured with the MTT assay. 2x104 fibroblasts were cultured in 1 mL of

DMEM media with 10% FCS for 1 day and 2x104 keratinocytes were co-

cultured with 2x104 i3T3 for two days in 1 mL of Greens medium. Then, 10

mg.mL-1 of KGM extract was added into the medium. Cells from passage 5-9

were used.

10 100 1k 10k 100k 1M 10M

-25

n.a

-50

-60

Effect on

keratinocytes

(%)

g/mol

-40

10

144

240

Ethanol

extracted

low MW

KGM

1 U.mL-1

B-mannanase

hydrolyzed

KGM

Ethanol

extracted

high MW

KGM

Effect on

fibroblasts

(%)

Molecular weight

KGM

Figure 7. Binding of Con A-FITC (green) to fibroblasts and keratinocytes

in the presence of D-mannose. Cell nuclei are labelled with DAPI (blue).

(A-D) 2x104 fibroblasts (seeded in 1mL of 10% DMEM) or (E-H) 5x104

keratinocytes (co-cultured with 2 x 104 i3T3 in 1 mL of Greens media)

were cultured for 3 days then an amount of D-mannose was added to the

culturse. After an hour of blocking the MR, the unbound mannose was

washed and Con A-FITC was added to the culture. Con A shares the same

receptor with D-mannose. Photographs show dose dependent inhibition of

Con A binding to fibroblasts. In keratinocytes, this inhibition was only

seen at 40 mg.mL-1. Scale bar: 100 m.

D-mannose

C) A) B) D)

G) E) F) H)

CONTROL 1 mg.ml-1 10 mg.ml-1 20 mg.ml-1

Ke

rati

no

cyte

s

Fib

rob

lasts

40 mg.ml-1

I)

0 1 2 3 4 5 60.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 1 2 3 4 5 60.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0.0

0.2

0.4

0.6

0.8

1.0

Co

ntr

ol

20

mg

.ml-

1

Ma

nn

ose

10

mg

.ml-

1

Ma

nn

ose

1 m

g.m

l-1

Ma

nn

ose

KG

M

KG

M +

20

mg

.ml-

1

Ma

nn

ose

KG

M +

1 m

g.m

l-1

Ma

nn

ose

KG

M +

10

mg

.ml-

1

Ma

nn

ose

MT

T a

bso

rb

an

ce a

t 5

40

nm

***

***

***

**

***

Control

10g.mL-1 Con A

50g.mL-1Con A

100g.mL-1 Con A

D)

A)

Time (Days)

B)

***

Control

KGM

KGM + 10g.mL-1 Con A

KGM + 50g.mL-1Con A

KGM + 100g.mL-1Con A

C)

Time (Days)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

KG

M +

10

mg

.ml-

1

Ma

nn

ose

KG

M +

5 m

g.m

l-1

Ma

nn

ose

KG

M +

1 m

g.m

l-1

Ma

nn

ose

KG

M

10

mg

.ml-

1

Ma

nn

ose

5 m

g.m

l-1

Ma

nn

ose

1 m

g.m

l-1

Ma

nn

ose

Co

ntr

ol

MT

T a

bso

rb

an

ce a

t 5

40

nm

******

Figure 8. (A) Effect of mannose on fibroblast metabolic and KGM stimulated

fibroblast metabolic activity at day 3 measured by the MTT assay. (B) Effect of

mannose on keratinocyte metabolic activity and on KGM stimulated metabolic

activity measured by the MTT assay. 5x104 keratinocytes were co-cultured with

i3T3 in 1 mL of Greens medium in a 12 well plate before being supplemented

with medium containing mannose or mannose with an extract of KGM (from

A.konjac Koch) at 10 mg.mL-1 for five days. In A, C, and D 2 x 104 fibroblasts

were cultured in 1 mL of DMEM medium with 10% FCS in a 12 well plate

before being supplemented with medium containing mannose or mannose with

an extract of KGM (from A.konjac Koch) at 10 mg.mL-1. (C) Effect of Con A

on fibroblast metabolic activity and (D) inhibition of the effect of KGM on

fibroblast metabolic activity by Con A after 1, 3, and 5 of culture.

Results shown are mean+SD, n=3 expts ,each with triplicate cultures,

***p

Glucomannan

Ker

atin

ocy

tes

F

ibro

bla

sts

Con A FITC stained

mannose receptors and

lectins on cell surface.

Live/Dead staining

showing the effect of

KGM. (Scale 200m)

Amorphophallus

konjac

27

Highlights

Species of Amorphophallus with at least 50% GM content stimulated fibroblasts and inhibited keratinocytes

The biological activity of KGM on fibroblasts is dependent on high molecular weight components

KGM maintained fibroblast and adipose derived stem cell metabolic activity under media starvation conditions