World Anti-Cancer Congress and Exhibition, 11 – 12 January 2014, Universiti Sains Malaysia, Pulau Pinang

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Anti-Proliferative Activity of Natural Rubber Latex Sera on

Human Cancer Cell Lines

E. Sunderasan1#

, Lam K.L.2, Yang K.L.

1,2, Norazreen Abd. Rahman

1, Ong M.T

2#

Natural rubber latex extracts are becoming increasingly visible in pharmaceutical and

therapeutical research. Anti-proliferative activity against a number of human cancer-origin cell

lines has been detected in latex B- and C-serum. The present report describes fractionation of

latex B- and C-serum using cell viability assay as a guide to narrow down on the active

constituents. Dialysis of B- and C-serum against distilled water caused some of its constituents

to precipitate, then a brief centrifugation was employed to separate their fractions – dialysed B-

serum precipitate (DBP), dialysed B-serum supernatant (DBS), dialysed C-serum precipitate

(DCP), and dialysed C-serum supernatant (DCS). Boiling of B- and C-serum resulted in

extensive precipitation, and then a brief centrifugation separated the boiled B- and C-serum

precipitate from their supernatant. Anti-proliferative activity markedly increased in dialysed B-

serum precipitate and dialysed C-serum fractions but diminished in the dialysed B-serum

supernatant and boiled B- and C-serum fractions. Among the active fractions, DCS revealed the

highest anti-proliferative activity, particularly against HepG2 - hepatocellular adenocarcinoma

cells. Molecular and cellular analyses indicated that dialysed C-serum fractions induce

autoschizic type of death to HepG2 cells.

Key words: Latex C-serum, dialysis, cell viability assay, cancer cell lines, cancer cell death

1Rubber Research Institute of Malaysia, Malaysian Rubber Board, P.O. Box 10150, K. Lumpur, Malaysia 2Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Minden, P. Pinang, Malaysia # Correspondence: sunderasan@lgm.gov.my; omt@usm.my

Natural rubber latex is a milky white sticky emulsion (cytoplasm) that exudes upon wounding of

articulated laticiferous canals in the soft bark of Hevea brasiliensis tree. Fresh latex, after tap-

ping from the tree, may be divided into three main fractions by high-speed centrifugation; these

are a white upper layer, an aqueous phase (C-serum) and the so-called 'bottom fraction'1,2

. The

top layer comprises rubber micro-particles stabilised by an adsorbed layer of protein and

phospholipids. The C-serum contains most of the soluble substances normally found in plant

cells, including amino acids, proteins, carbohydrates, organic acids, inorganic salts and

nucleotidic materials. The 'bottom fraction' consists largely of lutoid particles but also contains

varying amounts of other organelles or particulate components of normal plant cells having a

density greater than that of the C-serum3. The fluid content of the 'bottom fraction' is called B-

serum4. So far, investigations on natural rubber latex sera (B- and C- serum) and its constituents

have revealed multiple biological activities such as anti-fungal, anti-inflammatory, anti-oxidant,

World Anti-Cancer Congress and Exhibition, 11 – 12 January 2014, Universiti Sains Malaysia, Pulau Pinang

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and lipolytic properties5–13

. Potential anti-cancer therapeutic application of natural rubber latex

was first suggested when B- and C-serum were shown to exert specific anti-proliferative activity

against a malignant cell line14

. This is encouraging as latex sera is a relatively low-cost resource

particularly in the rubber-producing regions vis-à-vis its potential use as an anti-cancer

therapeutic.

The present study is aimed at investigating anti-proliferative activity of natural rubber latex B-

and C-serum, dialysed B- and C-serum fractions, and boiled B- and C-serum fractions. An array

of human cancer-origin adherent cell lines was employed in the initial tests; subsequent cell

viability assay were focused on hepatocellular adenocarcinoma (HepG2) and foreskin fibroblast

(Hs27) - a non-cancer-origin cell line that served as control. An attempt was also made to

delineate the molecular mechanism that incapacitates HepG2, the cancer cell line of interest.

