- Email: [email protected]
Human hemokinin-1 and human hemokinin-1(4–11), mammalian tachykinin peptides, suppress proliferation and induce differentiation in HL-60 cells
Peptides 30 (2009) 1514–1522
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Human hemokinin-1 and human hemokinin-1(4–11), mammalian tachykinin peptides, suppress proliferation and induce differentiation in HL-60 cells You-Li Zhao a,1, Yan Tao a,1, Cai-Yun Fu c, Zi-Qing Kong a, Qiang Chen a, Rui Wang a,b,* a Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Institute of Biochemistry and Molecular Biology, School of Basic Medical Sciences, State Key Laboratory of Applied Organic Chemistry, Lanzhou University, 222 Tian Shui South Road, Lanzhou 730000, China b State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong c College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 March 2009 Received in revised form 29 April 2009 Accepted 29 April 2009 Available online 9 May 2009
Human hemokinin-1 (h HK-1) and its truncated form h HK-1(4–11) are mammalian tachykinin peptides encoded by the TAC4 gene identified in human, and the biological functions of these peptides have not been well investigated. The tachykinins have shown immuno-regulatory activities in humans. In the present study, we investigated the effects of h HK-1 and h HK-1(4–11) on the proliferation and differentiation of a human promyelocyte leukemia cell line, HL-60. It is noteworthy that h HK-1 (1– 300 mM) displayed inhibitory effects on the proliferation of HL-60 cells in a dose- and time-dependent manner. The effect of suppressing proliferation induced by these peptides was accompanied by an accumulation of cell cycle in the S phase. Moreover, this peptide induced differentiation of HL-60 cells by significantly increasing the NBT-reduction activity. The effects induced by h HK-1(4–11) on HL-60 cells were similar to that of h HK-1, indicating that it is the active fragment of h HK-1. However these effects induced by h HK-1 or h HK-1(4–11) were not antagonized by the NK1 receptor antagonist SR140333 or the NK2 receptor antagonist SR48968. All the results indicated that h HK-1 and h HK-1(4–11) were able to significantly inhibit proliferation and induce differentiation and S phase arrest of a human promyelocyte leukemia cell line HL-60, which may not be mediated through the activation of classical tachykinin NK1 receptors and tachykinin NK2 receptors. Our observations also implied that h HK-1 and h HK-1(4–11) could act as immunomodulatory factors in cancer chemotherapy. ß 2009 Elsevier Inc. All rights reserved.
Keywords: h HK-1 and h HK-1(4–11) HL-60 Differentiation Proliferation inhibition SR140333 and SR48968
1. Introduction The mammalian tachykinins [27,42] are a family of evolutionary conserved peptides, which share the common C-terminal motif FXGLM-NH2 [5] (Table 1). For many years, the family consisted of three peptides that include substance P, neurokinin A and neurokinin B. Substance P and neurokinin A are products of the TAC1 gene, while neurokinin B is a product of the TAC2 (TAC3 in the human) gene [32]. More recently putative novel members of the tachykinin family, including hemokinins, have been cloned in the mouse [44], rat and human [15,29], which are encoded by TAC4, the third mammalian tachykinin gene. In human, the TAC4 gene can
* Corresponding author at: Institute of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Lanzhou University, 222 Tian Shui South Road, Lanzhou, Gansu 730000, China. Tel.: +86 931 8912567/852 34003755; fax: +86 931 8911255/852 23649932. E-mail address: [email protected] (R. Wang). 1 Both authors contributed equally to this work. 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.04.024
generate four distinct mRNAs and encodes hemokinin-1, endokinin A and endokinin B, which exhibit structural homology with known members of the tachykinin family [29,35]. Furthermore, human hemokinin-1 has the potential to yield a truncated form, hemokinin1(4–11), based on monobasic cleavage at N-terminal [15]. The biological actions of these tachykinins are mediated by at least three different transmembrane G-protein coupled receptors, designated tachykinin NK1, NK2 and NK3 receptors, which have been cloned and characterized pharmacologically [37]. The NK1 is the preferred receptor for substance P and hemokinin-1, the NK2 for neurokinin A, and the NK3 receptor for neurokinin B [3,6]. Each ligand can therefore interact with all receptors with varying affinity [31]. Many studies have investigated the binding affinity and agonist efficacy of h HK-1 at each of the three-mammalian tachykinin receptors. The result show that h HK-1 and h HK-1(4– 11) bind to the human NK1 receptor, with 14 and 70 times lower affinities, respectively, relative to SP [15]. Tachykinins are involved in a wide variety of biological actions such as smooth muscle contraction, vasodilatation, pain
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522 Table 1 Amino acid sequences of the tachykinin peptides substance P, neurokinin A, neurokinin B, hemokinin-1. Tachykinin
Amino acid sequence
Substance P Neurokinin A Neurokinin B Hemokinin-1 (mouse and rat) Hemokinin-1 (human) Hemokinin-1 (4–11) (human)
R
R T
P H D S G
K K M R K
P T H T A A
Q D D R S S
Q S F Q Q Q
F F F F F F
F V V Y F F
G G G G G G
L L L L L L
M-NH2 M-NH2 M-NH2 M-NH2 M-NH2 M-NH2
transmission, neurogenic inflammation, activation of the immune system and stimulation of endocrine gland secretion [18]. Many studies have revealed that tachykinins and their receptors are also expressed in non-neuronal cells such as immune cells and glial cell types [21,27,30]. Moreover, SP is known to have a regulatory effect on cells of the immune system. SP, act as a immunomodulatory agent, activates the oxidative metabolism of neutrophils [41], the degranulation of mast cells [38], monocyte chemotaxis [19,39] and modulates lymphocyte proliferation [33,34], antibody synthesis [16,43], and macrophage function [22]. However, little if anything is known about the effects of this immunomodulatory peptide on the proliferation and differentiation of human promyelocyte leukemia cells and since the discovery of human hemokinin-1, only four citations are available, two focused on the regulation of the contractile responses of human hemokinin-1 in human uterus [35] and mouse uterus [32], and the other focused on the regulation of pain after intracerebroventricular administration of human hemokinin-1 and human hemokinin-1(4–11) [11], and another focused on cardiovascular responses after intravenous administration of human hemokinin-1 and its truncated form hemokinin-1(4–11) [14]. In the present study, the effects of SP, h HK-1 and its truncated form h HK-1(4–11) on the proliferation and differentiation of HL60 are first investigated. The antiproliferation and differentiation effects induced by h HK-1 and its truncated form h HK-1(4–11) were determined by measuring cell viability, cell cycle phase distribution and NBT-reduction activity. The roles of NK1 receptors antagonists SR140333 and NK2 receptors antagonists SR48968 were also investigated. 2. Materials and methods 2.1. Chemicals The SP, h HK-1 and h HK-1(4–11) peptides used in this study were synthesized in our laboratory by the solid-phase peptide synthesis method and purified by high-performance liquid chromatography (HPLC). SP, h HK-1 and h HK-1(4–11) were dissolved in deionized water. The antagonists SR140333 or (S)1(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl) piperidin-3-yl]ethyl)-4-phenyl-1-azoniabicyclo [2.2.2] octane chloride and SR48968 or (S)-N-methyl-N[4-(4-acetylamino-4phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide were generous gifts from Sanofi-Synthelabo Recherche (Montpellie, France), and they were dissolved in deionized water with 6% DMSO (dimethyl sulphoxide) and further diluted with deionized water. Control trials were performed in the presence of corresponding concentration of DMSO to rule out any possible nonspecific action of this solvent. Nitrotetrazolium blue chloride (NBT) (Zetan Chemical, PR China) was dissolved in deionized water. Phorbol 12-myristate 13-acetate (TPA) (Sigma) was dissolved in deionized water with 1% CHCl3 and further diluted with deionized water.
