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Anticancer mechanism of peptide P18 in human leukemia K562 cells
Chengkang Tang,a,b Ximing Shao,a Binbin Sun,a Wenli Huang,a Feng Qiu,a Yongzhu Chen,a Ying-kang Shi,b
Er-yong Zhang,b Chen Wangc and Xiaojun Zhao*a,d
Received 5th October 2009, Accepted 11th December 2009
First published as an Advance Article on the web 7th January 2010
DOI: 10.1039/b920762g
Studies on the anticancer mechanism of peptide P18 in human
leukemia K562 cells revealed that P18 causes the death of
most K562 cells by depolarizing plasma membrane potential
and enhancingmembrane permeability, rather than activating
the classical apoptosis pathway. The mechanistic studies
indicate that disrupting plasma membrane is an effective
approach to kill cancer cells and help design more effective
peptide analogues in future cancer therapies.
Over the past 50 years, tremendous efforts have beenmade to treat
cancers, but unfortunately cancer is still one of the major causes
of mortality in the world. Cancer is a highly complex disease
caused by different genetic mutations and multiple molecular
alterations,1,2 and traditional anticancer drugs generally do not
differentiate between cancerous and normal cells, leading the
inefficiency and severe side effects.3 Therefore, the characteristics
of promising anticancer drugs should have excellent curative
properties and favorable toxicity profiles.4 Recently, antibiotic
peptides, which play an important role in the host defense
and innate immunity of insects, amphibians and mammals,5–10
have been developed as new anticancer drugs. Some of these
antibiotic peptides have shown attractive anticancer activity at
concentrations that are nontoxic toward normal mammalian
cells.11,12 Hence, antibiotic peptides may be a class of effective
agents for cancer treatment.8,13–16
P18 (KWKLFKKIPKFLHLAKKF) (Fig. 1), an antibiotic
peptide, is designed from the hybrid of cecropin A17 and magainin
2,18 which were isolated from Hyalaphora cecropia pupae and
the skin of the African clawed frog Xenopus Iaevis, respectively.
Previous studies have shown that P18 is highly active against
bacteria and fungi,19,20 the selective inhibitions of P18 against LPSaInstitute
for Nanobiomedical Technology and Membrane Biology, West
China Hospital, Sichuan University, Chengdu, 610041, China; Fax: +86-
28-8516-4072; Tel: +86-28-8516-4069
bDepartment of Thoracic and Cardiovascular Surgery,West China Hospital,
Sichuan University, Guo Xue Xiang 37, Chengdu, 610041, China
cNational Center of Nanoscience and Technology, Beijing 100080, China
dCenter for Biomedical Engineering, NE47-379, Massachusetts Institute of
Technology, Cambridge, MA, 02139-4307, USA. E-mail: xiaojunz@mit.edu
Fig. 1 The chemical structure of peptide P18 (KWKLFKKIPKFLHLAKKF).
stimulated inflammatory responses make P18 become a promising
anti-inflammatory agent.21 Most interestingly, P18 has significant
anticancer activity with low side effects,20,22 and these properties
make P18 a promising agent for cancer treatment. However, the
mechanism by which P18 exerts its anticancer activity and causes
the death of cancer cells is not well understood. Here, the primary
goal is to investigate the anticancer mechanism of P18 in human
leukemia K562 cells.
In this study, peptide P18 was synthesized by Shanghai Bootech
Bioscience & Technology Co., Ltd. with high purity (≥95%).
Crude peptide was purified by reverse-phase high-pressure liquid
chromatography (RP-HPLC). The identity of P18 was confirmed
by electrospray ionization mass spectrometry in an ion trap mass
spectrometer (Finnigan LCQ, San Jose, CA, USA). The working
solutions of P18 were prepared at different concentrations with
sterile water (18 MX; Millipore Milli-Q system).
We first examined the cytotoxicity of P18 by using the MTT
assay.23 MTT assay showed that P18 molecules have high anticancer
activities, causing remarkable dose-dependent death of
human cancer K562 cells (IC50 = 8.28 mM) (Fig. 2). Meanwhile,
P18 showed relatively low cytotoxicity against normal fibroblast
cell line NIH 3T3 (Fig. 2). These results indicated that P18 had
significant anticancer activity with low side effect.
Fig. 2 The cytotoxicity of P18 against human leukemia cell K562 and
fibroblast cell NIH3T3, bars, SD.
984 | Org. Biomol. Chem., 2010, 8, 984–987 This journal is © The Royal Society of Chemistry 2010
It is well known that aspartate-specific cysteine proteases
(caspases) play critical roles in the initiation and execution of
apoptotic pathways.24 Caspase-8 is one of the initiator caspases
for the death receptor pathway of apoptosis,25 and caspase-
9 is associated with the mitochondria-dependent pathway of
apoptosis,26 whereas caspase-3 plays critical roles in execution of
apoptosis as one of the executioner caspases.27 Therefore, we have
chosen caspase-3, 8 and 9 as molecular markers and examined
whether P18-mediated cell death is through the classical caspasedependent
apoptosis. After incubation with 20 mM P18 solution
or control solution (sterile water), K562 cells were harvested and
lysed, and the changes of caspases were detected by a Western
blot. We found that P18 treatment did not cause the cleavage of
procaspase-8 (Fig. 3a), 9 (Fig. 3b), 3 (Fig. 3c), and did not generate
active subunits (i.e. caspase-8, 9, 3).Western blot analysis showed
that P18 kills K562 cells without activating the caspase-dependent
apoptosis pathway.
Fig. 3 P18-induced K562 cell death is unrelated to the classical apoptosis
 ...........

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