MATERIALS AND METHODS

Preparation of latex B- and C-serum

Latex was collected from field-grown RRIM 600 trees at the Rubber Research Institute of

Malaysia Research Station, Sungai Buloh. To prepare latex B- and C-serum, fresh latex

collected in chilled flasks was fractionated by centrifugation at 44,000 × g for 2 hours, at 4ºC.

The latex separates into three distinct parts on high-speed centrifugation, as shown in figure 1.

To prepare whole C-serum (WC), the upper layer (rubber cream), was carefully removed and

WC was prepared from the remaining supernatant, based on a method previously described1,2

.

Latex B-serum was prepared from the bottom fraction of the centrifuged latex based on a method

previously described15

. Briefly, after removal of the rubber cream and C-serum, the sediment at

the bottom of the centrifuge tube was collected and re-suspended in 0.4 M mannitol to aid the

removal of remnant C-serum while retaining the lutoid particles intact. The cleansed bottom

fraction was recovered after another centrifugation and subjected to alternate freezing and

thawing (four times) to rupture the lutoid particles. The clear brownish fluid from the lutoid

particles, the whole B-serum (WB) was recovered by centrifugation. Both WB and WC were and

lyophilized until completion and kept in a desiccator for subsequent use.

Figure 1. Fractionation of fresh latex by high speed centrifugation. The positions of rubber

cream, C-serum and bottom fraction are marked.

Rubber cream

C-serum

Bottom fraction

World Anti-Cancer Congress and Exhibition, 11 – 12 January 2014, Universiti Sains Malaysia, Pulau Pinang

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Preparation of dialysed B- and C-serum fractions

A portion of the lyophilized WB and WC were reconstituted with 1 × phosphate-buffered saline

(PBS; AMRESCO, Solon, OH, USA). Serial dilutions of WB and WC were performed to

prepare working concentrations ranging from 2 to 2,000 µg/ml.

The subsequent fractions of WB and WC were prepared by dialysis using SnakeSkin™ (Pierce,

IL, USA) tubing with molecular weight cut-off 3,000 Da, against distilled water for 48 hours at

about 5°C. A whitish precipitate was recovered from dialysed B and C-serum by centrifugation

at 20,000 × g for 30 minutes; the precipitates (DBP and DCP) and the supernatants (DBS and

DCS) were then lyophilized and kept desiccated until further use. Working solutions for

dialysed fractions were prepared as described above.

Preparation of boiled B- and C-serum fractions

WB and WC were placed in a boiling water bath for 10 minutes. A centrifugation procedure at

20,000 × g for 30 minutes was employed to separate the boiled B- and C-serum fractions. The

recovered boiled B-serum precipitate (BBP), boiled B-serum supernatant (BBS), boiled C-serum

precipitate (BCP) and boiled C-serum supernatant (BCS) fractions, were then lyophilized until

completion. Reconstitution and working solutions of the lyophilized BBP, BBS, BCP and BCS

were as described above for WB and WC.

Cell culture

Cell line Hs27 (CRL-1634) – of human foreskin fibroblasts, that is not of malignant origin,

HepG2 (CRL-11997) – hepatocellular adenocarcinoma, MCF7 (HTB-22) and MDA MB231

(HTB26) – both breast cancer-origin cell lines, HeLa (CCL-2) – cervical cancer-origin cell line,

Caov-3 (CCL-247) – ovarian cancer-origin cell line, and HCT116 (CCL-247) – colorectal

adenocarcinoma cell line, were sourced from the American Type Culture Collection (Rockville,

MD, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco®

;

Invitrogen, Carlsbad, CA, USA) supplemented with foetal calf serum (10% v/v), penicillin (100

units/ml), streptomycin (100 µg/ml) and amphotericin B (0.025 µg/ml) (Gibco®

; Invitrogen);

Hs27 cells were supplemented with additional 4.5 g/l glucose. Cultures were maintained at 37°C

in a water-saturated atmosphere containing 5% CO2. Cell counts were performed using a

Neubauer haemocytometer under light microscope and living cells were identified by Trypan

blue (Gibco®

; Invitrogen) staining method. Approximately 2,500 cells were seeded in each well

of a 96-well culture plate and were starved in DMEM under cell culture incubation conditions

for 48 h prior to cell viability assay.