1515
2.2. Cells culture HL-60 cells were cultured in RPMI-1640 medium (Gibco-BRL, USA) supplemented with heat-inactivated 10% neonatal bovine serum (NBS) (Sijiqing Biotech, PR China), 2 mM L-glutamine, penicillin (100 IU/mL) and streptomycin (100 mg/mL). The cells were incubated in a humidified incubator containing 5% CO2 at 37 8C. The peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood. The peripheral venous blood was drawn from healthy volunteers. Blood samples were collected into sterile bags each containing 25 mL of citratephosphate dextrose anticoagulant and diluted to 1:2 with phosphate buffer saline (PBS). The PBMCs were separated by density gradient centrifugation at 2000 rpm for 20 min on FicollIsopaque (density, 1.077 g/mL). The isolated PBMCs were washed twice, counted by Trypan blue exclusion and suspended in RPMI-1640 growth medium supplied with 15% NBS. The cells were incubated in a humidified incubator containing 5% CO2 at 37 8C. 2.3. Effects of h HK-1 and h HK-1(4–11) on the proliferation of HL-60 cells The cytotoxic activities of SP, h HK-1 and its truncated form h HK-1(4–11) were evaluated in HL-60 using the MTT assay [24]. This assay is based on the reduction of the yellow colored 3-(40 , 50 -dimethylthiazol-20 -yl)-2, 5-diphenyl-tetrazolium bromide (MTT) by mitochondrial dehydrogenase of metabolically active cells to a purple-blue formazan. Cells were plated in 96-well plates (3 104 cells/mL) for cells in 100 mL of medium. SP, h HK1 and h HK-1(4–11) were dissolved in deionized water, and then they were added to each well at a final concentration of 1– 300 mM, respectively. The cells were exposed to the drugs for 24 h, 48 h, 72 h and 96 h. The control group received the same amount of deionized water. Thereafter, the plates were centrifuged and the medium was replaced by fresh medium (100 mL) containing 5 mg/mL MTT. After 4 h incubation, the MTT formazan product was dissolved in 150 mL DMSO, and absorbance was measured using a multiplate reader (Spectra Count, Packard, Ont., Canada). The effects of each drug were quantified as the percentage of control absorbance of reduced dye at 570 nm. As for the Trypan blue dye exclusion assay, cells were seeded at density of 3 104 cells/mL onto a 12-well plate for 24 h, then SP, h HK-1 and h HK-1(4–11) were added to medium at various indicated times and concentrations, respectively. After incubation, cells exposed to 0.2% Trypan blue were counted in a hemocytometer. 2.4. Effects of h HK-1 and h HK-1(4–11) on cell cycle distribution of HL-60 cells Cells were collected, washed, suspended in cold PBS, fixed in 75% ethanol at 20 8C overnight, washed and resuspended in PBS with RNAase (0.1 mg/mL). Cellular DNA was stained with PI and cell samples were analyzed on Becton Dickson Flow Cytometer (BD Biosciences, USA) using CELL Quest software (Verity Software House, Inc., Topsham, ME). 2.5. NBT reducing activity As a marker of cell differentiation, the reduction of NBT in HL60 cells was determined by a previously described method [2,9,20]. HL-60 cells were plated at a density of 3 104 cells/mL, and treated with h HK-1 or h HK-1(4–11) (1–300 mM) for 72 h. Briefly, after each treatment, cells at a density of 1 106 cells/
1516
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
mL were incubated for 30 min in RPMI-1640 growth medium with 10 mg/mL of TPA and 1 mg/mL of NBT at 37 8C. Then, the cells were collected and suspended in DMSO. The absorbance (OD) of the NBT-reduction product (formazane solutions) was measured at 570 nm in a kinetic microplate reader. 2.6. Tachykinins receptors antagonists investigation To investigate whether the antiproliferative effects induced by h HK-1 or h HK-1(4–11) were mediated through the activation of tachykinin NK1 or NK2 receptors, SR140333 (a selective NK1 receptor antagonist) or SR48968 (a selective NK2 receptor antagonist) was used at a range of concentrations from 0.1 mM to 30 mM. 2.7. Statistical analysis Data obtained from different experiments were presented as mean S.E.M from at least three independent experiments and evaluated by analysis of variance (ANOVA) or Student’s t-test, using a significance level of 5%.
3. Results 3.1. Effects of h HK-1 and h HK-1(4–11) on the proliferation in HL-60 cells To determine the effects of these tachykinin peptides on cell viability, HL-60 cells were treated with increasing concentrations of SP, h HK-1 and h HK-1(4–11) for 24 h, 48 h, 72 h, 96 h, respectively, and cell viability was assessed by the MTT assay. As shown in Fig. 1, a significant increase in antiproliferation effect was detected in a dose- and time-dependent manner after treatment with 1 mM, 3 mM, 10 mM, 30 mM, 100 mM and 300 mM h HK-1 or h HK-1(4–11). The antiproliferative effect reached at 36%, 34%, 70% and 21% after 24 h, 48 h, 72 h and 96 h exposure time treated with h HK-1 (300 mM), respectively (Fig. 1B), while 300 mM of h HK1(4–11) could lead to an increase on the antiproliferative effect of 49%, 44%, 60% and 59% after 24 h, 48 h, 72 h and 96 h exposure time, respectively (Fig. 1C). The antiproliferation effect of h HK-1 and h HK-1(4–11) after 72 h of incubation displayed the most potency compared with those of after 24 h, 48 h and 96 h incubation. Moreover, it was showed that the effect of SP was
Fig. 1. Effect of SP, h HK-1 and h HK-1(4–11) on cell growth in HL-60 cells in vitro. HL-60 cells were treated with the indicated concentrations of SP (A), h HK-1 (B) and h HK1(4–11) (C) for 24 h, 48 h, 72 h and 96 h and cell viability was determined using the MTT assay. The cellular growth of HL-60 cells was inhibited significantly in a dose- and time-dependent manner. The results are presented as the mean S.E.M of at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, statistically significant differences SP (A), h HK-1 (B) or h HK-1(4–11) (C) vs the control. Control: untreated cells. (D) Comparison of the potency of antiproliferation effect of 72 h among SP, h HK-1 and h HK-1(4–11) (#P < 0.05, ##P < 0.01, statistically significant differences between h HK-1 vs SP; *P < 0.05 and **P < 0.01, statistically significant differences between h HK-1(4–11) vs SP).
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
much weaker than h HK-1 or h HK-1(4–11) (Fig. 1D), and SP exerted hardly a marked effect on the proliferation of HL-60 cells (Fig. 1A). The inhibition of 100 mM h HK-1, h HK-1(4–11) and SP was 62.85 9.15%, 49.26 7.25% and 8.18 2.09%, respectively (Fig. 1D). Furthermore, morphological assessment of HL-60 cultures revealed that there were no significantly visible morphological differences before and after treatment with these peptides (Fig. 2). 3.2. Effects of h HK-1 and h HK-1(4–11) on the proliferation of PBMCs We also investigated the effects of SP, h HK-1 and h HK-1(4– 11) on the proliferation of PBMCs. SP, h HK-1 and h HK-1(4–11) displayed no inhibitory effect on the proliferation of PBMCs within the concentration range from 1 mM to 300 mM (data not shown). This suggested that SP, h HK-1 and h HK-1(4–11) had no potential side effects on normal blood cells even though they exhibited significant inhibitory effects on the proliferation of HL60 within the concentration range from 1 mM to 300 mM.
1517
3.3. Effects of h HK-1 and h HK-1(4–11) on the cell cycle distribution of HL-60 cells We also studied the cell cycle distribution of HL-60 cells after the treatment with h HK-1 and h HK-1(4–11) (200 mM and 300 mM) for 72 h (Fig. 3). The percentage of HL-60 cells in the S phase was increased from 28.8% (vehicle-treated cells, control group) to 56.4% and 57.4% after the treatment with 200 mM and 300 mM h HK-1 for 72 h, respectively. However, the percentage of the G0/G1 phase was decreased from 53.4% (vehicle-treated cells, control group) to 39.7% and 40.2% after treatment with 200 mM and 300 mM h HK-1 for 72 h, respectively (Fig. 3A and C). Similar results were also observed on h HK-1(4–11). The percentage of HL-60 cells in the S phase was increased from 27.3% (vehicle-treated cells, control group) to 48.3% and 52.2% after treatment with 200 mM and 300 mM h HK-1(4–11) for 72 h, respectively. Also, the percentage of the G0/G1 phase was decreased from 55.8% (control cells) to 45.8% and 44.0% after
Fig. 2. Photomicrographs of HL-60 cells after 72 h of incubation with h HK-1 and h HK-1(4–11) (1–300 mM) showed the concentration-dependent inhibitory effect of h HK-1 (A) and h HK-1(4–11) (B) on cell proliferation. HL-60 cells were plated at a density of 3 104 cells/mL and counted with a hemocytometer. Cell density decreased in a concentration-dependent manner as the concentration of h HK-1 and h HK-1(4–11) are increased from 1 mM to 300 mM. There is no significantly visible change in cell morphology of HL-60 cells treated with h HK-1 and h HK-1(4–11).
1518
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
treatment with 200 mM and 300 mM h HK-1(4–11) for 72 h, respectively (Fig. 3B and D). These results indicated that h HK-1 and h HK-1(4–11) could promote cell cycle accumulation in the S phase of HL-60 cells. These results were consistent with the
results of cell numbers and viability assays. Therefore, the HL-60 cell growth inhibition exerted by h HK-1 and h HK-1(4–11) was probably due to the inhibition of cellular progression to S phases.
