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Indian Journal of Biochemistry & Biophysics
Vol. 51, April 2014, pp. 127-134
Anti-proliferative and apoptotic properties of a peptide from the seeds of
Polyalthia longifolia against human cancer cell lines
S Rupachandra and D V L Sarada*
Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur-603203, India
Received 17 August 2013; revised 10 March 2014
The peptides produced enzymatically from various plants have shown various biological activities including cytotoxicity.
Different types of cytotoxic peptides have been reported from the seeds and leaves of Violaceae, Rubiaceae and Annonaceae
families. In this study, we report purification and characterization of peptide(s) showing cytotoxic activity against A549 and
HeLa cancer cell lines from the seeds of Polyalthia longifolia (Annonaceae). Seed proteins of P. longifolia were extracted
and hydrolyzed using trypsin. The enzyme hydrolysate was applied on to a Sephadex G10 column and eluted using
Tris-HCl buffer (pH 7.5). Two fractions F1 and F2 were obtained, of which F2 showed significant cytotoxic activity against
lung (A549) cancer cells at 10 µg/mL and cervical (HeLa) cancer cell lines at 30 µg/mL, as revealed by the MTT assay.
DNA fragmentation was observed in the tested cancer cell lines treated with F2 peptide at a concentration of 10 µg/mL and
30 µg/mL, respectively. Further, increased number of apoptotic cells was observed in sub-G0 phase of cell cycle of A549
and HeLa cell lines, when treated with 10 µg/mL and 30 µg/mL of F2, as revealed by the flow cytometric analyses. FTIR
spectrum of F2 peptide detected the presence of stretching vibrations of carboxylic acid OH residue with peak at 3420 cm-1
and carbonyl (C=O) groups at 1636 cm-1, respectively. RP-HPLC analysis of F2 peptide showed a single peak at a retention
time of 12.8 min detected at 280 nm, depicting the purity of F2 to be more than 90%. LC-ESI-MS/MS analysis showed the
average theoretical mass of F2 to be 679.8 using m/z ratios. In conclusion, the findings suggest that F2 peptide is an
effective inducer of apoptosis of cancer cells, thus offers an important strategy in the development of cancer therapeutics.
Keywords: Cytotoxicity, Flow cytometer, LC-ESI- MS/MS analysis, MTT assay.
Plant-derived compounds and their semi-synthetic
as well as synthetic analogs serve as major source
of pharmaceuticals for human diseases. The
development of more selective agents focused on
targeted delivery of imaging probes and drugs to
different tumour sites is the current trend in cancer
diagnosis and therapies. In the cancer drug discovery
program, a paradigm based on ethno botanical and
ethno pharmacological data is beneficial for
identifying potential anticancer molecules than mass
screening of plant species.
Plants containing pharmacologically active
polypeptides have been used in natural product-based
drug discovery programs. Peptides are small amino
acid sequences that can be isolated to bind to a
predetermined target and are potentially capable of
inhibiting individual signalling components, which
are essential in cancer development and progression1.
Peptides allow structural changes to incorporate
protective substitutions, chiral derivatives, non-natural
——————
*Corresponding author:
Mobile +91 9884876619
E-mail: [email protected]
amino acids and other modifications aiming at
increased stability, efficiency and resistance to
proteolysis2,3. Studies have shown that proteins and
peptides from plants exhibit marked inhibitory effects
on the proliferation of various tumour cell lines,
including murine leukaemia, lung carcinoma, cervical
carcinoma, rat osteoblast like sarcoma, human
nasopharyngeal carcinoma and ovarian neoplasm4. In
recent years, plant peptides with cytotoxic activity
have gained the potential for research towards the
development of anticancer drugs.
Lunasin is a novel and promising chemopreventive
peptide derived from soybean seeds. The cancer
preventive properties in vitro make lunasin a perfect
candidate to study an in vivo cancer-preventive
activity5. In search of natural sources of lunasin
besides soybean6, the isolation and bioassay of lunasin
from barley7and wheat8 have also been reported.
Recently, a partially purified peptide from Euphorbia
hirta Linn. with molecular mass less than 3 kDa,
has been found to possess significant cytotoxicity
against KATO-III, a gastric carcinoma cell line9.