Measurement of cell proliferation inhibition

Cytotoxic effects were measured using standard 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl

tetrazolium bromide (MTT) assay (Sigma Chemical Company, St. Louis, MO, USA) after cell

treatment with WC or its fractions, for a pre-determined time points i.e. 24, 48 and 72 h; the

assay was developed based on the method described by Mossman16

. Two thousand five hundred

cells were seeded into 96-well plates and cultured in a CO2 incubator at 37°C. After 48 h, cells

were treated with serially-diluted concentrations of WC, DCP, DCS, BCP, and BCS, and kept

again in the CO2 incubator at 37°C. Cell morphology was observed using reverse phase-contrast

microscopy (ApoTome; Carl Zeiss MicroImaging GmbH, Jena, Germany). Absorbance (OD) at

570 nm was read using a spectrophotometric plate reader (Multiskan spectrum; Thermo Electron

Co., Waltham, MA, USA) and proportions of surviving cells were calculated as:

World Anti-Cancer Congress and Exhibition, 11 – 12 January 2014, Universiti Sains Malaysia, Pulau Pinang

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OD of drug-treated sample - OD of blank × 100%

OD of control - OD of blank

Dose–response curves were constructed using Probit analysis17

to obtain LC50 and LC80 values.

All experimental data were derived from at least three independent experiments.

Real-time quantitative RT-PCR analysis

Expression levels of Bax, Bcl-2 and caspase-3 mRNA were analysed using the real-time

quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technique. Experimental

cells were treated with concentrations 0 and LC50 of WC, DCS and DCP. Cells were then

harvested at 48 h and total RNA was isolated using TriPure isolation reagent according to the

manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany). Integrity and purity of

RNA were spectrophotometrically verified by measuring absorption ratio, OD 260 nm/OD 280

nm. Complementary DNA (cDNA) was synthesized with 1 µg total RNA using Transcriptor

First Strand cDNA Synthesis Kit according to the manufacturer’s instructions (Roche

Diagnostics). The reaction was performed at 25°C for 10 min, followed by 50°C for 60 min,

heated to 95°C for 5 min and chilled at 4°C using a MyCycler Thermal Cycler System (Bio-Rad

Laboratories, Hercules, CA, USA). cDNA stock was maintained at 20 ng/µl concentration and -

20°C until further use. Primer sequences for amplification of Bcl-2, Bax and caspase-3 genes by

real-time PCR were designed and synthesized by Primer Design Ltd. (Hampshire, UK). Primer

sequence for GADPH gene was designed using Primer 3 Input (version 0.4.0), and synthesized at

1st BASE Pte. Ltd. (Singapore).

Quantification of gene expression was performed using an ABI Prism 7700 Sequence Detector

(Perkin-Elmer Applied Biosystems, Foster, CA, USA). PCR solution (20 µl) was composed of

1× FastStart Universal SYBR Green Master (ROX) (Roche Diagnostics) containing 2.5 µl

cDNA of treated and untreated samples, and 0.3 µM of each primer (Table 1). Thermal cycle

parameters were the following: 1 cycle at 95°C for 10 min followed by 40 cycles at 95°C for 15

s, 60°C for 30 s, and 72°C for 15 s. In addition, real-time reaction of the products was checked

by melting point analysis, after each reaction. Standard curves for Bax, Bcl-2, caspase-3 and

GADPH were generated using serial dilution of cDNA derived from the cell lines. GADPH was

monitored as reference gene and Bcl-2, Bax and caspase-3 expression levels were normalized

with respect to GADPH transcript and calculated by the 2-ΔΔCt

method18

. A two-sided P-value

lower than 0.05 was considered to be statistically significant.

