AbstractAn anti-cancer peptide was purified from the Mercenaria (Meretrix meretrix Linnaeus) by the method of chromatography on Sephadex G-25 and FPLC, and its molecular weight was determined to be 3147Da by the way of MALDI-TOF mass spectrum. The effects of this peptide on human gastric gland carcinoma cells (BGC-823) and their cytoskeletal morphology were investigated. The results showed that the peptide could inhibit the proliferation of BGC-823 cells and obviously destroy the skeletal structures of the cells. When the concentration of the peptide reached 4.0μg/ml, the inhibition percentage of the cell growth was about 60%. The effects of this anticancer peptide on the activities of superoxide dismutase (SOD), alkaline phosphatase (ALP) and tyrosinase were studied. The results showed that the peptide activated ALP and SOD, but inhibit the tyrosinase activity. When the concentration of the peptide reached to 0.5μg/ml, the relative activities of SOD, ALP and tyrosinase were determined to be 188.5%, 122.0% and 27.5%, respectively

Chen, Qing-Xi

Leng, Bo

Liu, Xiao-Dan

Elsevier - Publisher Connector 

Inhibitory effects of anticancer peptide from Mercenaria on the BGC-823 cells and several enzymes 

FEBS 29262 FEBS Letters 579 (2005) 1187–1190Inhibitory eﬀects of anticancer peptide from Mercenaria on the
BGC-823 cells and several enzymes
Bo Leng, Xiao-Dan Liu, Qing-Xi Chen*
The Key Lab of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University,
Xiamen 361005, Peoples Republic of China
Received 16 November 2004; revised 30 December 2004; accepted 31 December 2004
Available online 19 January 2005
Edited by Vladimir SkulachevAbstract An anti-cancer peptide was puriﬁed from the Merce-
naria (Meretrix meretrix Linnaeus) by the method of chroma-
tography on Sephadex G-25 and FPLC, and its molecular
weight was determined to be 3147 Da by the way of MALDI-
TOF mass spectrum. The eﬀects of this peptide on human gastric
gland carcinoma cells (BGC-823) and their cytoskeletal mor-
phology were investigated. The results showed that the peptide
could inhibit the proliferation of BGC-823 cells and obviously
destroy the skeletal structures of the cells. When the concentra-
tion of the peptide reached 4.0 lg/ml, the inhibition percentage of
the cell growth was about 60%. The eﬀects of this anticancer
peptide on the activities of superoxide dismutase (SOD), alkaline
phosphatase (ALP) and tyrosinase were studied. The results
showed that the peptide activated ALP and SOD, but inhibit
the tyrosinase activity. When the concentration of the peptide
reached to 0.5 lg/ml, the relative activities of SOD, ALP and
tyrosinase were determined to be 188.5%, 122.0% and 27.5%,
respectively.
 2005 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
Keywords: Anticancer peptide; Human gastric gland
carcinoma cell; Superoxidedismutase; Alkaline phosphatase;
Tyrosinase; Eﬀect; Meretrix meretrix Linnaeus1. Introduction
Meretrix meretrix Linnaeus is a kind of popular ocean mol-
lusks, the values of which are shown not only in its abundance
in nutrition, but also in the use of medical resource. Mercena-
ria has been reported to show eliminate cyst, detoxiﬁcation
and anti-cancer activity [1–3]. Its extract is used in the treat-
ment of cancer for ages. Moreover, Mercenaria can be used
to decrease the level of blood sugar and blood fat [4,5], and
it also has the physiological function of protecting the body
cells from mutation and decrepitude [6]. Schmeer et al. [7] ex-
tracted a reagent from Mercenaria and named it as ‘‘Mercen-Abbreviations: BGC-823, human gastric gland carcinoma cells; SOD,
superoxide dismutase; ALP, alkaline phosphatase; FPLC, ﬂow-per-
formance liquid chromatography; MTT, 3-(4,5-di-methylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide; H.E., haematoxylin and eosin
*Corresponding author. Fax: +86 592 2185487.
E-mail address: chenqx@jingxian.xmu.edu.cn (Q.-X. Chen).
0014-5793/$30.00  2005 Federation of European Biochemical Societies. Pu
doi:10.1016/j.febslet.2004.12.089ene’’, which was demonstrated to have anticancer activity on
L1210, Krebs 2-breast carcinoma, Bittner mammary tumor
and so on. However, the molecular characteristic and action
mechanism of ‘‘Mercenene’’ are unknown. Because the com-
mon edible Mercenaria is abundant in nature and has never