Fig. 3. Changes of cell cycle distribution of HL-60 cells after treatment with h HK-1 and h HK-1(4–11). HL-60 cells were plated at a density of 3 104 cells/mL, and cultured in mediums containing h HK-1 and h HK-1(4–11) at various concentrations (200 mM and 300 mM). After incubation, the cell cycle distribution was measured by flow cytometry. (A and C) Cells treated with h HK-1 for 72 h, while (B and D): cells treated with h HK-1(4–11) for 72 h. Control: untreated cells. Values represent the mean S.E.M. of data from three different experiments. *P < 0.05 vs. control and **P < 0.01 vs. control.
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
1519
Fig. 3. (Continued ).
3.4. Induction of NBT-reduction activity of HL-60 by h HK-1 and h HK-1(4–11) We further assessed NBT-reduction in the HL-60 cells treated with h HK-1 and h HK-1(4–11) to determine whether they induced the differentiation of the cells (Fig. 4). Rarely were NBT positive cells observed throughout the 72 h incubation with the control cells. h HK-1 and h HK-1(4–11) (30 mM) significantly induced the generation of NBT-reducing activity, and the absorbance of the NBT-reduction product (OD570 nm) increased from 0.376 (control) to 0.632 (300 mM h HK-1) (Fig. 4A) and from 0.541 (control) to 1.124 (300 mM h HK-1(4–11)) (Fig. 4B) at 72 h, respectively. More and more cells became NBT positive as the concentration of h HK-1 and h HK-1(4–11) increased. 3.5. Effects of tachykinin receptors antagonists To explore whether the tachykinin NK1 receptor or the tachykinin NK2 receptor is involved in h HK-1 or h HK-1(4–11)induced antiproliferation responses, the non-peptide NK1 receptor antagonist SR140333 or the non-peptide NK2 receptor antagonist SR48968 is added 45 min before h HK-1 or h HK-1(4–11), respectively. From Fig. 5, we could see that although the high concentration of SR140333 and SR48968 (10 mM) inhibited the proliferation of HL-60, the antiproliferation induced by h HK-1 or h HK-1(4–11) were not antagonized by the lower concentration of SR140333 (Fig. 5C and E) and SR48968 (Fig. 5D and F), indicating that the antiproliferation induced by h HK-1 or h HK-1(4–11) may not be mediated through the activation of tachykinin NK1 receptors and tachykinin NK2 receptors at the HL-60 cells. 4. Discussion
Fig. 4. Effects of h HK-1 and h HK-1(4–11) on the differentiation of HL-60 cells were determined by NBT-reduction. HL-60 cells were plated at a density of 3 104 cells/ mL, and were treated with h HK-1 and h HK-1(4–11) (10–300 mM) for 72 h. h HK-1 and h HK-1(4–11) significantly induced the generation of NBT-reduction activity in a concentration-dependent manner. (A) Cells treated with h HK-1 for 72 h, (B) cells treated with h HK-1(4–11) for 72 h. Values represent the mean S.E.M. of data from three different experiments. *P < 0.05 vs. control and **P < 0.01 vs. control.
Recently, Kurtz et al. reported the isolation and characterization of the human TAC4 gene, which encodes human hemokinin-1 and its N-terminally truncated form named human hemokinin-1(4– 11) [15]. Using various in vitro and in vivo investigations, it has been shown that HK-1 peptides behave as SP-like peptides [3,4,15,29,44]. SP is a ubiquitous, 11 amino acid peptide that has been associated with many physiological processes in the cardiovascular, nervous, respiratory and immune systems [36]. SP and its preferred receptor, NK1, have been identified in immune cells [27] and implicated in immunomodulation and inflammation [21,44]. The expression of neuropeptides and their receptors has
1520
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
Fig. 5. The effects of SR140333 (A, C and E) and SR48968 (B, D and F) on the inhibitory effects of SP, h HK-1 and h HK-1(4–11) on the proliferation of HL-60 cells. The cell viability was determined by using the MTT assay. The results are presented as the mean S.E.M of at least three independent experiments.
gained increasing interest in the field of tumor biology since these peptides were shown to be capable of influencing cell differentiation and proliferation [4]. However, we have very little insight into the effects of this immunomodulatory peptide on the proliferation and differentiation of human promyelocyte leukemia cells. In the present investigation, the effects of SP, h HK-1 and h HK-1(4–11) on the proliferation and differentiation of human leukemia cell lines HL-60 were confirmed. In our present investigation, h HK-1 caused a significant doseand time-dependent suppression of the proliferation in HL-60 cells
within a concentration range from 1 mM to 300 mM. The suppression induced by h HK-1 was accompanied by an accumulation of cell cycle in the S phase. The cell cycle related mechanisms may take effect in the HL-60 cell cycle accumulation induced by h HK-1. A common goal of cancer therapy is to restore normal growth control in transformed tissues [7]. One area that has been intensively studied in recent years is biological modifiers of cancer growth which are designed to retard proliferation [28], to induce differentiation of these cells to a quiescent, nondividing stage [17,40]. Cell differentiation is disrupted in the acute
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
leukaemia and many solid tumors [10]. In the present study, h HK1 was able to induce the differentiation of HL-60 cells. This may provide important implications that h HK-1 could act as a valuable human leukaemia therapy agent because of its potent effect on the differentiation of human leukaemia cells. h HK-1(4–11) is the truncated version of h HK-1 based on the monobasic cleavage site following a lysine in the +3 position [15]. Cleavage at that site is likely as the sequence conforms to a number of the consensus rules for non-proline-directed monobasic processing, namely a single basic residue (lysine) with an additional basic residue at the 3 position (lysine) and a short side chain amino acid (alanine) in the +1 position relative to the cleavage site [8]. Our present data showed that h HK-1(4–11) produced dose- and time-related antiproliferation effects which were equally effective with but slightly less potent than h HK-1. Moreover, h HK-1(4–11) could also induce a S arrest of the cell cycle and increase the NBT-reduction activity of HL-60 cells significantly, similar to that of h HK-1. All above results indicated that as the active fragment of human hemokinin-1, h HK-1(4–11) could also act as a valuable human leukaemia therapy agent. Our results show that the antiproliferation induced by SP, h HK1 or h HK-1(4–11) could not be antagonized by NK1 receptor antagonist SR140333 and the NK2 receptor antagonist SR48968, indicating that the antiproliferation induced by SP, h HK-1 or h HK1(4–11) may not be mediated through the activation of tachykinin NK1 receptors and tachykinin NK2 receptors. This is consistent with the results of studies by Kavelaars and co-workers that there are multiple pathways for SP to activate the immune system, apart from the classical NK1 receptor-mediated pathway, SP can also stimulate cells via the non-NK1 receptor as well as via receptorindependent routes [12,13]. A receptor-independent mechanism of action of SP can be explained by the fact that the peptide has a cationic amphiphilic nature [25,26]. It has been shown that interaction of cationic amphiphilic peptides with a negatively charged cell membrane result in insertion of the peptide into the membrane [13]. Mousli et al. [25,26] have suggested that the peptide then acquires a membrane-spanning conformation, which enables direct interaction with proteins in the cytosolic compartment. Considering that cancer treatment has severe side effects, it is very important to determine how harmful new drugs may be to normal dividing cells such as proliferating lymphocytes [1,23,45]. Interestingly, despite the fact that HL-60 leukemia cells and lymphocytes are derived from white blood cell lineages, h HK-1 and h HK-1(4–11) seems to be selective for tumor cells, since no toxicity against normal cells was observed in our assay. In summary, the body of data derived from our present experiments strongly indicates, for the first time, that h HK-1, the well-known immunomodulatory polypeptide, was able to significantly suppress the proliferation and induce differentiation and S phase arrest of HL-60 cells. Strikingly, the truncated version of h HK-1, h HK-1(4–11) has the similar effects on HL-60 cells as that of h HK-1. Moreover, the effects may not be mediated through the activation of classical tachykinin NK1 receptor and NK2 receptor. The combination of differentiation-induction and growth inhibition by h HK-1 and h HK-1(4–11) should not only make them act as immunomodulatory factors in cancer chemotherapy, but also facilitate the analysis of the role of the tachykinin peptides encoded by the newly identified TAC4 gene in lymphocytic leukemia and may open novel pharmacological interventions. Acknowledgments This study was supported by the grants from the National Natural Science Foundation of China (Nos. 20525206, 20772052, 20621091 and 90813012), the Specialized Research Fund for the