Table 1. The primers used for the amplification of the respective genes in the real-time

quantitative RT-PCR Gene Primer sequence PCR product

size (bp)

Bax Forward: 5′-ATG GAG CTG CAG AGG ATG AT-3′ Reverse: 5′-CAG TTG AAG TTG CCG TCA GA-3′

101

Bcl-2 Forward: 5′-GAG GTC ACG GGG GCT AAT T-3′ Reverse: 5′-GAG GCT GGG CAC ATT TAC TG-3′

88

Caspase-3 Forward: 5′-TGT AGA AAT GAT GAT GTG GAA GAA C-3′ Reverse: 5′-GCA GTT AAG TCA TCC GTG TAT ATC-3′

98

World Anti-Cancer Congress and Exhibition, 11 – 12 January 2014, Universiti Sains Malaysia, Pulau Pinang

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GADPH Forward: 5′-GAG TCA ACG GAT TTG GTC GT-3’

Reverse: 5′-TTG ATT TTG GAG GGA TCT CG-3′ 234

Fluorescence and transmitted light microscopy

Approximately 50,000 cells/well were seeded in a 24-well culture plate under nutrient starvation

condition in DMEM and placed in a water saturated CO2 incubator at 37°C. After 48 hours, the

cells were treated with LC50 concentrations of DCS and DCP and returned to the CO2 incubator

at 37 °C for another 48 hours. Cell without undergoing latex subfraction treatment were used as

control in this experiment. Cell fixation was conducted using 4% ice-cold paraformaldehyde for

20 min at 4 °C. Treated and untreated cells were rinsed with 0.01M PBS and incubated with

Hoechst 33258 (0.5μg/ml) staining for 45 min in the dark at room temperature. Cells were rinsed

with 0.01M PBS again and treated with RNase A (10µg/ml) for 30 min to remove cytoplasmic

RNA. Annexin V/ propidium iodide (PI) staining was performed using an Annexin-V-FLUOS

staining kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s

protocol after rinsing with 0.01M PBS. Treated and untreated cells were incubated with

Annexin V/PI (1 µg/ml) mix for 15 min in the dark at room temperature. Cells were observed

and results were analyzed using an ApoTome Fluorescence Microscope equipped with an

AxioCam MRc5 digital camera controlled by the AxioVision software package, version 4.2 from

Carl Zeiss MicroImaging GmbH (Jena, Germany).

Electrophoresis and protein detection

Two-dimensional electrophoresis was carried out with isoelectric focusing for the first dimension

on IPGphor (GE Healthcare Life Sciences, Piscataway, NJ, USA), and SDS-PAGE for the

second dimension with Mini-Protean II apparatus (Bio-Rad Laboratories, Hercules, CA, USA),

according to the manufacturers’ instructions. Briefly, the 7 cm IPG strips, pH 3-10 L, containing

100 µg samples were dissolved in IPG buffer (with freshly added 18 mM DTT) were placed in

porcelain strip-holders. The strips were overlaid with ~1 ml mineral oil and rehydration was

performed at 20ºC for 16 h. This was followed by isoelectric focusing at constant voltage of 500

V for 30 minutes, 1000 V for 1 h and thereafter at 5000 V for 1 h. After isoelectric focusing, the

strips were soaked in equilibration buffer for 10 minutes before loading on to 12.5% SDS-

polyacrylamide slab gels and electrophoresed at constant current of 30 mA, for 45 minutes.

Upon completion, the gels were stained with Coomassie brilliant blue R250 to detect the

separated proteins.

Statistical analysis

Data are presented as mean ± SEM of triplicate determinations, except when results of plots are

shown, in which case, a representative experiment is depicted in each figure. Comparisons

between multiple groups were performed using one-way ANOVA with Tukey and Duncan

corrections. Statistical significance was indicated when P < 0.05.

