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A polypeptide from Chlamys farreri abolishes UV-induced apoptosis in murine thymocytes in vitro
Journal of Photochemistry and Photobiology B: Biology 84 (2006) 189–196 www.elsevier.com/locate/jphotobiol
A polypeptide from Chlamys farreri abolishes UV-induced apoptosis in murine thymocytes in vitro Chun-Ling Yan
b
a,b
, Ru-Yong Yao c, Li-Yan Jing d, Yue-Jun Wang e, Wan-Shun Liu a, Chun-Bo Wang b,*
a Marine Life Sciences College, Ocean University of China, Qingdao 266003, China Department of Pharmacology, Medical College, Qingdao University, 38, Dongzhou Road, Qingdao 266021, China c Affiliated Hospital of Qingdao University, Qingdao 266003, China d Tai’an Public Health School, Taian 271000, China e Yellow Sea Fishery Research Institute, Qingdao 266071, China
Received 7 January 2006; received in revised form 10 February 2006; accepted 17 February 2006 Available online 4 May 2006
Abstract Previously we reported that a polypeptide from Chlamys farreri (PCF) was a potent photoprotective agent against ultraviolet (UV) irradiation in vitro. To understand the mechanism by which PCF protects cells from irradiation, we studied anti-apoptotic effects of PCF against UV irradiation on the murine thymocytes in vitro. MTT and flow cytometric analysis assays showed that 2 h pretreatment with PCF completely abolished UV induced cell death. TEM examination showed that PCF fully protected the ultrastructure of thymocytes exposed to UV irradiation. Lipid peroxidation and intracellular reactive oxygen species assays indicated that PCF efficiently blocked production of reactive oxygen intermediates induced by UV irradiation. Further, PCF protected UV-irradiated thymocytes from losing mitochondrial transmembrane potential and DNA fragmentation. Based on these observations we propose that PCF is a potent antiapoptotic factor, which protects cells from irradiation at multiple steps. 2006 Elsevier B.V. All rights reserved. Keywords: Polypeptide from Chlamys farreri; Ultraviolet; Apoptosis; Murine thymocytes
1. Introduction Skin cancer is the most common cancer in fair-skinned populations. The incidence of skin cancers is rising and thus poses a threat to public health [1,2]. Exposure to ultraviolet (UV) radiation such as sun or other commercial indoor tanning equipment plays a key role for development of the skin caner – both melanoma and non-melanoma. It is known that sunburns and excessive UV exposures cause cumulative damage which triggers a multitude of molecular changes, including defects in DNA repair [3], growth arrest
*
Corresponding author. Tel.: +86 532 299 1202; fax: +86 532 299 1009. E-mail address: [email protected] (C.-B. Wang).
1011-1344/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2006.02.013
[4], apoptosis [5,6], changes of gene expressions [7–9], and reduced cellular immune responses [10,11]. Recent studies also show that a photodynamic action from UV irradiation can produce reactive oxygen intermediates that can indirectly affect a variety of cellular targets [12,13]. On the other hand, antioxidant reagents can block thymocyte apoptosis caused by gamma radiation [14,15]. In searching for potential blocker for such reactive oxygen intermediates, we isolated a marine antioxidant polypeptide from Chlamys farreri (a Chinese scallop), named PCF, a octal peptide (Mr = 879) consists of Pro, Asn, Ser, Thr, Arg, Hyl, Cys, and Gly. Previously, we reported that PCF had a potent antioxidant activity [16], and we have also showed that this peptide can protect hairless mouse skin from UV irradiation [17]. To understand the mechanism by which PCF protect skin from UV
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irradiation effects, in this study we investigated the potential role of PCF in improving immune system. Specifically we studied its anti-apoptotic role of PCF in murine thymocytes exposed to UV light. 2. Materials and methods 2.1. Agent PCF (purity >96%) was isolated from gonochoric Chinese scallop C. farreri that has been served as seafood for several 1000 years. The extraction yield of PCF was 1.14 g kg 1 C. farreri by our techniques. PCF was purified and analyzed by HPLC, dissolved in sterile deionized water, stored at 4 C.
2.4. Transmission electron microscopy At the end of the culture, cells from control, UV alone and UV + 5.68 mmol L 1 PCF were washed two times with PBS and fixed with 2.5% glutaraldehyde for 30 min at 4 C. Followed by washing 3 times with 0.1 M sodium cacodylate buffer, and post-fixed with 1% osmium tetroxide in 0.1 m sodium cacodylate buffer for 1 h. The cultures were then dehydrated with ethanol series and processed for embedding in epon resin (Poly/bed 812 Polysciences Int.) for 2 h. Complete polymerization is performed in a 60 C oven for 24 h. Thin sections are cut using a diamond knife (70 nm). The ultra thin sections were stained with uranium acetate and plumbum citrate then examined under a JEM-1200EX electron microscope [19] (JEOL Company, Japan).