1521
Doctoral Program in Higher Education Institutions and the Chang Jiang Program of the Ministry of Education of China. References [1] Anazetti MC, Melo PS, Duran N, Haun M. Comparative cytotoxicity of dimethylamide-crotonin in the promyelocytic leukemia cell line (HL-60) and human peripheral blood mononuclear cells. Toxicology 2003;188:261–74. [2] Baehner RL, Nathan DG. Quantitative nitroblue tetrazolium test in chronic granulomatous disease. N Engl J Med 1986;278:971–6. [3] Bellucci F, Carini F, Catalani C, Cucchi P, Lecci A, Meini S, et al. Pharmacological profile of the novel mammalian tachykinin, hemokinin 1. Br J Pharmacol 2002;135:266–74. [4] Berger A, Paige CJ. Hemokinin-1 has substance P-like function in U-251 MG astrocytoma cells: a pharmacological and functional study. J Neuroimmunol 2005;164:48–56. [5] Berger A, Tran AH, Paige CJ. Co-regulated decrease of Neurokinin-1 receptor and Hemokinin-1 gene expression in monocytes and macrophages after activation with pro inflammatory cytokines. J Neuroimmunol 2007;187:83– 93. [6] Camarda V, Rizzi A, Calo G, Guerrini R, Salvadori S, Regoli D. Pharmacological profile of hemokinin 1: a novel member of the tachykinin family. Life Sci 2002;71:363–70. [7] Campbell MJ, Reddy GS, Koeffler HP. Vitamin D3 analogs and their 24-Oxo metabolites equally inhibit clonal proliferation of a variety of cancer cells but have differing molecular effects. J Cell Biochem 1997;66:413–25. [8] Devi L. In: Fricker LD, editor. Peptide biosynthesis and processing. Florida: CRC Press; 1991. p. 175–98. [9] Eugenia Riveiro M, Shayo C, Monczor F, Fernandez N, Baldi A, De Kimpe N, et al. Induction of cell differentiation in human leukemia U-937 cells by 5-oxygenated-6,7-methylenedioxycoumarins from Pterocaulon polystachyum. Cancer Lett 2004;210:179–88. [10] Fan YZ, Chang H, Yu Y, Liu J, Zhao L, Yang DJ, et al. Thymopentin (TP5), an immunomodulatory peptide, suppresses proliferation and induces differentiation in HL-60 cells. BBA Mol Cell Res 2006;1763:1059–66. [11] Fu CY, Zhao YL, Dong L, Chen Q, Ni JM, Wang R. In vivo characterization of the effects of human hemokinin-1 and human hemokinin-1(4–11), mammalian tachykinin peptides, on the modulation of pain in mice. Brain Behav Immun 2008;22:850–60. [12] Jeurissen F, Kavelaars A, Korstjens M, Broeke D, Franklin RA, Gelfand EW, et al. Monocytes express a non-neurokinin substance P receptor that is functionally coupled to MAP kinase. J Immunol 1994;152:2987–94. [13] Kavelaars A, Jeurissen F, Von Frijtag Drabble Kunzel J, Van Roijen JH, Rijkers GT, Heijnen CJ. Substance P induces a rise in intracellular calcium concentration in human T lymphocytes in vitro: evidence of a receptor-independent mechanism. J Neuroimmunol 1993;42:61–70. [14] Kong ZQ, Fu CY, Chen Q, Wang R. Cardiovascular responses to intravenous administration of human hemokinin-1 and its truncated form hemokinin1(4–11) in anesthetized rats. Eur J Pharmacol 2008;590:310–6. [15] Kurtz MM, Wang R, Clements MK, Cascieri MA, Austin CP, Cunningham BR, et al. Identification, localization and receptor characterization of novel mammalian substance P-like peptides. Gene 2002;296:205–12. [16] Laurenzi MA, Person MAA, Dalsgaard CJ, Haegerstrand A. The neuropeptide substance P stimulates production of interleukin 1 in human blood monocytes: activated cells are preferentially influenced by the neuropeptide. Scand J lmmunol 1990;31:529–33. [17] Liu L, Shack S, Stetler-Stevenson WG, Hudgins WR, Samid D. Differentiation of cultured human melanoma cells induced by the aromatic fatty acids phenylacetate and phenylbutyrate. J Invest Dermatol 1994;103:335–40. [18] Longmore J, Hill RG, Hargreaves RJ. Neurokinin-receptor antagonists: pharmacological tools and therapeutic drugs. Can J Physiol Pharm 1997;75:612– 21. [19] Lotz M, Vaughn JH, Carson DA. Effects of neuropeptides on production of inflammatory cytokines by human monocytes. Science 1988;241:1218–21. [20] Lucas DL, Tanuma S, Davies PJ, Wright DG, Johnson GS. Maturation of human promyelocytic leukemia cells induced by nicotinamide: evidence of a regulatory role for ADP-ribosylation of chromosomal proteins. J Cell Physiol 1984;121:334–40. [21] Maggi CA. The effects of tachykinins on inflammatory and immune cells. Regul Pept 1997;70:75–90. [22] McGillis J, Mitsuhashi M, Payan D. Immunologic properties of substance P. In: Ader R, Felten DL, Cohen N, editors. Psychoneuroimmunology. 2nd ed., San Diego, CA: Academic Press; 1991. p. 209–23. [23] Montenegro RC, De Vasconcellos MC, Bezerra FS, Andrade-Neto M, Pessoa C, De Moraes MO, et al. Pisosterol induces monocytic differentiation in HL-60 cells. Toxicol In Vitro 2007;21:795–800. [24] Mosmann T. Rapid colorimetric assays for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;16:55–63. [25] Mousli M, Bronner C, Landry Y, Bockaert J, Rouot B. Direct activation of GTPbinding regulatory proteins (G-proteins) by substance P and compound 48/80. FEBS Lett 1990;259:260–2. [26] Mousli M, Bued JL, Bronner C, Rouot B, Landry Y. G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides. Trends Pharmacol Sci 1990;11:358–62.
1522
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
[27] Nelson DA, Bost KL. Non-neuronal mammalian tachykinin expression. Front Biosci 2004;9:2166–76. [28] Novichenko N, Konno S, Nakajima Y, Hsieh TC, Xu W, Turo K, et al. Growth attenuation in a human prostate cell line mediated by a phorbol ester. Proc Soc Exp Biol Med 1995;209:152–6. [29] Page NM, Bell NJ, Gardiner SM, Manyonda IT, Brayley KJ, Strange PG, et al. Characterization of the endokinins: human tachykinins with cardiovascular activity. Proc Natl Acad Sci U S A 2003;100:6245–50. [30] Palma C, Maggi CA. The role of tachykinins via NK1 receptors in progression of human gliomas. Life Sci 2000;67:985–1001. [31] Patacchini R, Maggi CA. Peripheral tachykinin receptors as targets for new drugs. Eur J Pharmacol 2001;429:13–21. [32] Patak E, Pennefather JN, Candenas ML, Kerr K, Exintaris B, Ziccone S, et al. Functional characterisation of hemokinin-1 in mouse uterus. Eur J Pharmacol 2008;601:148–53. [33] Payan DG. Neuropeptides and inflammation. Annu Rev Med 1989;40:341– 52. [34] Payan DG, Levine JD, Goetzl EJ. Modulation of immunity and hypersensitivity by sensory neuropeptides. J Immunol 1983;132:1601–4. [35] Pennefather JN, Patak E, Ziccone S, Lilley A, Pinto FM, Page NM, et al. Regulation of the stimulant actions of neurokinin A and human hemokinin-1 on the human uterus: a comparison with histamine. Biol Reprod 2006;75:334–41. [36] Pernow B. Substance P. Pharmacol Rev 1983;35:85–141.
[37] Regoli D, Drapeau G, Dion S, D’Orle0 ans-Juste P. Pharmacological receptors for substance P and neurokinins. Life Sci 1987;40:109–17. [38] Repke H, Bienert M. Mast cell activation a receptor-independent mode of substance P action. FEBS Lett 1987;221:236–40. [39] Ruff MR, Wahl SM, Pert CB. Substance P receptor mediated chemotaxis of human monocytes. Peptides 1985;6:107–11. [40] Samid D, Shack S, Myers CE. Selective growth arrest and phenotypic reversion of prostate cancer cells in vitro by nontoxic pharmacological concentrations of phenylacetate. J Clin Invest 1993;91:2288–95. [41] Serra MC, Bazzoni F, Della Bianca V, Greskowiak M, Rossi F. Activation of human neutrophils by substance P. Effect on oxidative metabolism, cytosolic Ca2+ concentration and inositol phosphate formation. J Immunol 1988;141:2118–24. [42] Severini C, Improta G, Falconieri-Erspamer G, Salvadori S, Erspamer V. The tachykinin peptide family. Pharmacol Rev 2002;54:285–322. [43] Stanisz AM, Befus D, Bienenstock J. Differential effects of vasoactive intestinal peptide, substance P, and somatostatin on immunoglobulin synthesis and proliferations by lymphocytes from Peyer’s patches, mesenteric lymph nodes, and spleen. J Immunol 1986;136:152–6. [44] Zhang Y, Lu L, Furlonger C, Wu GE, Paige CJ. Hemokinin is a hematopoieticspecific tachykinin that regulates B lymphopoiesis. Nat Immunol 2000;1:392–7. [45] Zuco V, Supino R, Righetti SC, Cleris L, Marchesi E, Passerine Gambacorti C, et al. Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer Lett 2002;175:17–25.