Also, the peptides derived from the high oleic acid
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INDIAN J. BIOCHEM. BIOPHYS., VOL. 51, APRIL 2014
soybean meals have shown cell growth inhibition of
human colon (HCT-116, Caco-2), liver (HepG-2) and
lung (NCL-H1299) cancer cell lines10.
Annonaceae plants are a rich source of bioactive
substances, such as flavonoids, alkaloids and
acetogenins, extracted from the seeds and other
parts of these plants with antitumor properties. Many
cyclic peptides have been isolated from the species of
Annona genus, including A. glabra11 and A. squamosa12.
Polyalthia longifolia chinensis Benth. & Hook. F,
commonly known as Devdaru is a tall tree native
to India. The bark of P. longifolia (Annonaceae)
is traditionally known to lower blood pressure,
stimulate respiration and treat skin diseases, diabetes
and hypertension13. In addition to antimicrobial
compounds14, cytotoxic constituents, such as a halimane
diterpene-3β, 5β, 16α-trihydroxyhalima-13(14)-en-15,
16-olide (1) and an oxoprotoberberine alkaloid-8oxopolyalthiaine (2) have been isolated from the
methanolic extract of P. longifolia15. In the present
study, we report purification and characterization of
peptide(s) from the seeds of P. longifolia, which
show cytotoxic activity against A549 and HeLa
cancer cell lines.
Materials and Methods
Materials
Seeds of Polyalthia longifolia were collected in the
month of May and authenticated by Dr. P Jayaraman,
from Plant Anatomy Research Centre, Chennai.
Buffers used for FPLC and HPLC analysis were of
analytical grade and were procured from SigmaAldrich, St.Louis, MO, USA. Tumour cell lines A549
(human lung adenocarcinoma epithelial cell line) and
HeLa (human cervical adenocarcinoma epithelial cell
line) used for cytotoxicity assay were procured from
National Centre for Cell Science, Pune.
Protein extraction and purification
Seeds were washed with distilled water and shadedried. The dried seeds were ground to fine powder
and the proteins were extracted with extraction buffer
(10 mM Na2HPO4, 15 mM NaH2PO4, 10 mM KCl,
2 mM EDTA, pH 7.0) by stirring overnight at 4ºC16.
The crude protein extract was filtered using
the muslin cloth and centrifuged at 10,000 rpm for
10 min. The supernatant obtained was digested using
1 mL of trypsin solution (5 mg/mL in 0.1 M Tris-HCl
containing 0.03 M NaCl, pH 8.0) and incubated at
37ºC overnight. The enzyme was inactivated by
heating in a boiling water bath for 10 min.
The trypsin hydrolysate was filtered through 0.45
µm filter and used for further study. Ammonium
sulphate was added to a concentration of 80%
saturation in the filtrate and incubated overnight
at 4ºC. The precipitate was centrifuged at 12,000 rpm
for 20 min and the pellet was dissolved in Tris-HCl
buffer (pH 7.5). The protein concentration was
determined according to Lowry et al17 using bovine
serum albumin as the standard. The pellet containing
the trypsin hydrolysate was applied on to fast protein
liquid chromatography (FPLC, Akta purifier GE
Healthcare) using Sephadex G-10 gel filtration
column (1.5 x 50 cm) equilibrated with 50 mM
Tris-HCl buffer (pH 7.5)18. The peptide fractions F1
and F2 were eluted using the same buffer at a flow
rate of 0.1 mL/min.
Evaluation of cytotoxic activity
The isolated peptides were further used for
cytotoxicity activity against lung (A549) and cervical
(HeLa) cancer cell lines. Vero (green monkey kidney)
cells which are the normal growing cells were used as
non-cancerous control cells to examine the inhibitory
action of the peptide fraction (F2) in the in vivo
system. All the cells (Vero, A549 and HeLa) used for
this study were cultured in Dulbecco’s modified eagle
medium (GIBCO), supplemented with 10% fetal
bovine serum (GIBCO) and 1% antibiotic antimycotic
solution at 37ºC in a humidified atmosphere of 5%
CO2. The A549 cells were seeded at a density of 3500
cells/well, while HeLa and Vero cells were seeded at
a density of 3000 cells/well in a 96 well microtitre
plates in a total volume of 200 µL/well.
