Spirulina
Platensis & Lenkemia
Abstract
Introduction
Material and methods
Results
References
Spirulina Platensis & lenkemia
Inhibitory effect of phycocyanin from Spirulina platensis
on the growth of human leukemia K562 cells
Yufeng Liu, Lizhi Xu, Ni Cheng, Lijun Lin & Chengwu
Zhang
Medical School, Nanjing University, Nanjing, 210093, PR of China
Department of Biological Science & Technology, Nanjing University,
Nanjing, 210093, PR China
Department of Applied Biochemistry, Nanjing University of Chemical
Technology, Nanjing, 210009, PR of China
(* Author for correspondence; e-mail ziq@nju.edu.en)
Received 18 January 2000; revised 22 February 2000; accepted
2 Match 2000
Key words: Bcl-2, C-myc, growth inhibition, G1 arrest,
K562 cells, Phycocyanin, Spirulina platensis
Abstract
The effect of phycocyanin from Spirulina platensis on
the growth of human chronic myelogenous leukemia-blast crisis K562
cells was studied by semi-solid agar assay and cell viability measurement.
Phycocyanin significantly inhibited the growth of K562 cells in a
dose-dependent manner. The IC50 value of the phycocyanin was 72.5
mg L-1. After the K562 cells were cultured with phycocyanin for 6
days, flow cytometric assays showed that more K562 cells were blocked
to progress through S-phase and arrested at G1 phase. DNA fragmentation
assay indicated that there was no ladder of DNA fragments of approximately
200-bp multiples, indicating that apoptosis had not occurred. Western
blot analysis showed that Bcl-2 protein was expressed, but its level
remained unchanged, whereas the expression level of c-myc increased.
These findings suggest that phycocyanin may be able to inhibit the
growth of K562 cells by pathways other than apoptosis, and that changed
a expression pattern of the c-myc protein may be involved in such
inhibition.
Introduction
There has recently been a surge of interest in marine
bioresources, particularly seaweeds, as sources of bioactive substances
(Sadanori et al., 1993). Among the studies, it has been shown that
preparations of seaweed such as polysaccharide, peptide and phycobiliprotein
can affect the multiplication of tumor cells (Schwartz et al., 1986;
Noda et al., 1989; Riou et al., 1996). Many extracts from seaweeds
were found to possess bioactivity against murine immunocytes after
they were cultured with murine immunocytes at concentrations from
30 to 100 mg L-1 (Sadanori et al., 1993). The phycocyanin from cyanobacteria
(C-phycocyanin, C-PC) has similar features, including a hepatoprotective
effect (Vadiraja et al., 1998) and antioxidant and anti-inflammatory
properties (Romay et al., 1998). Daily ingestion of a small dosage
of C-PC could maintain or accelerate lymphocytic functions to prevent
malignancy such as cancer or to inhibit its growth or recurrence (Dainippon
Ink & Chemicals, 1983). C-PC enhanced the laser cytotoxic effect
for cancer laser therapy after tumor cells had been cultured with
250 mg L-1 C-PC (Morcos, 1988).
The aim of the present study was to elucidate further
the potential pathways by which C-PC can affect the multiplication
of tumor cells in vitro and to explore the possibility that C-PC can
be used as a potential antitumor substance. Human leukemia K 562 cells
were used as the target.
Materials and methods
Phycocyanin
The C-PC (Kindly provided by Dr Zhang Chenwu) used in this study was
purified from Spirulina (Arthrospira) Platensis. The C-PC had a purity
of 4.71 (A520/A277), its absorption maximum was at 620 mm and isoelectric
point was 4.8. SDS-PAGE showed that the C-PC yielded two coloured
protein bands with molecular masses of 15.0 KD and 14.5 KD, respectively,
which suggests that the minimal molecular mass of the C-PC was 29.5
KD. The C-PC was 29.5 KD. The C-PC contained 14 amino acids and lacked
bistidine(His), Proline (Pro) and methionine (Met) (Zhang et al .,1996)
Cell culture
Human chronic myelogenous leukemia-blast crsis K562 cells were cultured
in RPMI-1640 medium (Gibco, BRL) supplemented with 10% fetal bovine
serum (FBS: Gibco, BRL), 5 mM glutamine (Gibco), 5% antibiotic and
antimycotic solution (30000 unites L-1 penicillin and 30 mg L-1 streptomycin,
at pH 7.2: Gibco). Cells were maintained at 37C in a humidified atmosphere
of 5% CO2. Exponentially growing cells were collected and prepared
for the experiments.