Contents lists available at ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Human hemokinin-1 and human hemokinin-1(4–11), mammalian tachykinin peptides, suppress proliferation and induce differentiation in HL-60 cells You-Li Zhao a,1, Yan Tao a,1, Cai-Yun Fu c, Zi-Qing Kong a, Qiang Chen a, Rui Wang a,b,* a Key Laboratory of Preclinical Study for New Drugs of Gansu Province, Institute of Biochemistry and Molecular Biology, School of Basic Medical Sciences, State Key Laboratory of Applied Organic Chemistry, Lanzhou University, 222 Tian Shui South Road, Lanzhou 730000, China b State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong c College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 March 2009 Received in revised form 29 April 2009 Accepted 29 April 2009 Available online 9 May 2009
Human hemokinin-1 (h HK-1) and its truncated form h HK-1(4–11) are mammalian tachykinin peptides encoded by the TAC4 gene identified in human, and the biological functions of these peptides have not been well investigated. The tachykinins have shown immuno-regulatory activities in humans. In the present study, we investigated the effects of h HK-1 and h HK-1(4–11) on the proliferation and differentiation of a human promyelocyte leukemia cell line, HL-60. It is noteworthy that h HK-1 (1– 300 mM) displayed inhibitory effects on the proliferation of HL-60 cells in a dose- and time-dependent manner. The effect of suppressing proliferation induced by these peptides was accompanied by an accumulation of cell cycle in the S phase. Moreover, this peptide induced differentiation of HL-60 cells by significantly increasing the NBT-reduction activity. The effects induced by h HK-1(4–11) on HL-60 cells were similar to that of h HK-1, indicating that it is the active fragment of h HK-1. However these effects induced by h HK-1 or h HK-1(4–11) were not antagonized by the NK1 receptor antagonist SR140333 or the NK2 receptor antagonist SR48968. All the results indicated that h HK-1 and h HK-1(4–11) were able to significantly inhibit proliferation and induce differentiation and S phase arrest of a human promyelocyte leukemia cell line HL-60, which may not be mediated through the activation of classical tachykinin NK1 receptors and tachykinin NK2 receptors. Our observations also implied that h HK-1 and h HK-1(4–11) could act as immunomodulatory factors in cancer chemotherapy. ß 2009 Elsevier Inc. All rights reserved.
Keywords: h HK-1 and h HK-1(4–11) HL-60 Differentiation Proliferation inhibition SR140333 and SR48968
1. Introduction The mammalian tachykinins [27,42] are a family of evolutionary conserved peptides, which share the common C-terminal motif FXGLM-NH2 [5] (Table 1). For many years, the family consisted of three peptides that include substance P, neurokinin A and neurokinin B. Substance P and neurokinin A are products of the TAC1 gene, while neurokinin B is a product of the TAC2 (TAC3 in the human) gene [32]. More recently putative novel members of the tachykinin family, including hemokinins, have been cloned in the mouse [44], rat and human [15,29], which are encoded by TAC4, the third mammalian tachykinin gene. In human, the TAC4 gene can
* Corresponding author at: Institute of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Lanzhou University, 222 Tian Shui South Road, Lanzhou, Gansu 730000, China. Tel.: +86 931 8912567/852 34003755; fax: +86 931 8911255/852 23649932. E-mail address: [email protected] (R. Wang). 1 Both authors contributed equally to this work. 0196-9781/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2009.04.024
generate four distinct mRNAs and encodes hemokinin-1, endokinin A and endokinin B, which exhibit structural homology with known members of the tachykinin family [29,35]. Furthermore, human hemokinin-1 has the potential to yield a truncated form, hemokinin1(4–11), based on monobasic cleavage at N-terminal [15]. The biological actions of these tachykinins are mediated by at least three different transmembrane G-protein coupled receptors, designated tachykinin NK1, NK2 and NK3 receptors, which have been cloned and characterized pharmacologically [37]. The NK1 is the preferred receptor for substance P and hemokinin-1, the NK2 for neurokinin A, and the NK3 receptor for neurokinin B [3,6]. Each ligand can therefore interact with all receptors with varying affinity [31]. Many studies have investigated the binding affinity and agonist efficacy of h HK-1 at each of the three-mammalian tachykinin receptors. The result show that h HK-1 and h HK-1(4– 11) bind to the human NK1 receptor, with 14 and 70 times lower affinities, respectively, relative to SP [15]. Tachykinins are involved in a wide variety of biological actions such as smooth muscle contraction, vasodilatation, pain
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522 Table 1 Amino acid sequences of the tachykinin peptides substance P, neurokinin A, neurokinin B, hemokinin-1. Tachykinin
Amino acid sequence
Substance P Neurokinin A Neurokinin B Hemokinin-1 (mouse and rat) Hemokinin-1 (human) Hemokinin-1 (4–11) (human)
R
R T
P H D S G
K K M R K
P T H T A A
Q D D R S S
Q S F Q Q Q
F F F F F F
F V V Y F F
G G G G G G
L L L L L L
M-NH2 M-NH2 M-NH2 M-NH2 M-NH2 M-NH2
transmission, neurogenic inflammation, activation of the immune system and stimulation of endocrine gland secretion [18]. Many studies have revealed that tachykinins and their receptors are also expressed in non-neuronal cells such as immune cells and glial cell types [21,27,30]. Moreover, SP is known to have a regulatory effect on cells of the immune system. SP, act as a immunomodulatory agent, activates the oxidative metabolism of neutrophils [41], the degranulation of mast cells [38], monocyte chemotaxis [19,39] and modulates lymphocyte proliferation [33,34], antibody synthesis [16,43], and macrophage function [22]. However, little if anything is known about the effects of this immunomodulatory peptide on the proliferation and differentiation of human promyelocyte leukemia cells and since the discovery of human hemokinin-1, only four citations are available, two focused on the regulation of the contractile responses of human hemokinin-1 in human uterus [35] and mouse uterus [32], and the other focused on the regulation of pain after intracerebroventricular administration of human hemokinin-1 and human hemokinin-1(4–11) [11], and another focused on cardiovascular responses after intravenous administration of human hemokinin-1 and its truncated form hemokinin-1(4–11) [14]. In the present study, the effects of SP, h HK-1 and its truncated form h HK-1(4–11) on the proliferation and differentiation of HL60 are first investigated. The antiproliferation and differentiation effects induced by h HK-1 and its truncated form h HK-1(4–11) were determined by measuring cell viability, cell cycle phase distribution and NBT-reduction activity. The roles of NK1 receptors antagonists SR140333 and NK2 receptors antagonists SR48968 were also investigated. 2. Materials and methods 2.1. Chemicals The SP, h HK-1 and h HK-1(4–11) peptides used in this study were synthesized in our laboratory by the solid-phase peptide synthesis method and purified by high-performance liquid chromatography (HPLC). SP, h HK-1 and h HK-1(4–11) were dissolved in deionized water. The antagonists SR140333 or (S)1(2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl) piperidin-3-yl]ethyl)-4-phenyl-1-azoniabicyclo [2.2.2] octane chloride and SR48968 or (S)-N-methyl-N[4-(4-acetylamino-4phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide were generous gifts from Sanofi-Synthelabo Recherche (Montpellie, France), and they were dissolved in deionized water with 6% DMSO (dimethyl sulphoxide) and further diluted with deionized water. Control trials were performed in the presence of corresponding concentration of DMSO to rule out any possible nonspecific action of this solvent. Nitrotetrazolium blue chloride (NBT) (Zetan Chemical, PR China) was dissolved in deionized water. Phorbol 12-myristate 13-acetate (TPA) (Sigma) was dissolved in deionized water with 1% CHCl3 and further diluted with deionized water.