The isolated peptide fractions F1 and F2 were
added at a concentration ranging from 10-1000
µg/mL and the cells were incubated for 24 h. The
morphology of treated cells was observed using a
phase contrast microscope (Leica, Wetzlar, Germany).
MTT assay19 was also performed to evaluate the
cytotoxic activity of peptide fractions F1 and F2.
To each of the wells, 50 µL (5 mg/mL) of 0.5% MTT
was added and incubated for 4 h. The formazan
crystals formed were dissolved in dimethyl sulfoxide
(DMSO) and the absorbance was read at 570 nm
using a microplate reader (Bio-Rad, CA). Seeded
wells without added peptides served as blanks.
Doxorubicin (Sigma) was used as positive control.
Untreated A549 and HeLa cells were used as control.
The percentage inhibition of cell viability was
determined by using the formula:
Acontrol - Atreatment/Acontrol x 100, where ‘A’ denotes
the absorbance read at 570 nm.
RUPACHANDRA & SARADA : ANTI-PROLIFERATIVE AND APOPTOTIC PROPERTIES OF A PEPTIDE
DNA Fragmentation analysis
Based on the results obtained in the MTT assay,
DNA fragmentation analysis was carried out to
evaluate the mechanism of cell death20 in cancer
cell lines treated with peptide fraction F2. The A549
cells were seeded at a density of 3500 cells/well and
HeLa cells of 3000 cells/well in a 6-well titre
plate containing 2 mL of minimal essential medium
and incubated at 37°C for 48 h in a CO2 incubator
with 5% CO2. A549 and HeLa cells were treated with
F2 fraction at a concentration of 10 µg/mL and
30 µg/mL and incubated for 24 h in 5% CO2
incubator. The media was discarded, followed by the
addition of 1 mL of TPVG (trypsin, PBS, versene,
and glucose) and 1mL of phosphate buffered saline
(PBS) to each well. The contents of wells were
collected and DNA isolation was performed21 using
DNA isolation kit (Genei, India). The pattern of bands
for DNA fragmentation was visualized on 1% agarose
gel under UV light and photographed.
Cell cycle analysis
A549 and HeLa cells were seeded in 6-well culture
plates at a density of 0.3 × 106 cells/well and incubated
at 37°C, 5% CO2 in an incubator for 24 h. The cells
were then treated with 10 µg/mLand 30 µg/mL of F2,
respectively and incubated at 37°C, 5% CO2 for 24 h
and 48 h. The treated cells were trypsinized after 24 h
and 48 h, washed with fresh medium and centrifuged
at 1000 rpm for 5 min at 4°C. The supernatant was
discarded and the cells were suspended in 300 µL of
PBS and 700 µL of ethanol. The cells were then
washed and centrifuged again at 1500 rpm for 10 min
at 4°C. The supernatant was discarded and cells
were resuspended in 600 µL of PBS containing 0.5%
Triton X-100, 20 µg of RNase and incubated at room
temperature for 1 h. After incubation, 40 µg/mL of
propidium iodide (1 mg/mL stock) was added to the
wells and incubated at room temperature for 45 min in
dark. The cells were observed in a flow cytometer
(FACSCalibur, Beckton Dickinson), equipped with an
air cooled argonlaser providing 15 mW at 488 nm
(blue laser) with standard filter setup22. About 10,000
events were acquired and the percentage of each
phase of the cell cycle was analyzed using Cell Quest
Pro software (Becton Dickinson, USA).
129
was homogenized with KBr in a mortar and pestle.
The sample mixtures were then pressed by vacuum
hydraulic at 1.2 psi to obtain a transparent pellet.
The pellet was scanned in the infrared absorption
region between 400 cm-1 and 4000 cm-1 with a
resolution of 4 cm-1.
Reverse-phase HPLC analysis
The purity of peptide fraction F2 was tested using
HPLC analyses performed on an Agilent series 1100
HPLC system fitted with a reversed-phase high
performance liquid chromatography (RP-HPLC)
cartridge, C18 column (250 x 4.6 mm, 5 µm, 300 Å),
with solution A - 0.1% trifluoro acetic acid (TFA) and
solution B - Acetonitrile (90%) as the mobile phase at
a flow rate of 1 mL/min and detected at 280 nm.