2.2. Thymocyte culture and treatment 2.5. Measurement of apoptosis Thymocytes were collected after mincing the murine thymus and were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal calf serum, 100 U ml 1 penicillin, and 100 mg ml 1 streptomycin at 37 C in a humidified incubator containing 5% CO2. The cells were plated at a density of 1–5 · 106 cells ml 1 in 24-well plates. Cells were divided into five groups including: (1) Control (no exposure to UV light), (2) UV alone (model), (3) UV + 1.42 mmol L 1 PCF, (4) UV + 2.84 mmol L 1 PCF, and (5) UV + 5.68 mmol L 1 PCF. Before UV irradiation, PCF was added into the medium at a final concentration for 2 h followed by two times of PBS washing. Cells, with a very thin layer of PBS were irradiated for 10 s under UVA and UVB lamps (Beijing Normal University, China). The wavelength range of UVA lamp was 315–400 nm with a peak wavelength at 365 nm, and the wavelength range of UVB lamp was 280–315 nm with a peak wavelength at 297 nm. The total dosage irradiated to these cells, measured by an IL700 radiometer (International Light Inc.), was 6.16 mJ cm 2. After irradiation, the cells were incubated for additional 6 h, and processed for the following experiments. 2.3. MTT test for cell viability Cells were seeded in microtiter plates (96 wells) at a density of 2 · 106 cells per well. Two hours after UV irradiation, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) [18] was added into medium, and cells were incubated for additional 4 h. MTT reacted with dehydrogenase enzymes in metabolically active cells, which yielded a blue formazan product. The formazan product was dissolved in DMSO (Sigma) and the absorbance was determined with an enzyme-linked immunosorbent assay in a plate reader (Shanghai Medical Instrument, China) at 490 nm and the background readings were automatically subtracted. The results were expressed as percentage of the measurements obtained from untreated controls.
Flow cytometric analysis was performed for quantification of cell death by apoptosis. Due to DNA degradation and subsequent leakage from cells, apoptotic cells can be detected via their diminished staining with DNA-specific fluorochromes such as propidium iodide (PI) [20]. Thymocytes (1 · 106 cells ml 1) were harvested and then cells were washed twice with cold PBS and subsequently stained with propidium iodide (Becton Dickinson Company, San Diego, CA, USA) before being analyzed on FACS Advantage flow cytometer. DNA fluorescence was analyzed by quantitative flow cytometry using CellQuest Software. Analyzing hypodiploid areas identified the percentage of apoptotic cells, 10,000 cells in each group were analyzed. Apoptosis was also quantified by measuring DNA fragmentation, which is one of the later steps during the apoptotic program [21] and by agarose gel electrophoresis as previously described [22]. Briefly, 1 · 106 ml 1 cells were harvested and centrifuged at 1000g for 10 min. Cell pellets were suspended in Tris–HCl 10 mmol L 1 (PH 7.4), edetic acid 10 mmol L 1, 0.5% Triton X-100 and proteinase K 40 lg L 1 (Merck,USA) at 37 C for 2 h. The lysate was extracted with 0.5% NaCl 5 M and 50% 2-propanol and incubated overnight at 20C, and centrifuged at 7000g for 20 min. The supernatant was precipitated with 70% ethanol and centrifuged. The pellets was dried and suspended in Tris–HCl 10 mmol L 1 (PH 7.4), edetic acid 1 mmol L 1, and RNase A 40 lg L 1 (sigma, USA) at 37 C for 60 min, electrophoresis was performed in 1.5% agarose gels in Tris–borate buffer at 50 V cm 1 for 30 min and stained with 0.1% ethidium bromide, then visualized under 305 nm ultraviolet (UV) illumination, and photographed. 2.6. Lipid peroxidation and intracellular reactive oxygen species (ROS) assays Malondialdehyde (MDA), a terminal product of lipid peroxidation, was measured to estimate the extent of lipid
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peroxidation in cells. The measurements of MDA and ROS formation were performed following the instruction of reagent kits (Nanjing Institute of Jiancheng Biological Engineering, China) [23,24].
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mental concentration of PCF (1.42 mmol L 1, 1 1 2.84 mmol L , and 5.68 mmol L ) for 2 h, significantly increases the cell survival rate at all tested concentrations. Particularly, at the concentration of 5.68 mmol L 1, PCF completely abolish the effect of UV on cell death.