1515
2.2. Cells culture HL-60 cells were cultured in RPMI-1640 medium (Gibco-BRL, USA) supplemented with heat-inactivated 10% neonatal bovine serum (NBS) (Sijiqing Biotech, PR China), 2 mM L-glutamine, penicillin (100 IU/mL) and streptomycin (100 mg/mL). The cells were incubated in a humidified incubator containing 5% CO2 at 37 8C. The peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood. The peripheral venous blood was drawn from healthy volunteers. Blood samples were collected into sterile bags each containing 25 mL of citratephosphate dextrose anticoagulant and diluted to 1:2 with phosphate buffer saline (PBS). The PBMCs were separated by density gradient centrifugation at 2000 rpm for 20 min on FicollIsopaque (density, 1.077 g/mL). The isolated PBMCs were washed twice, counted by Trypan blue exclusion and suspended in RPMI-1640 growth medium supplied with 15% NBS. The cells were incubated in a humidified incubator containing 5% CO2 at 37 8C. 2.3. Effects of h HK-1 and h HK-1(4–11) on the proliferation of HL-60 cells The cytotoxic activities of SP, h HK-1 and its truncated form h HK-1(4–11) were evaluated in HL-60 using the MTT assay [24]. This assay is based on the reduction of the yellow colored 3-(40 , 50 -dimethylthiazol-20 -yl)-2, 5-diphenyl-tetrazolium bromide (MTT) by mitochondrial dehydrogenase of metabolically active cells to a purple-blue formazan. Cells were plated in 96-well plates (3 104 cells/mL) for cells in 100 mL of medium. SP, h HK1 and h HK-1(4–11) were dissolved in deionized water, and then they were added to each well at a final concentration of 1– 300 mM, respectively. The cells were exposed to the drugs for 24 h, 48 h, 72 h and 96 h. The control group received the same amount of deionized water. Thereafter, the plates were centrifuged and the medium was replaced by fresh medium (100 mL) containing 5 mg/mL MTT. After 4 h incubation, the MTT formazan product was dissolved in 150 mL DMSO, and absorbance was measured using a multiplate reader (Spectra Count, Packard, Ont., Canada). The effects of each drug were quantified as the percentage of control absorbance of reduced dye at 570 nm. As for the Trypan blue dye exclusion assay, cells were seeded at density of 3 104 cells/mL onto a 12-well plate for 24 h, then SP, h HK-1 and h HK-1(4–11) were added to medium at various indicated times and concentrations, respectively. After incubation, cells exposed to 0.2% Trypan blue were counted in a hemocytometer. 2.4. Effects of h HK-1 and h HK-1(4–11) on cell cycle distribution of HL-60 cells Cells were collected, washed, suspended in cold PBS, fixed in 75% ethanol at 20 8C overnight, washed and resuspended in PBS with RNAase (0.1 mg/mL). Cellular DNA was stained with PI and cell samples were analyzed on Becton Dickson Flow Cytometer (BD Biosciences, USA) using CELL Quest software (Verity Software House, Inc., Topsham, ME). 2.5. NBT reducing activity As a marker of cell differentiation, the reduction of NBT in HL60 cells was determined by a previously described method [2,9,20]. HL-60 cells were plated at a density of 3 104 cells/mL, and treated with h HK-1 or h HK-1(4–11) (1–300 mM) for 72 h. Briefly, after each treatment, cells at a density of 1 106 cells/
1516
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
mL were incubated for 30 min in RPMI-1640 growth medium with 10 mg/mL of TPA and 1 mg/mL of NBT at 37 8C. Then, the cells were collected and suspended in DMSO. The absorbance (OD) of the NBT-reduction product (formazane solutions) was measured at 570 nm in a kinetic microplate reader. 2.6. Tachykinins receptors antagonists investigation To investigate whether the antiproliferative effects induced by h HK-1 or h HK-1(4–11) were mediated through the activation of tachykinin NK1 or NK2 receptors, SR140333 (a selective NK1 receptor antagonist) or SR48968 (a selective NK2 receptor antagonist) was used at a range of concentrations from 0.1 mM to 30 mM. 2.7. Statistical analysis Data obtained from different experiments were presented as mean S.E.M from at least three independent experiments and evaluated by analysis of variance (ANOVA) or Student’s t-test, using a significance level of 5%.
3. Results 3.1. Effects of h HK-1 and h HK-1(4–11) on the proliferation in HL-60 cells To determine the effects of these tachykinin peptides on cell viability, HL-60 cells were treated with increasing concentrations of SP, h HK-1 and h HK-1(4–11) for 24 h, 48 h, 72 h, 96 h, respectively, and cell viability was assessed by the MTT assay. As shown in Fig. 1, a significant increase in antiproliferation effect was detected in a dose- and time-dependent manner after treatment with 1 mM, 3 mM, 10 mM, 30 mM, 100 mM and 300 mM h HK-1 or h HK-1(4–11). The antiproliferative effect reached at 36%, 34%, 70% and 21% after 24 h, 48 h, 72 h and 96 h exposure time treated with h HK-1 (300 mM), respectively (Fig. 1B), while 300 mM of h HK1(4–11) could lead to an increase on the antiproliferative effect of 49%, 44%, 60% and 59% after 24 h, 48 h, 72 h and 96 h exposure time, respectively (Fig. 1C). The antiproliferation effect of h HK-1 and h HK-1(4–11) after 72 h of incubation displayed the most potency compared with those of after 24 h, 48 h and 96 h incubation. Moreover, it was showed that the effect of SP was
Fig. 1. Effect of SP, h HK-1 and h HK-1(4–11) on cell growth in HL-60 cells in vitro. HL-60 cells were treated with the indicated concentrations of SP (A), h HK-1 (B) and h HK1(4–11) (C) for 24 h, 48 h, 72 h and 96 h and cell viability was determined using the MTT assay. The cellular growth of HL-60 cells was inhibited significantly in a dose- and time-dependent manner. The results are presented as the mean S.E.M of at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001, statistically significant differences SP (A), h HK-1 (B) or h HK-1(4–11) (C) vs the control. Control: untreated cells. (D) Comparison of the potency of antiproliferation effect of 72 h among SP, h HK-1 and h HK-1(4–11) (#P < 0.05, ##P < 0.01, statistically significant differences between h HK-1 vs SP; *P < 0.05 and **P < 0.01, statistically significant differences between h HK-1(4–11) vs SP).
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
much weaker than h HK-1 or h HK-1(4–11) (Fig. 1D), and SP exerted hardly a marked effect on the proliferation of HL-60 cells (Fig. 1A). The inhibition of 100 mM h HK-1, h HK-1(4–11) and SP was 62.85 9.15%, 49.26 7.25% and 8.18 2.09%, respectively (Fig. 1D). Furthermore, morphological assessment of HL-60 cultures revealed that there were no significantly visible morphological differences before and after treatment with these peptides (Fig. 2). 3.2. Effects of h HK-1 and h HK-1(4–11) on the proliferation of PBMCs We also investigated the effects of SP, h HK-1 and h HK-1(4– 11) on the proliferation of PBMCs. SP, h HK-1 and h HK-1(4–11) displayed no inhibitory effect on the proliferation of PBMCs within the concentration range from 1 mM to 300 mM (data not shown). This suggested that SP, h HK-1 and h HK-1(4–11) had no potential side effects on normal blood cells even though they exhibited significant inhibitory effects on the proliferation of HL60 within the concentration range from 1 mM to 300 mM.
1517
3.3. Effects of h HK-1 and h HK-1(4–11) on the cell cycle distribution of HL-60 cells We also studied the cell cycle distribution of HL-60 cells after the treatment with h HK-1 and h HK-1(4–11) (200 mM and 300 mM) for 72 h (Fig. 3). The percentage of HL-60 cells in the S phase was increased from 28.8% (vehicle-treated cells, control group) to 56.4% and 57.4% after the treatment with 200 mM and 300 mM h HK-1 for 72 h, respectively. However, the percentage of the G0/G1 phase was decreased from 53.4% (vehicle-treated cells, control group) to 39.7% and 40.2% after treatment with 200 mM and 300 mM h HK-1 for 72 h, respectively (Fig. 3A and C). Similar results were also observed on h HK-1(4–11). The percentage of HL-60 cells in the S phase was increased from 27.3% (vehicle-treated cells, control group) to 48.3% and 52.2% after treatment with 200 mM and 300 mM h HK-1(4–11) for 72 h, respectively. Also, the percentage of the G0/G1 phase was decreased from 55.8% (control cells) to 45.8% and 44.0% after
Fig. 2. Photomicrographs of HL-60 cells after 72 h of incubation with h HK-1 and h HK-1(4–11) (1–300 mM) showed the concentration-dependent inhibitory effect of h HK-1 (A) and h HK-1(4–11) (B) on cell proliferation. HL-60 cells were plated at a density of 3 104 cells/mL and counted with a hemocytometer. Cell density decreased in a concentration-dependent manner as the concentration of h HK-1 and h HK-1(4–11) are increased from 1 mM to 300 mM. There is no significantly visible change in cell morphology of HL-60 cells treated with h HK-1 and h HK-1(4–11).
1518
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
treatment with 200 mM and 300 mM h HK-1(4–11) for 72 h, respectively (Fig. 3B and D). These results indicated that h HK-1 and h HK-1(4–11) could promote cell cycle accumulation in the S phase of HL-60 cells. These results were consistent with the
results of cell numbers and viability assays. Therefore, the HL-60 cell growth inhibition exerted by h HK-1 and h HK-1(4–11) was probably due to the inhibition of cellular progression to S phases.
Fig. 3. Changes of cell cycle distribution of HL-60 cells after treatment with h HK-1 and h HK-1(4–11). HL-60 cells were plated at a density of 3 104 cells/mL, and cultured in mediums containing h HK-1 and h HK-1(4–11) at various concentrations (200 mM and 300 mM). After incubation, the cell cycle distribution was measured by flow cytometry. (A and C) Cells treated with h HK-1 for 72 h, while (B and D): cells treated with h HK-1(4–11) for 72 h. Control: untreated cells. Values represent the mean S.E.M. of data from three different experiments. *P < 0.05 vs. control and **P < 0.01 vs. control.