Liquid chromatography-electrospray ionization-mass spectrometry
(LC-ESI-MS/MS) analysis
The purified F2 fraction was dissolved in 50%
methanol and 50% water of HPLC grade, infused
into a Finnigan TSQ Quantum ultra AM mass
spectrometer (Thermo, USA) at a flow rate of 30 µL
per min, operated in the positive electrospray
ionization (ESI + ve) mode via the electrospray
interface24.
Statistical analysis
The results were expressed as mean ± S.E.M.
Comparisons between treated groups and control were
performed with the Dunnett’s multiple comparison
test using Graph Pad Prism software.
Results and Discussion
Purification and characterization of peptide
The protein content of crude supernatant from
the seeds of P. longifoliawas found to be 4.27 mg/mL,
as determined according to Lowry et al17. The peptide
fractions obtained from the trypsin hydrolysate of
crude supernatant were precipitated up to 80%
saturation limit with ammonium sulphate and
further purified using Sephadex G-10 gel filtration
chromatography. Two peptide fractions, namely peaks
1 and 2 designated as F1 and F2, respectively were
eluted from Sephadex G-10 chromatography (FPLC,
Aktaand GE) and detected at 280 nm (Fig. 1). Two
mL of each fraction was collected and tested for
cytotoxic activity against A549 and HeLa cells.
FTIR analysis
Cytotoxic activity of peptides
Identification of functional groups of F2 fraction
was performed using Shimadzu Fourier transform
infrared spectrophotometer23. The peptide fraction
Cytotoxicity is one of the chemotherapeutic
hallmarks of anticancer activity25. MTT assay is a
well-established in vitro model for cytotoxicity
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INDIAN J. BIOCHEM. BIOPHYS., VOL. 51, APRIL 2014
against cancer cell lines and is used as one of
the conventional methods for the screening of
compounds with potential anticancer properties26. We
used MTT assay to screen the in vitro anticancer
activity of the purified peptide fractions using A549
and HeLa cell lines.
The F1 fraction eluted at retention time of 25 min
didn’t show cytotoxic effect on selected cell lines.
However, fraction F2 eluted at retention time of
50 min showed significant inhibition on the proliferation
of A549 (Fig. 2A) and HeLa (Fig. 2B) cells treated
with a concentration ranging from 10-1000 µg/mL
(Table 1), warranting further characterization of F2
fraction.
Morphological changes showing the apoptotic
phenomenon was observed in A549 (Fig. 2A) and
HeLa cells (Fig. 2B), where the nucleus was
degenerated into discretely spherical fragments of
Table 1—Growth inhibitory effects of peptide (F2 fraction) from
seeds of P. longifolia against human cancer cell lines along with
positive control
[Cell inhibition was measured by MTT assay and the values are
expressed as mean ± SEM. Values were analyzed by using Dunnett’s
multiple comparison tests]
Sample
conc.
(µg/mL)
F2 peptide
10
30
100
300
1000
Growth inhibition (%)
A549
58.33 ± 6.46****
69.36 ± 3.77****
78.34 ± 1.33****
77.12 ± 3.36****
80.91 ± 3.86****
HeLa
Vero
29.89 ± 1.98**** 10.01 ± 1.06****
51.12 ± 0.39**** 11.24 ± 1.77****
74.85 ± 0.89**** 14.35 ±1.13****
78.09 ± 1.69**** 16.12 ± 0.36****
82.32 ± 1.35**** 19.21 ± 0.86****
Doxorubicin
****
Fig. 1—Size-exclusion chromatogram (Sephadex G-10) of
the peptides obtained by trypsin digestion from the seeds of
P. longifolia
0.1
52.82 ± 1.15
46.38 ± 0.43**** 40.32 ±1.15****
****
0.3
70.92 ± 2.81
55.85 ± 1.56**** 51.83 ± 1.81****
****
1
85.17 ± 1.37
62.10 ± 1.65**** 75.17 ±1 .35****
****
3
87.35 ± 4.33
84.89 ± 2.97**** 89.72 ± 0.24****
****
10
99.11 ± 0.53
99.54 ± 0.46**** 92.37 ± 0.82****
****p< 0.0001, ***p< 0.001 and **p< 0.01 indicate significant
difference compared to positive control.