suﬀered from cancer, this clam is undoubtedly a potential anti-
cancer medicine resource. The aim of the present paper is,
therefore, to report the extraction, puriﬁcation and identiﬁca-
tion of the anticancer peptide from Mercenaria. Furthermore,
the eﬀects of this peptide on the cytoskeletal morphology of
human gastric gland carcinoma cells (BGC-823) cells are car-
ried out. We also discuss the anticancer mechanism of this pep-
tide by researching its eﬀect on several enzymes relevant to the
cancers. The study presented here demonstrates that the pep-
tide from Mercenaria has reliable anticancer function, which
oﬀers a clue for looking for novel and natural anticancer
remedy.2. Materials and methods
2.1. Materials
Mercenaria was purchased at local market; superoxide dismutase
(SOD), alkaline phosphatase (ALP) and mushroom tyrosinase were
purchased from Sigma; the haematoxylin and eosin Y (H.E.) were pur-
chased from BDH; the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) was from Fluka. All other reagents were
local products of analytical grade. The water used was re-distilled
and ion free.
2.2. Extraction and puriﬁcation
The body from Mercenaria (Meretrix meretrix Linnaeus) was
homogenized and extracted with 25% alcohol for 2 h at 4 C; then,
the extraction was centrifugated at 15 000 · g for 30 min (4 C). The
supernatant was gathered and lyophilized to be powder for storage.
When puriﬁed, the powder was redissolved in water at the concentra-
tion of 0.5 g/ml and further chromatographed on Sephadex G-25 col-
umn (2.5 · 60 cm), which was eluted with water. The ﬂow rate was
controlled at 30 ml/h and 2.5 ml per tube was collected. The fraction
with anti-cancer activity was loaded onto ﬂow-performance liquid
chromatography (FPLC) with Sephadex G-75 column (16 · 600 mm,
22–44 lm, from Pharmacia, Sweden), pre-equilibrated and eluted with
50 mM Tris–HCl (pH 7.5) buﬀer. The ﬂow rate was 0.5 ml/min and
1.5 ml per tube was collected. Fractions with anticancer activity were
pooled, concentrated and desalted on FPLC with Sephadex G-25 col-
umn (1.5 · 50 cm) equilibrated and eluted with water.
2.3. Protein and peptide determination
The concentration of protein and the peptide were determined by the
Bradford methods. The molecular weight of the peptide was deter-
mined with MALDI-TOF MS, which was performed with a BIFLEX
III mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany),blished by Elsevier B.V. All rights reserved.
1188 B. Leng et al. / FEBS Letters 579 (2005) 1187–1190using a 337-nm nitrogen laser. 0.5–1.0 ll of sample (5–20 pmol) and
0.5 ll of a-cyano-4-hydroxycinnamic acid (10 mg/ml in water) were
mixed on the target plate and dried with a gentle stream of air. Spectra
were obtained in the reﬂected mode at an accelerating voltage of 19 kV
and a reﬂecting voltage of 23 kV. Deﬂection of the low mass ions was
used to enhance the target protein signal. External calibration (from
Bruker) was performed for measurement. Mass determination was
averaged from three sample-standard pairs.
2.4. Cell culture
BGC-823 cells were used to test the anticancer activity of the peptide
fraction. The cells were cultured in the Roswell Park Memorial Insti-
tute tissue 1640 medium containing 1% non-essential amino acid mix-
ture solutions, 10% newborn bovine serum, 100 IU/ml of penicillin and
100 lg/ml of streptomycin with humidiﬁed atmosphere of 5% CO2 and
95% air at 37 C. Cell numbers were monitored by directly counting
with haemacytometer. The inhibitory rate and the variety of cytoskel-
etal morphology were assessed by the way of MTT [8] and H.E. [9],
respectively.
2.5. Determination of enzymes activity
The enzyme activities of SOD, ALP and tyrosinase were determined
by the methods of Chen [10], Stewert [11] and Chen [12], respectively.
The eﬀects of the peptide on these enzymes activities were deter-
mined as a percentage of their control activities. All results are ex-
pressed as arithmetic means ± S.D. of three independent tests.20 40 60 80 100
Fraction No.
0.0
0.3
0.6
0.9
1.2
O
D 2
80
Peak 3
A B
Fig. 1. Column chromatography of anticancer peptide from
Fig. 2. The determination of the molecular weight of3. Results
3.1. Puriﬁcation of the anticancer peptide from Mercenaria
The crude extraction was applied to the column of Sephadex
G-25. The elution proﬁle of peptide was shown in Fig. 1A.