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
1519
Fig. 3. (Continued ).
3.4. Induction of NBT-reduction activity of HL-60 by h HK-1 and h HK-1(4–11) We further assessed NBT-reduction in the HL-60 cells treated with h HK-1 and h HK-1(4–11) to determine whether they induced the differentiation of the cells (Fig. 4). Rarely were NBT positive cells observed throughout the 72 h incubation with the control cells. h HK-1 and h HK-1(4–11) (30 mM) significantly induced the generation of NBT-reducing activity, and the absorbance of the NBT-reduction product (OD570 nm) increased from 0.376 (control) to 0.632 (300 mM h HK-1) (Fig. 4A) and from 0.541 (control) to 1.124 (300 mM h HK-1(4–11)) (Fig. 4B) at 72 h, respectively. More and more cells became NBT positive as the concentration of h HK-1 and h HK-1(4–11) increased. 3.5. Effects of tachykinin receptors antagonists To explore whether the tachykinin NK1 receptor or the tachykinin NK2 receptor is involved in h HK-1 or h HK-1(4–11)induced antiproliferation responses, the non-peptide NK1 receptor antagonist SR140333 or the non-peptide NK2 receptor antagonist SR48968 is added 45 min before h HK-1 or h HK-1(4–11), respectively. From Fig. 5, we could see that although the high concentration of SR140333 and SR48968 (10 mM) inhibited the proliferation of HL-60, the antiproliferation induced by h HK-1 or h HK-1(4–11) were not antagonized by the lower concentration of SR140333 (Fig. 5C and E) and SR48968 (Fig. 5D and F), indicating that the antiproliferation induced by h HK-1 or h HK-1(4–11) may not be mediated through the activation of tachykinin NK1 receptors and tachykinin NK2 receptors at the HL-60 cells. 4. Discussion
Fig. 4. Effects of h HK-1 and h HK-1(4–11) on the differentiation of HL-60 cells were determined by NBT-reduction. HL-60 cells were plated at a density of 3 104 cells/ mL, and were treated with h HK-1 and h HK-1(4–11) (10–300 mM) for 72 h. h HK-1 and h HK-1(4–11) significantly induced the generation of NBT-reduction activity in a concentration-dependent manner. (A) Cells treated with h HK-1 for 72 h, (B) cells treated with h HK-1(4–11) for 72 h. Values represent the mean S.E.M. of data from three different experiments. *P < 0.05 vs. control and **P < 0.01 vs. control.
Recently, Kurtz et al. reported the isolation and characterization of the human TAC4 gene, which encodes human hemokinin-1 and its N-terminally truncated form named human hemokinin-1(4– 11) [15]. Using various in vitro and in vivo investigations, it has been shown that HK-1 peptides behave as SP-like peptides [3,4,15,29,44]. SP is a ubiquitous, 11 amino acid peptide that has been associated with many physiological processes in the cardiovascular, nervous, respiratory and immune systems [36]. SP and its preferred receptor, NK1, have been identified in immune cells [27] and implicated in immunomodulation and inflammation [21,44]. The expression of neuropeptides and their receptors has
1520
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
Fig. 5. The effects of SR140333 (A, C and E) and SR48968 (B, D and F) on the inhibitory effects of SP, h HK-1 and h HK-1(4–11) on the proliferation of HL-60 cells. The cell viability was determined by using the MTT assay. The results are presented as the mean S.E.M of at least three independent experiments.
gained increasing interest in the field of tumor biology since these peptides were shown to be capable of influencing cell differentiation and proliferation [4]. However, we have very little insight into the effects of this immunomodulatory peptide on the proliferation and differentiation of human promyelocyte leukemia cells. In the present investigation, the effects of SP, h HK-1 and h HK-1(4–11) on the proliferation and differentiation of human leukemia cell lines HL-60 were confirmed. In our present investigation, h HK-1 caused a significant doseand time-dependent suppression of the proliferation in HL-60 cells
within a concentration range from 1 mM to 300 mM. The suppression induced by h HK-1 was accompanied by an accumulation of cell cycle in the S phase. The cell cycle related mechanisms may take effect in the HL-60 cell cycle accumulation induced by h HK-1. A common goal of cancer therapy is to restore normal growth control in transformed tissues [7]. One area that has been intensively studied in recent years is biological modifiers of cancer growth which are designed to retard proliferation [28], to induce differentiation of these cells to a quiescent, nondividing stage [17,40]. Cell differentiation is disrupted in the acute
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
leukaemia and many solid tumors [10]. In the present study, h HK1 was able to induce the differentiation of HL-60 cells. This may provide important implications that h HK-1 could act as a valuable human leukaemia therapy agent because of its potent effect on the differentiation of human leukaemia cells. h HK-1(4–11) is the truncated version of h HK-1 based on the monobasic cleavage site following a lysine in the +3 position [15]. Cleavage at that site is likely as the sequence conforms to a number of the consensus rules for non-proline-directed monobasic processing, namely a single basic residue (lysine) with an additional basic residue at the 3 position (lysine) and a short side chain amino acid (alanine) in the +1 position relative to the cleavage site [8]. Our present data showed that h HK-1(4–11) produced dose- and time-related antiproliferation effects which were equally effective with but slightly less potent than h HK-1. Moreover, h HK-1(4–11) could also induce a S arrest of the cell cycle and increase the NBT-reduction activity of HL-60 cells significantly, similar to that of h HK-1. All above results indicated that as the active fragment of human hemokinin-1, h HK-1(4–11) could also act as a valuable human leukaemia therapy agent. Our results show that the antiproliferation induced by SP, h HK1 or h HK-1(4–11) could not be antagonized by NK1 receptor antagonist SR140333 and the NK2 receptor antagonist SR48968, indicating that the antiproliferation induced by SP, h HK-1 or h HK1(4–11) may not be mediated through the activation of tachykinin NK1 receptors and tachykinin NK2 receptors. This is consistent with the results of studies by Kavelaars and co-workers that there are multiple pathways for SP to activate the immune system, apart from the classical NK1 receptor-mediated pathway, SP can also stimulate cells via the non-NK1 receptor as well as via receptorindependent routes [12,13]. A receptor-independent mechanism of action of SP can be explained by the fact that the peptide has a cationic amphiphilic nature [25,26]. It has been shown that interaction of cationic amphiphilic peptides with a negatively charged cell membrane result in insertion of the peptide into the membrane [13]. Mousli et al. [25,26] have suggested that the peptide then acquires a membrane-spanning conformation, which enables direct interaction with proteins in the cytosolic compartment. Considering that cancer treatment has severe side effects, it is very important to determine how harmful new drugs may be to normal dividing cells such as proliferating lymphocytes [1,23,45]. Interestingly, despite the fact that HL-60 leukemia cells and lymphocytes are derived from white blood cell lineages, h HK-1 and h HK-1(4–11) seems to be selective for tumor cells, since no toxicity against normal cells was observed in our assay. In summary, the body of data derived from our present experiments strongly indicates, for the first time, that h HK-1, the well-known immunomodulatory polypeptide, was able to significantly suppress the proliferation and induce differentiation and S phase arrest of HL-60 cells. Strikingly, the truncated version of h HK-1, h HK-1(4–11) has the similar effects on HL-60 cells as that of h HK-1. Moreover, the effects may not be mediated through the activation of classical tachykinin NK1 receptor and NK2 receptor. The combination of differentiation-induction and growth inhibition by h HK-1 and h HK-1(4–11) should not only make them act as immunomodulatory factors in cancer chemotherapy, but also facilitate the analysis of the role of the tachykinin peptides encoded by the newly identified TAC4 gene in lymphocytic leukemia and may open novel pharmacological interventions. Acknowledgments This study was supported by the grants from the National Natural Science Foundation of China (Nos. 20525206, 20772052, 20621091 and 90813012), the Specialized Research Fund for the