Fig. 2—Photomicrographs (40x) of A549 cells (A) and HeLa cells (B) treated with different concentrations of peptide
(F2 fraction) ranging from 10-1000 µg
RUPACHANDRA & SARADA : ANTI-PROLIFERATIVE AND APOPTOTIC PROPERTIES OF A PEPTIDE
highly condensed chromatin, suggesting that fraction
F2 induced A549 and HeLa cell apoptosis. Further,
A549 and HeLa cells were also treated with
doxorubicin, which served as a positive control with
concentration ranging from 0.1-10 µg/mL (Table 1).
Addition of peptide (F2) showed 58% growth
inhibition of A549 cells at 10 µg/mL and 51%
inhibition of proliferation of HeLa cells at 30 µg/mL,
depicting the cytotoxic effect of the peptide F2,when
compared with the cell control (untreated A549 and
HeLa cells), showing 100% cell growth. Further,
Vero cells exhibited negligible inhibition when treated
with F2 at 10-1000 µg/mL, indicating the absence of
cytotoxic effect of F2 in Vero cells (Table 1).
The results obtained in this study were in
agreement with the apoptotic activity of a small
peptide (with a molecular mass of 1,050 Da) purified
from Cycas revoluta seeds against human epidermoid
cancer (Hep2) and colon carcinoma cells (HCT15)27.
Similarly, microcolin A, a linear peptide isolated
from Lyngbyama juscula is found to inhibit cell
proliferation via the induction of cell apoptosis,
suggesting it as potentially active against human
cancer cells28. Previous studies have also reported the
cytotoxic effect of vitri A, varv A, varv E peptides
isolated from Viola tricolor29 and viphi A peptide
from Viola philippica30 of Violaceae family against
MM966, HeLa and BGC-823 cells.
A novel anticancer peptide derived from rice bran
enzymatic hydrolysate is found to possess growth
inhibitory properties on colon, breast, lung and liver
cancer cells31. Dianthin peptides, isolated from
Dianthus superbus have shown potent cytotoxic effect
against the Hep G2 cancer cell line32. Kahalalide O,
a cyclopeptide from the Sacoglossan Elysiaornata is
reported to possess significant inhibitory effects on
P-388 cells and human lung cancer A549 cells33.
A tridecapeptide from Papaver somniferum inhibits
the proliferation of human liver (Bel-7402) and
mammary gland cancer cell lines (MCF7)34.
DNA fragmentation analysis
Apoptosis, a kind of cellular death is characterized
by the early activation of endogenous proteases,
cell shrinkage and DNA fragmentation35. The nuclear
DNA of apoptotic cells shows a characteristic
laddering pattern of oligonucleosomal fragments36.
In this study, the effect of the purified F2 fraction
on the induction of apoptosis in A549 and HeLa cells
was analysed. Agarsose gel electrophoresis of DNA
isolated from A549 cells treated with 10 µg/mL
131
Fig. 3—Agarose gel depicting DNA fragmentation of A549
cells (A) and HeLa cells (B) showing marker (M) - 1500 bp
ladder, control (c) - untreated cells, L1 – 10 µg of F2
fraction for A549 cells and 30 µg for HeLa cells
concentration of F2 peptide (Fig. 3A, lane 1) showed
a ladder pattern, indicating cell death due to DNA
fragmentation. Similar fragmentation pattern of DNA
was observed with HeLa cells treated with 30 µg/mL
of F2 peptide (Fig. 3B, lane 1). These results were
similar with the previous study which reported DNA
fragmentation, indicating the cell death of the nuclear
DNA of HeLa cells treated with isolated proteins
from the leaves of Mirabilis jalapa at IC50 value of
0.65 mg/mL37.