RESULTS

Initial cell viability assay showed that whole B-serum (WB) and C-serum (WC) exerted anti-

proliferative activity on a number of cancer-origin cell lines. The anti-proliferative activity

improved after dialysis, while the killing of cancer-origin cells was observed in the dialysed B-

World Anti-Cancer Congress and Exhibition, 11 – 12 January 2014, Universiti Sains Malaysia, Pulau Pinang

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serum precipitate (DBP), a higher killing activity was discerned with dialysed C-serum

supernatant (DCS) compared to dialysed C-serum precipitate (DCP). On the other hand, anti-

proliferative activity diminished in the boiled B- and C-serum fractions i.e. boiled B-serum

supernatant (BBS), boiled B-serum precipitate (BBP), boiled C-serum supernatant (BCS) and

boiled C-serum precipitate (BCP) (Table 2). It was also discerned that among the cancer-origin

cell lines, HepG2 was the most susceptible, while HCT116 was the most resistant to the

treatments. These findings justified further evaluation of WB, DBP, WC, DCS and DCP

fractions in view of targeting HepG2 - the most susceptible cancer-origin cell line.

Table 2. A summary of anti-proliferative activity of latex B- and C-serum and its fractions

WB DBS DBP BBS BBP

Control cell line Hs27 X X X X X

Cervical cancer HeLa X X √ X X

Liver cancer HepG2 √ X √√ X X

Breast cancer MCF-7 X X √

(72H)

X X

Breast cancer

MDA-

MB231 √ (72H)

X √ X X

Ovarian cancer Caov-3 X X √

(72H)

X X

Colorectal cancer HCT116 X X √ X X

WC DCS DCP BCS BCP

Control cell line Hs27 X X X X X

Cervical cancer HeLa √ (72H)

√ √ (72H)

√ (72H)

X

Liver cancer HepG2 √ √√ √ √ (72H)

X

Breast cancer MCF-7 X √ √ (72H)

√ (72H)

X

Breast cancer

MDA-

MB231 √ √√ √ X X

Ovarian cancer Caov-3 X √ X X X

Colorectal cancer HCT116 X √ (72H)

X X X

Oral cancer CAL27 X √ √ (72H)

X X

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X not susceptible

√√ very susceptible

√ susceptible

√ (72H)

susceptible at high concentrations (at 72 hours post-

treatment)

Specific anti-proliferative activity of WB, WC, DBP, DCS and DCP fractions on

HepG2 cells

Cell-based MTT assay with the pre-determined concentrations of WB, DBP, WC, DCS and DCP

showed that proliferation of Hs27 was not affected, whereas that of HepG2 were inhibited within

the test concentration range (0 - 1 µg/ml) at 48 h post-treatment (Fig. 2). Although HepG2 is of

malignant origin, a significant degree of difference in viability was discerned at all-time points

i.e. 24, 48 and 72 h post-treatment (data not shown), which reflects on the specificity of the test

fractions in exerting anti-proliferative activity against the cancer-origin cell line.

0%

20%

40%

60%

80%

100%

120%

Ce

ll V

iab

ility

(%

)

Concentrations (µg/ml) Hs27 HepG2

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0%

20%

40%

60%

80%

100%

120%C

ell

Via

bili

ty (

%)

Concentrations (µg/ml) Hs27 HepG2

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

120.0%

Ce

ll vi

abili

ty (

%)

Concentration (ng/ml)

MTT assay (WC_0-1000ng/ml) at 48 hours

Hs27 cells HepG2 cells

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

120.0%

140.0%

Ce

ll vi

abili

ty (

%)

Concentration (ng/ml)

MTT assay (DCS_0-1000ng/ml) at 48 hours

Hs27 cells HepG2 cells

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Figure 2. Viability pattern of HepG2 and Hs27 cells treated with WB, WBP, WC, DCP or

DCS up to 1000 ng/ml, at 48 h post-treatment.

Among the test fractions, HepG2 cells were most susceptible to DCS. LC50 and LC80 calculated

using Probit analysis of WC, DCS and DCP, for HepG2 and HCT116 at different time points

post-treatment are shown in Table 3. Within the C-serum fractions, it was evident that LC50 and

LC80 values of DCS for HepG2 were consistently lower than those obtained after DCP treatment,

especially at 72 h; indicating a higher potency residing in the DCS fraction (see Fig. 3).