1521
Doctoral Program in Higher Education Institutions and the Chang Jiang Program of the Ministry of Education of China. References [1] Anazetti MC, Melo PS, Duran N, Haun M. Comparative cytotoxicity of dimethylamide-crotonin in the promyelocytic leukemia cell line (HL-60) and human peripheral blood mononuclear cells. Toxicology 2003;188:261–74. [2] Baehner RL, Nathan DG. Quantitative nitroblue tetrazolium test in chronic granulomatous disease. N Engl J Med 1986;278:971–6. [3] Bellucci F, Carini F, Catalani C, Cucchi P, Lecci A, Meini S, et al. Pharmacological profile of the novel mammalian tachykinin, hemokinin 1. Br J Pharmacol 2002;135:266–74. [4] Berger A, Paige CJ. Hemokinin-1 has substance P-like function in U-251 MG astrocytoma cells: a pharmacological and functional study. J Neuroimmunol 2005;164:48–56. [5] Berger A, Tran AH, Paige CJ. Co-regulated decrease of Neurokinin-1 receptor and Hemokinin-1 gene expression in monocytes and macrophages after activation with pro inflammatory cytokines. J Neuroimmunol 2007;187:83– 93. [6] Camarda V, Rizzi A, Calo G, Guerrini R, Salvadori S, Regoli D. Pharmacological profile of hemokinin 1: a novel member of the tachykinin family. Life Sci 2002;71:363–70. [7] Campbell MJ, Reddy GS, Koeffler HP. Vitamin D3 analogs and their 24-Oxo metabolites equally inhibit clonal proliferation of a variety of cancer cells but have differing molecular effects. J Cell Biochem 1997;66:413–25. [8] Devi L. In: Fricker LD, editor. Peptide biosynthesis and processing. Florida: CRC Press; 1991. p. 175–98. [9] Eugenia Riveiro M, Shayo C, Monczor F, Fernandez N, Baldi A, De Kimpe N, et al. Induction of cell differentiation in human leukemia U-937 cells by 5-oxygenated-6,7-methylenedioxycoumarins from Pterocaulon polystachyum. Cancer Lett 2004;210:179–88. [10] Fan YZ, Chang H, Yu Y, Liu J, Zhao L, Yang DJ, et al. Thymopentin (TP5), an immunomodulatory peptide, suppresses proliferation and induces differentiation in HL-60 cells. BBA Mol Cell Res 2006;1763:1059–66. [11] Fu CY, Zhao YL, Dong L, Chen Q, Ni JM, Wang R. In vivo characterization of the effects of human hemokinin-1 and human hemokinin-1(4–11), mammalian tachykinin peptides, on the modulation of pain in mice. Brain Behav Immun 2008;22:850–60. [12] Jeurissen F, Kavelaars A, Korstjens M, Broeke D, Franklin RA, Gelfand EW, et al. Monocytes express a non-neurokinin substance P receptor that is functionally coupled to MAP kinase. J Immunol 1994;152:2987–94. [13] Kavelaars A, Jeurissen F, Von Frijtag Drabble Kunzel J, Van Roijen JH, Rijkers GT, Heijnen CJ. Substance P induces a rise in intracellular calcium concentration in human T lymphocytes in vitro: evidence of a receptor-independent mechanism. J Neuroimmunol 1993;42:61–70. [14] Kong ZQ, Fu CY, Chen Q, Wang R. Cardiovascular responses to intravenous administration of human hemokinin-1 and its truncated form hemokinin1(4–11) in anesthetized rats. Eur J Pharmacol 2008;590:310–6. [15] Kurtz MM, Wang R, Clements MK, Cascieri MA, Austin CP, Cunningham BR, et al. Identification, localization and receptor characterization of novel mammalian substance P-like peptides. Gene 2002;296:205–12. [16] Laurenzi MA, Person MAA, Dalsgaard CJ, Haegerstrand A. The neuropeptide substance P stimulates production of interleukin 1 in human blood monocytes: activated cells are preferentially influenced by the neuropeptide. Scand J lmmunol 1990;31:529–33. [17] Liu L, Shack S, Stetler-Stevenson WG, Hudgins WR, Samid D. Differentiation of cultured human melanoma cells induced by the aromatic fatty acids phenylacetate and phenylbutyrate. J Invest Dermatol 1994;103:335–40. [18] Longmore J, Hill RG, Hargreaves RJ. Neurokinin-receptor antagonists: pharmacological tools and therapeutic drugs. Can J Physiol Pharm 1997;75:612– 21. [19] Lotz M, Vaughn JH, Carson DA. Effects of neuropeptides on production of inflammatory cytokines by human monocytes. Science 1988;241:1218–21. [20] Lucas DL, Tanuma S, Davies PJ, Wright DG, Johnson GS. Maturation of human promyelocytic leukemia cells induced by nicotinamide: evidence of a regulatory role for ADP-ribosylation of chromosomal proteins. J Cell Physiol 1984;121:334–40. [21] Maggi CA. The effects of tachykinins on inflammatory and immune cells. Regul Pept 1997;70:75–90. [22] McGillis J, Mitsuhashi M, Payan D. Immunologic properties of substance P. In: Ader R, Felten DL, Cohen N, editors. Psychoneuroimmunology. 2nd ed., San Diego, CA: Academic Press; 1991. p. 209–23. [23] Montenegro RC, De Vasconcellos MC, Bezerra FS, Andrade-Neto M, Pessoa C, De Moraes MO, et al. Pisosterol induces monocytic differentiation in HL-60 cells. Toxicol In Vitro 2007;21:795–800. [24] Mosmann T. Rapid colorimetric assays for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;16:55–63. [25] Mousli M, Bronner C, Landry Y, Bockaert J, Rouot B. Direct activation of GTPbinding regulatory proteins (G-proteins) by substance P and compound 48/80. FEBS Lett 1990;259:260–2. [26] Mousli M, Bued JL, Bronner C, Rouot B, Landry Y. G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides. Trends Pharmacol Sci 1990;11:358–62.
1522
Y.-L. Zhao et al. / Peptides 30 (2009) 1514–1522
[27] Nelson DA, Bost KL. Non-neuronal mammalian tachykinin expression. Front Biosci 2004;9:2166–76. [28] Novichenko N, Konno S, Nakajima Y, Hsieh TC, Xu W, Turo K, et al. Growth attenuation in a human prostate cell line mediated by a phorbol ester. Proc Soc Exp Biol Med 1995;209:152–6. [29] Page NM, Bell NJ, Gardiner SM, Manyonda IT, Brayley KJ, Strange PG, et al. Characterization of the endokinins: human tachykinins with cardiovascular activity. Proc Natl Acad Sci U S A 2003;100:6245–50. [30] Palma C, Maggi CA. The role of tachykinins via NK1 receptors in progression of human gliomas. Life Sci 2000;67:985–1001. [31] Patacchini R, Maggi CA. Peripheral tachykinin receptors as targets for new drugs. Eur J Pharmacol 2001;429:13–21. [32] Patak E, Pennefather JN, Candenas ML, Kerr K, Exintaris B, Ziccone S, et al. Functional characterisation of hemokinin-1 in mouse uterus. Eur J Pharmacol 2008;601:148–53. [33] Payan DG. Neuropeptides and inflammation. Annu Rev Med 1989;40:341– 52. [34] Payan DG, Levine JD, Goetzl EJ. Modulation of immunity and hypersensitivity by sensory neuropeptides. J Immunol 1983;132:1601–4. [35] Pennefather JN, Patak E, Ziccone S, Lilley A, Pinto FM, Page NM, et al. Regulation of the stimulant actions of neurokinin A and human hemokinin-1 on the human uterus: a comparison with histamine. Biol Reprod 2006;75:334–41. [36] Pernow B. Substance P. Pharmacol Rev 1983;35:85–141.
[37] Regoli D, Drapeau G, Dion S, D’Orle0 ans-Juste P. Pharmacological receptors for substance P and neurokinins. Life Sci 1987;40:109–17. [38] Repke H, Bienert M. Mast cell activation a receptor-independent mode of substance P action. FEBS Lett 1987;221:236–40. [39] Ruff MR, Wahl SM, Pert CB. Substance P receptor mediated chemotaxis of human monocytes. Peptides 1985;6:107–11. [40] Samid D, Shack S, Myers CE. Selective growth arrest and phenotypic reversion of prostate cancer cells in vitro by nontoxic pharmacological concentrations of phenylacetate. J Clin Invest 1993;91:2288–95. [41] Serra MC, Bazzoni F, Della Bianca V, Greskowiak M, Rossi F. Activation of human neutrophils by substance P. Effect on oxidative metabolism, cytosolic Ca2+ concentration and inositol phosphate formation. J Immunol 1988;141:2118–24. [42] Severini C, Improta G, Falconieri-Erspamer G, Salvadori S, Erspamer V. The tachykinin peptide family. Pharmacol Rev 2002;54:285–322. [43] Stanisz AM, Befus D, Bienenstock J. Differential effects of vasoactive intestinal peptide, substance P, and somatostatin on immunoglobulin synthesis and proliferations by lymphocytes from Peyer’s patches, mesenteric lymph nodes, and spleen. J Immunol 1986;136:152–6. [44] Zhang Y, Lu L, Furlonger C, Wu GE, Paige CJ. Hemokinin is a hematopoieticspecific tachykinin that regulates B lymphopoiesis. Nat Immunol 2000;1:392–7. [45] Zuco V, Supino R, Righetti SC, Cleris L, Marchesi E, Passerine Gambacorti C, et al. Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer Lett 2002;175:17–25.