Semi-solid agar culture
The concentration of base agarose was 0.5% which contained 16% RPMI-1640
(4X), 20% FBS. The concentration of super agarose was 0.3%, which
contained K562 cells (4 x 106 L-1) and different concentrations of
C-PC (40, 80 and 160 mg L-1), supplemented with 16% RPMI-1640 and
20% FBS. Cells without C-PC were used as a control. After incubation
for 4, 6, 8, 10 or 12 days at 37C in the humidified 5% CO2-air mixture,
colony numbers were counted.
Cell viability
K562 cells were inoculated at a density of 2 x 108 cells L-1 in 100-mm
dishes; C-PC was added into the dishes at the final concentrations
of 20, 40, 80 and 160 mg L-1. K562 cells cultured without C-PC was
used as control. After the cells had been cultured for 1, 2, 3, 4,
5 or 6 days, their cviability was measured by the XTT (2, 3-bis [2-methoxy-4-nitro-5-sulphopheny1]-2H-tetraz
olium-5-carboxanilide, sodium salt) (Sigma) dye reduction assay. XTT
powder was dissolved in phosphate buffer saline (PBS, Gibco) to form
a stock solution (1000 mg L -1), which was syringe-filtered and stored
at 4?C. XTT working solution was freshly prepared prior to use by
mixing 1 ml XTT stock solution with 5 ml 5 M PMS (phenazine methosulphate
[N-methyldibenzopyrazine methyl sulphate]) (Sigma). The cells were
incubated at 37?C for 4h. The absorbance of each well was measured
at 450 nm using a microtitre plate reader. Cell viability was expressed
as follows: A450 of treated cells / A450 of untreated cells.
Flow cytometric assay
After the K562 cells had been cultured with C-PC for 6 days, they
were washed three times with cold phosphate buffer saline (PBS, Gibco)
and then fixed overnight in cold 70% ethanol. After washing twice
with PBS, these cells were gently resuspended in 1 ml DNA staining
reagent (PBS, pH7.4, containing 0.1% Triton X-100, 0.1 mM FDTA, pH7.4,
50 mg L-1 RNase A [50 units mg -1], 50 mg L-1 propidium iodide) for
over one hour at room temperature. Flow cytometric assay was performed
using a FACS calibur instrument (Becton Dickinson). The percentage
of K562 cells in each cycle phase was analyzed by ModFITLT for mac
v1.01 software (B.D. company).
DNA fragmentation assay
After the C-PC treated K562 had been washed twice with cold PBS, they
were lysed with a lysis buffer (50 mM Tris-HCI, pH8.0, 2 mM EDTA,
10 mM NaCI, 2% SDS, 50 mg L-1 proteinase K) at 50 ?C for more than
4 hours and then chilled on ice. Proteins were precipitated by saturated
NaCI and removed by centrifugation (15000 g, 10 min). Two volumes
of 100% ethanol were added to precipitate DNA. Finally, the DNA pellet
was suspended in TE (Tris-EDTA, pH8.0) buffer containing 20 mg L-1
Rnase. The mixture was incubated at 37C for 0.5 b before electrophoresis
in 1.5 % agarose. The gel was viewed under ultraviolet illumination.
Western blot assay
The K562 cells cultured with C-PC for 6 days were washed three time
with cold PBS, lysed with a lysis buffer (50 mM Tris-HCI, pH 6.8,
2% SDS, 100 mM DTT) and were boiled for 10 minutes. After centrifugation,
the solutions were collected and the protein concentrations were determined
by Coomassie blue staining. Samples were electrophoresed by 12% gradient
SDS-PAGE gel. Electrophoretically separated proteins were transferred
onto nitrocellulose paper (Schleicher and Schuell, Keene, NH) using
a wet blotting device (Bio-Rad, Richmond, CA) to nitrocellulose paper
with a current of 0.20 A for 4 h at 4C. The nitrocellulose paper was
than blocked overnight with a blocking solution (PBS, pH 7.4, containing
5% non-fat dried milk powder, 0.1% Tween 20) at 4C. This was followed
by an incubation with anti-Bel-2 monoclonal antibody (1:500, Cambridge
Research Biochemical) or c-myc monoclonal antibody (1:500, DAKO) for
two hours at room temperature. Washing the nitrocellulose paper was
then incubated in goat anti-mouse secondary antibody (1:1000, Sigma)
for two hours at room temperature. A detection reagent (ECL, Western
blotting detection Kit, Amersham, Buckinghamshire, England) was added
onto the membrane. To detect the presence of Bel-2 or c-cmyc protein,
the membrane was exposed to X-ray film (Fuji) for 1 min. 1 kb prestained
protein molecular weight standards (high range, Gibco) were used to
mark the size of the unknown protein.
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