Table 3. Probit analysis results showing the LC50 and LC80 at various time points post-treatment

with WC, DCS and DCP for HepG2 and HCT 116, with 95% confidence level

WC DCS DCP

24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

HepG2 LC50 N/A 888.6 ng/ml 818.1 ng/ml 2.4 µg/ml 2.0ng/ml 0.06 ng/ml N/A 280 ng/ml 42.3 ng/ml

LC80 N/A 3.2 µg/ml 5.7 µg/ml 42.3 µg/ml 79.6 µg/ml 174 ng/ml N/A 3.5 µg/ml 362.3 ng/ml

HCT116 LC50 N/A N/A N/A N/A 64.6 µg/ml 120.5 µg/ml N/A N/A N/A

LC80 N/A N/A N/A N/A 1.8 mg/ml N/A N/A N/A N/A

N/A denotes that the value was too high and therefore not included.

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

120.0%

140.0%

Ce

ll vi

abili

ty (

%)

Concentration (ng/ml)

MTT assay (DCP_0-1000ng/ml) at 48 hours

Hs27 cells HepG2 cells

World Anti-Cancer Congress and Exhibition, 11 – 12 January 2014, Universiti Sains Malaysia, Pulau Pinang

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Figure 3. Viability pattern of HepG2 cells treated with the different latex C-serum fractions i.e.

WC, DCS and DCP, at 72 h post-treatment.

Deciphering the susceptibility of HepG2 cells to WC-, DCP- and DCS-fractions

Generally, malignant cells treated with potent anti-cancer drugs undergo apoptosis – a type of

programmed cell death. In this study, real-time qRT-PCR revealed that pro-apoptotic genes such

as Bax and caspase-319

were not up-regulated in WC, DCP or DCS treated HepG2 cells. Instead,

expression of Bcl-2 remained high, although viability of HepG2 cells was compromised at 48

and 72 h. Bcl-2/Bax and Bcl-2/casp3 expression ratios in HepG2 cells also remained high

compared to those in Hs27 and HCT116 cells (Fig. 4). Expression ratios in these latter were

neither significantly different nor elevated between themselves. As Bcl-2 is an anti-apoptotic

gene20

, its high expression in HepG2 cells, and the concomitant low expression of Bax and

caspase-3 suggest that susceptibility to these treatments was not due to apoptosis.

In addition, agarose gel electrophoresis of total genomic DNA of WC-, DCP- and DCS- treated

HepG2 cells gave a smearing of ethidium bromide stain along the sample lanes (data not shown),

which indicates degraded DNA. The absence of distinct DNA fragmentation or laddering reflects

on the lack of caspase-induced endonuclease activity, otherwise would be evident in cells

undergoing apoptosis21

.

a.

6

5

4

3

2

1

0 WC-treated DCS-treated DCP-treated

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

120.0%

Ce

ll V

iab

ility

MTT assay on HepG2 cells at 72 h

WC

DCP

DCS

HepG2 HCT116 Hs27

Rel

ativ

e ex

pre

ssio

n

Bcl

-2/B

ax r

atio

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b.

8

7

6

5

4

3

2

1

0 WC-treated DCS-treated DCP-treated

Figure 4. Expression of Bcl-2/Bax (a) and that of Bcl-2/caspase-3 (b) in HepG2, HCT116 and

Hs27 cells after treatment with WC, DCS and DCP for 48 h.

HepG2 cells were positively stained with propidium iodide

DCS-treated cells were positively stained with Hoechst 33258 and PI but negatively stained with

Annexin V. However, DCP-treated cells were positively stained with Hoechst 33258, PI and

very slightly stained with Annexin V (Fig. 5). Morphology examination under microscope

revealed that DCS- and DCP-treated cells were dead or in the process of dying, with cells

detached from the coverslip surface, observation correlated to the cell nuclei stained with PI .

However, the non-treated control cells were positively stained for Hoechst 33258 and negatively

stained for both Annexin V and PI, indicating live cells.

Figure 5: DCS-treated HepG2 cells co-stained with Hoechst 33258 (blue) and Annexin-V/ PI

staining kit (green and red, respectively) at 48 hours post-treatment.