There were three protein peaks. Each peak was pooled and
tested its eﬀects on BGC-823 cells, respectively. The results
indicated that the Peak 3 had strong anticancer activity. The
sample of Peak 3 was further puriﬁed by FPLC eluted with
50 mM Tris–HCl (pH 7.5). Fig. 1B showed the elution proﬁle
and the main peak was pooled for further test.
3.2. Determination of the molecular weight
The molecular weight of Mercenaria anticancer peptide was
determined to be 3147.41 Da by MALDI-TOF Mass Spectrum
(shown in Fig. 2).
3.3. Inhibitory eﬀect of the peptide on BGC-823 cells
The eﬀects of the peptide on the growth of BGC-823 cells
were studied. The results (Fig. 3) suggested that the peptide
could inhibit the cells at a very low concentration and had a
signiﬁcant dose-dependent-inhibitory eﬀect. When the pep-0 5 10 15 20 25
V (ml)
0
1000
2000
3000
m
AU
Mercenaria on Sephadex G-25 (A) and on FPLC (B).
the peptide by MALDI-TOF Mass Spectrum.
0.0 0.2 0.4 0.6
[Peptide] (µg/ml)
100
120
140
160
180
200
R
el
at
iv
e 
ac
tiv
ity
 (%
)
0.0 0.2 0.4 0.6
0
20
40
60
80
100
1
2
3
Fig. 5. Eﬀects of the peptide on enzyme activities of SOD (curve 1),
ALP (curve 2) and tyrosinase (curve 3).
Fig. 3. The inhibitory eﬀect of the peptide on BGC-823 cells. Column
(a) shows the concentration of peptide from Mercenaria (lg/ml)
Column (b) shows the inhibitory percentage (%).
B. Leng et al. / FEBS Letters 579 (2005) 1187–1190 1189tides concentration was 4.0 lg/ml, its inhibitory rate on the
growth of BGC-823 cells reached 60%.
The changes of the cytoskeletal morphology of BGC-823
cells treated by the peptide have been investigated (as shown
in Fig. 4). The cells ability of sticking to the bottle surface de-
creased, and the cell ﬁgures bulked and became anomalistic.
Moreover, the colouration inside the cells was deeper and
the conﬁguration of karyon turned blurred. These results indi-
cated that the peptide could aﬀect the cytoskeletal morphology
of BGC-823 cells.
3.4. Eﬀects of the peptide on the activities of SOD, ALP and
tyrosinase
The eﬀects of the peptide on the activity of SOD, ALP and
tyrosinase were studied, respectively. The results showed in
Fig. 5. The peptide could activate SOD (curve 1) as well as
ALP (curve 2). With increasing the concentrations of the pep-
tide, the activities of SOD and ALP increased. When the con-
centration of the peptide reached 0.5 lg/ml, the relative
activities of SOD and ALP were 188.5% and 122.0%, respec-
tively. The activation of peptide on SOD activity was more ob-
servable than on ALP activity. However, the peptide inhibited
tyrosinase with concentration-dependent as shown in Fig. 5
(curve 3). The IC50 value, the concentration leading to 50%Fig. 4. Eﬀect of the peptide on the cytoskeletal morphology of BGC-823 cells
and (C) treated by the peptide at the concentration of 8.0 lg/ml.activity of the enzyme lost, of the peptide was determined to
be 0.16 lg/ml.4. Discussion
In recent years, more and more people paid highly attention
to ﬁnding novel anti-cancer drugs from natural organism.
Anti-cancer peptides have attracted concern recently due to
the characteristic of multi-function, high-sensitivity, stability
and so on. In this investigation, a peptide from Mercenaria
was found to strongly inhibit the growth of BGC-823 cells
and obviously destroy the cytoskeletal morphology of the cells.
Compared with other anti-cancer drugs, Mercenaria peptide
exhibits higher inhibitory eﬃciency on BGC-823 cells. Results
obtained indicated that Mercenaria peptide might be used as a
potent anti-cancer drug.
ALP, a kind of membrane-bound metalloenzyme, is related
to transport process [13]. Its activity is usually low or repressed
in normal stomach tissue and will increase once the cell is ma-
lignly transformed [14]. ALP was usually regarded as an
important index of diﬀerentiation of cancer cells [15].Mercena-
ria peptide has obvious active eﬀect on ALP, which suggests
that the peptide inhibit cancer by the way of accelerating the
diﬀerentiation of cancer cells.(A) control; (B) treated by the peptide at the concentration of 4.0 lg/ml
1190 B. Leng et al. / FEBS Letters 579 (2005) 1187–1190Reactive oxygen species (ROS) joins in generation as well as
in malign transformation of cancer cells [16,17]. If compounds
can enhance the level of anti-oxidation and clear the ROS in
