doi:10.1016/j.peptides.2007.06.014
Copyright © 2007 Published by Elsevier Inc.
Induction of apoptosis in human leukemia K562 cells by cyclic lipopeptide from Bacillus subtilis natto T-2
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C.L. Wanga, T.B. Ngb, 
, 
, F. Yuana, Z.K. Liu and F. Liua, c
aDepartment of Microbiology, College of Life Science, Nankai University, Tianjin, China
bDepartment of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China
cTianjin Key Laboratory of Microbial Functional Genomics, Tianjin, China
Received 27 March 2007;
revised 7 June 2007;
accepted 7 June 2007.
Available online 20 June 2007.
Abstract
A new cyclic lipopeptide (CLP) purified from Bacillus subtilis natto T-2 dose dependently inhibited growth in human leukemia K562 cells. The results of fluorescent staining indicated that CLP brought about apoptosis in K562 cells. Flow cytometric analysis also demonstrated that CLP caused dose-dependent apoptosis of K562 cells through cell arrest at G1 phase. Western blotting revealed that CLP-induced apoptosis in K562 cells was associated with caspase-3 and poly(ADP-ribose)polymerase (PARP) protein. It is estimated that CLP inhibited proliferation in K562 cells by inducing apoptosis.
Keywords: Apoptosis; Human leukemia K562 cells; Cyclic lipopeptide (CLP); Anti-tumor
Fig. 1. Elution profile of CLPs from Pharmadex LH 20 gel filtration column.
Fig. 2. HPLC chromatograms of CLPs.
Fig. 3. (A) ESI mass spectrum of purified CLP. (B) Mass spectrum of the purified CLP at m/z 1072.
Fig. 4. Cytotoxic effects of CLPs on K562 and HLF cells. The cells were treated with various concentrations (2, 4, 8, 16, 32, 64 μg/ml) of CLPs for 24, 36, and 48 h. Cell viability was determined by MTT assay and was expressed as the mean ± S.D. of three separate experiments n = 3 each of the three experiments (
) K562 cells treated for 48 h; (■) K562 cells treated for 36 h; (
) K562 cells treated for 24 h; (–) fibroblasts treated for 48 h; (*) fibroblasts treated for 36 h; (○) fibroblasts treated for 24 h.
Fig. 5. Nuclear morphology of K562 cells. (A) Untreated control cells and (B) cells treated with 16 μg/ml CLPs for24 h. The representative cells were counted as apoptosis in fluorescence microscopy with 200× magnification. Results presented are representative of three independent experiments.
Fig. 6. Effect of CLP on cell-cycle distribution of K562 cells (n = 3). (A) Cell-cycle analysis of K562 cells following treatment in the absence of CLP for 24 h; (B) cell-cycle analysis of K562 cells following treatment with 8 μg/ml CLP for 24 h; (C) cell-cycle analysis of K562 cells following treatment with 16 μg/ml CLP for 24 h; (D) cell-cycle analysis of K562 cells following treatment with 32 μg/ml CLP for 24 h.
Fig. 7. Effect of CLP on caspase-3 and proteins in K562 cells. (A) Treatment with CLP at various concentrations for 9 h and (B) treatment with CLP at 32 μg/ml. The data shown are representative of three independent experiments.
Table 1.
The effects of CLP on the caspase-3 activity of K562 cells (n = 3)
K562 cells were treated with CLP and with or without the cell-permeable broad-spectrum caspase inhibitor (Z-VAD-fmk, 200 mM) prior to CLP treatment. Caspase-3 activity was assayed by using fluorogenic peptide substrates. Data are means ± S.D. of three independent experiments.
Table 2.
Effect of lipopeptide on cell-cycle distribution of K562 cells (n = 3)
(A) Cell-cycle analysis of control K562 cells for 24 h; (B) cell-cycle analysis of K562 cells following treatment with8 μg/ml CLP for 24 h; (C) cell-cycle analysis of K562 cells following treatment with 16 μg/ml CLP for 24 h; (D) cell-cycle analysis of K562 cells following treatment with 32 μg/ml CLP for 24 h. Data are means ± S.D. of three independent experiments. *P < 0.05 in groups by Student's test.
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