HepG2 HCT116 Hs27 R

elat

ive

exp

ress

ion

Bcl

-2/C

asp

3 r

atio

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DISCUSSION

Cell viability assay showed that a number of adherent human cancer-origin cell lines were

susceptible to whole C-serum (WC). Further analysis revealed that viability of WC treated

HepG2 cells were markedly reduced at 48 h, whereas HCT116 and Hs27 cells remained

unaffected within the tested concentration range (0 - 1 µg/ml). Hs27 and HCT116 cells were also

not affected by dialysed C-serum fractions (DCP and DCS) in subsequent cell viability assays,

indicating that inactivity of WC against these cell lines was not due to the presence of small

inhibitory molecule(s) that are eliminable by dialysis. As both DCP and DCS elicited slightly

different anti-proliferative patterns (Fig. 2), these fractions could be harbouring different active

constituents. Moreover, the anti-proliferative activity improved after dialysis, with DCS eliciting

a higher activity compared to DCP (Fig. 3), which suggests enrichment and at least a partial

separation of the active constituent(s), and perhaps removal of low molecular weight inhibitor(s).

On the other hand, diminished anti-proliferative activity in the boiled C-serum fractions reflects

on the heat sensitive nature of the active-constituents. Thus it’s tempting to speculate that the

active constituent(s) are of protein-origin – the predominant macromolecules (>3,000 Da) that

are retained in the dialysed C-serum fractions. The diversity of proteins in the dialysed C-serum

fractions is shown in figure 6. Chromatographic fractionation to narrow down on the active-

constituents of DCP and DCS is in progress. Subsequent cell viability assay will prove if further

enriched sub-fractions were attainable, and may be used to compare its efficacy with that of

known anti-cancer agents.

pH 3 10 3 10

a

.

b

.

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Figure 6. Coomassie blue staining of two-dimensional polyacrylamide gel electrophoresis (2D-

PAGE) of dialysed B-serum precipitate (a), dialysed B-serum supernatant (b), dialysed C-serum

precipitate (c) and dialysed C-serum supernatant (d).

Probit analysis revealed that LC50 values on HepG2 were significantly reduced to 2 ng/ml for

DCS and 280 ng/ml for DCP, compared to 818 ng/ml for WC. The LC50 values obtained in this

study were far lower than the 30 µg/ml standard benchmarked by the American National Cancer

Institute for crude plant extracts22

. With the specificity of dialysed C-serum fractions to HepG2,

there seems to be a potential for application in the treatment of hepatocellular carcinomas. It has

been reported that only 10 – 20% of hepatocellular carcinomas can be removed completely under

surgery23,24

. If dialysed C-serum fractions were specifically targeted to malignant cells in the

liver, population of the primary cancer cells could be suppressed, and then eliminated by surgical

intervention, or even without surgery, hence facilitating recovery of healthy hepatocytes.

Furthermore, the activity of DCS and DCP was shown to be prolonged, as the number of living

cells continued to decrease over time, without the need for introduction of a fresh dose of the

dialysed C-serum fractions (Fig. 3). Thus, a minimal effective dose would suffice to destroy

target malignant cells.

In the efforts to decipher the mechanism that leads to the destruction of cancer cells, we have

performed qRT-PCR, DNA fragmentation assay and preliminary morphological assessment on

the WC-, DCP- and DCS-treated HepG2 cells15

. For the most part, the results did not support

involvement of the classical apoptosis cell-death signalling pathway. More recently, DCP- and

DCS-treated HepG2 cells were observed under scanning electron microscope (SEM), subjected

to cell cycle analysis by flow cytometry, as well as Hoechst, Propidium Iodide (PI) and Annexin

V staining. These additional cellular and morphological examinations suggest involvement of

autoschizic programmed death pathway of treated HepG2 cells.

ACKNOWLEDGEMENTS

This work was supported by the Malaysian Rubber Board (SEAC grant S11BTP0373) and

Universiti Sains Malaysia (USM incentive grant 1001/CIPPM/82111). The skilled technical

assistance of Mr. Mony Rajan and Ms. Azlina Azharuddin is gratefully acknowledged.

c

..

.

d

..

.

World Anti-Cancer Congress and Exhibition, 11 – 12 January 2014, Universiti Sains Malaysia, Pulau Pinang

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