Effect of cytotoxic peptide on A549 and HeLa cell cycle
Cell cycle modulation by various anticancer agents
from plants is gaining widespread attention in recent
years. A number of plant peptides have shown the
ability to induce apoptosis and play an important role
in cancer prevention and therapy. The percentage of
cells in each phase of cell cycle in A549 and HeLa
cells treated with the peptide F2 was determined by
flow cytometry38. The sub-G0 group of A549 cells
treated with F2 at 10 µg/mL depicted increased
apoptotic cell count as 2.03% and 2.89% at 24 h and
48 h, respectively (Fig. 4A). Similarly, an increased
number of apoptotic cells were observed in Sub-G0
group of HeLa cells treated with F2 at 30 µg/mL as
2.41% and 3.43% for 24 h and 48 h (Fig. 4B). These
results suggested that F2 caused apoptotic-associated
chromatin degradation in both the tested cancer cell
lines, as revealed by the increase in the number of
apoptotic cells in Sub-G0 phase from 24 to 48 h,
when compared with Sub-G0 phase of cell cycle in
control cells of both the cancer cell lines, indicating
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INDIAN J. BIOCHEM. BIOPHYS., VOL. 51, APRIL 2014
Fig. 4—Effect of F2 fraction on the cell cycle of A549 cells (A) and HeLa cells (B) for 24 and 48 h
Fig. 5—FTIR spectrograph of F2 fraction
the non-apoptotic population. Our findings in this
study were in accordance with the anti-proliferative
activity against human lymphoma cell (U937) of
peptide fraction from anchovy sauce at 500 µg/mL39.
Identification of functional groups by FTIR analysis
The FTIR spectrum of F2 showed the bands of
O-H stretching vibrations at 3420 cm-1, confirming
the presence of carboxylic acid OH residue in the
peptide (Fig. 5). Appearance of similar peak bands at
3379 cm-1 assigned to O-H stretching vibrations
Fig. 6—RP-HPLC of F2 fraction
indicated the presence of hydroxyl groups associated
with proteins in Ananus comosus peel40. The peak at
1636 cm-1 in the F2 indicated the presence of C=C and
carbonyl (C=O) stretching vibration. Some studies
have reported that amino acids, peptides and proteins
display IR absorption bands in the range of
800-2500 cm-1 and such bands include N-H and C=O41.
RP-HPLC analysis
RP-HPLC analysis of cytotoxic F2 fraction showed
a single peak (Fig. 6), indicating the presence of
RUPACHANDRA & SARADA : ANTI-PROLIFERATIVE AND APOPTOTIC PROPERTIES OF A PEPTIDE
133
Fig. 7—Identification of molecular mass of F2 fraction by LC-ESI-Q-TOF MS/MS
single peptide with a retention time of 12.8 min
detected at 280 nm.
Identification of peptide by LC-ESI-MS/MS
The cytotoxic peptide (F2) was subjected to
LC-ESI-MS/MS analysis to identify the peptide
ions obtained from the tryptic digest. The MS/MS
spectrum of a single charged ion with m/z at 679.8
is illustrated in Fig. 7, depicting the fragmentation
spectrum containing major ions at m/z 100.4, 122.4,
182.0, 209.3 and 326.4, respectively. Similar studies
have demonstrated that the molecular weight, amino
acid type and sequence of peptides play a key role
on the biological activity of the peptides42-44.
In conclusion, a cytotoxic peptide F2 was isolated
from the seeds of Polyalthia longifolia with average
mass of 679.8 which showed significant antiproliferative activity against lung (A549) cancer cells
at concentration of 10 µg/mL and cervical (HeLa)
cancer cell lines, at 30 µg/mL, respectively. However,
F1 fraction did not exhibit any cytotoxic activity
against the tested cell lines. Besides, F2 did not show
any inhibitory effects on the proliferation of Vero
cells. Further, treatment of A549 and HeLa cells at
10 µg/mL and 30 µg/mL with F2 peptide resulted
in DNA fragmentation, a significant feature of
apoptotic cell death. Morphologic changes in cellular
nuclei revealed that F2 induced A549 and HeLa
cell apoptosis. Thus, the results suggest that F2
peptide is an effective inducer of apoptosis of cancer
cells, which offers an important strategy to create new
anticancer therapeutics with promising potential in
clinical practice.
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