cancer cell, they may inhibit the cells growth. In our research,
Mercenaria peptide could activate the SOD, a very important
anti-oxidation enzyme, indicating that the peptide inhibited
the growth of BGC-823 cells by increasing the activity level
of SOD.
Abnormal accumulation of melanin pigments will induce
hyperpigmentation. Tyrosinase catalyzes the rate-limiting step
for the biosynthetic pathway of melanin pigments. It was re-
ported that Mercenaria extraction could evidently inhibit the
B16 melanoma and prolong the survival of mice by injecting
the extraction into the tumour [18]. Results in our research
proved that the peptide could eﬀectively inhibit the activity
of tyrosinase, which may be a proof why Mercenaria peptide
has inhibitory eﬀect on melanoma.
Acknowledgements: The present investigation was supported by Grant
2003N0052 of the Science and Technology Foundation of Fujian Prov-
ince and by the Project Sponsored by the Scientiﬁc Research Founda-
tion for the Returned Overseas Chinese Scholars, State Education
Ministry for Q.X.C.References
[1] Chen, H.Y., Cong, X.Q., Zhang, A. and Zhu, J.M. (1980) The
anticancer research of extraction from Mercenaria. Cancer Res.
Prev. Treat. 4, 3–7 (in Chinese).
[2] Zhang, J.X. and Xing, Y.P. (1990) Studies on anti-cancer activity
of Meretrix Meretrix nucleic acid. Oceanol. Et. Limnol. 21 (1),
88–91 (in Chinese).
[3] Dou, C.H., Huang, F., Huang, L.S., Li, P.Z. and Xiao, W.D.
(1999) An experimental study on the eﬀects of Meretrix polysac-
charide. J. Marine Drugs 2, 15–19 (in Chinese).
[4] Xu, X.L., Li, T.M. and Zhang, C.R. (1999) Study on antihyper-
glycemia and antihyper- lipemia of hydrolysate of Meretrix
Meretrix Linnaeus. J. Biochem. Pharm. 6, 298–299 (in Chinese).[5] Levy, R.I. (1985) Primary prevention of coronary heart disease by
lowering lipids: results and implications. Am. Heart J. 110 (5),
1116.
[6] He, Y.J., Wu, Q. and Zhu, R.F. (1995) Immunomodulating eﬀects
of the extract from clam Meretrix Meretrix on delayed hyper-
sensitivity in mice. J. Marine Drugs 3, 20–21, (in Chinese).
[7] Schmeer, M.R., Horton, D. and Tanimura, A. (1966) Mercenene,
a tumor inhibitor from Mercenaria: puriﬁcation and character-
ization studies. Life Sci. 5, 1169–1177.
[8] Klucar, J. and Al-Rubeai, M. (1997) G2 cell cycle arrest and
apoptosis are induced in Burkitts lymphoma cells by the
anticancer agent oracin. FEBS Lett. 400, 127–130.
[9] Hall, D.O. and Hawkins, S.E. (1975) Laboratory Manual of Cell
Biology, The English Universities Press Ltd., pp. 1–17.
[10] Chen, Q.X., Zhang, W. and Zheng, W.Z. (1996) Kinetics of
inhibition of alkaline phosphatase from green crab (Scylla
serrata) by N-bromosuccinimide. J. Protein Chem. 15 (4), 345–
350.
[11] Stewert, R.C. and Bewley, J.D. (1980) Lipid peroidation associ-
ated with accelerated aging of soybean axes. Plant Physiol. 65,
245–248.
[12] Chen, Q.X., Song, K.K., Wang, Q. and Huang, H. (2003)
Inhibitory eﬀects on mushroom tyrosinase by some alkylbenzal-
dehydes. J. Enzy. Inhib. Med. Chem. 18 (6), 491–496.
[13] Van Hoof, V. and De Broe, M.E. (1994) Interpretation and
clinical signiﬁcance of alkaline phosphatase isoenzyme patterns.
Crit. Rev. Clin. Lab. Sci. 31, 197–203.
[14] Wang, X.L., Ma, Y.E., Zhu, L.P. and Yang, Z.P. (1996) The
relationship between ALP and gastric cancer. J. Tumor Clin.
Heal. 3 (1), 28–29(in Chinese).
[15] Kim, Y.S. (1984) Eﬀects of sodium butyrate, dimethy sulfoxide
and retinoic acid or glycolipids on human retal adenocarcinoma
cells. J. Cancer Res. 44, 1648–1652.
[16] Chen, H.W. and Huang, H.C. (1998) Eﬀect of curcumin on cell
cycle progression and apoptosis in vascular smooth muscle cells.
J. Pharmacol. 124, 1029–1040.
[17] Hanif, R., Qiao, L. and Shiﬀ, S.J. (1997) Curcumin, a natural
plant phenolic food additive, inhibits cell proliferation and
induces cell cycle changes in colon adenocarcinoma cell lines by
a prostaglandin-independent pathway. J. Lab Clin. Med. 130,
576–584.
[18] Schmeer, A.C. (1979) Chemical characterization and biological
activity of an anticancer agent of marine origin. Physiol. Chem.
Phys. 11, 415–424.


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Inhibitory effects of anticancer peptide from Mercenaria on the BGC-823 cells and several enzymes

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