2.7. Detection of mitochondrial transmembrane potential The cells were washed two times with PBS, and then resuspended in PBS at a concentration of 1 · 109 cells L 1. Five hundred microliters of cell solution were transferred into a 5 ml culture tube and rhodamine 123 solution (Sigma, USA) was added with a final concentration of 1 lmol L 1. Cells were then incubated for 30 min at 37 C. At the end, cells were washed two times with PBS, and analyzed using Becton Dickinson FACS Vantage flow cytometer for rhodamine 123 fluorescence that indicates the level of mitochondrial transmembrane potential. For each sample, 10,000 events were collected and measured [25].
In further examination of morphological changes in irradiated thymocytes, we observed apoptotic characteristics in these cells: cytoplasmic vesiculation, condensation and margination of the nuclear chromatin (Fig. 2B) compared to the control (Fig. 2A). The ultrastructure of the cell pretreated with PCF appeared close to normal (Fig. 2C), suggesting that PCF can preserve cellular morphology against UV irradiation. 3.3. PCF effectively slows down apoptosis induced by UV irradiation
2.8. Statistical analysis All data were expressed as mean ± SD one-way analysis of variance (ANOVA) was performed. The dose–response data for PCF were analyzed with Student–Newman–Keuls’ test. Differences were considered significant if P-value <0.05. 3. Results 3.1. PCF improves survival rate of UV-radiated thymocytes The short-term MTT assay is a simple and reproducible method used for prediction of the cell survival rate [26]. With this chemosensitivity technique we showed that UV irradiation reduced cell survival rate of thymocytes 60% (Fig. 1). Pretreatment of thymocytes with PCF, with incre-
Cell viability (percentage of the control)
3.2. PCF protects ultrastructure of thymocyte exposed to UV irradiation
120
bcd 100
bc 80
a 60
In the irradiated cells undergoing apoptosis, DNA is degraded to low molecular weight DNA fragment with subsequent leakage from the cell leading to reduction of DNA content. Flow cytometry, a technique for analyzing DNA content, is valuable for identification of apoptotic cells. In this technique, apoptotic cells stained with a DNA specific fluorochrome PI will exhibit a special DNA peak, called the sub-G1 peak or apoptotic peak. In the control group the apoptotic cells count for 13.8% of total cell numbers (Fig. 3A). The apoptotic cells were sharply increased to 5-fold (65.2%, Fig. 3B) after UV-irradiation. Pretreatment of PCF significantly slowed down cell apoptosis lead by UV-irradiation to 14.6% at the concentration of 5.68 mmol L 1 (Fig. 3C), 33.4% at the concentration of 2.84 mmol L 1 (Fig. 3D) and 51.6% at the concentration of 1.42 mmol L 1 (Fig. 3E) separately. One of the important hallmarks of apoptosis is the DNA fragmentation of 180–200 bp in length, which appears in the typical ‘‘DNA laddering’’ pattern on DNA electrophoresis gel. As shown in Fig. 4, DNA ladders appeared in the cells exposed to UV. Two-hour pretreatment of PCF at the concentration of 5.68 mmol L 1 completely prevent appearances of DNA ladders (DNA fragmentation).
40
3.4. PCF efficiently blocked production of ROS induced by UV irradiation
20 0 control
UV alone
UV+1.42mM PCF
UV+2.84mM PCF
UV+5.68mM PCF
Fig. 1. Protective effect of PCF on UV-irradiated thymocytes. Thymocytes were pretreated with increasing concentrations of PCF (1.42 mmol L 1, 2.84 mmol L 1, 5.68 mmol L 1) before UV irradiation (6.16 mJ cm 2). Cell viability was assayed with MTT and expressed as percentage of control. All values are means ± SD: a, P < 0.05 vs. control; b, P < 0.01 vs. UV alone; c, P < 0.01 vs. UV + 1.42 mmol L 1 PCF; d, P < 0.01 vs. UV + 2.84 mmol L 1 PCF.
UV irradiation can also activate reactive oxygen intermediates that subsequently induce apoptosis. To test if PCF could slow down production of lipid peroxidation in UV-irradiated cells, we compared levels of MDA and ROS in control, and UV irradiated cells in the absence or presence of PCF with different dosages. Table 1 showed that UV significantly increases the levels of both MDA and ROS, while in pretreatment of PCF cells, a dose-dependent
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Fig. 2. Effect of PCF on the ultrastructure of UV-irradiated thymocytes. (A) Untreated control cells; (B) UV alone: cells showed apoptotic features, such as cytoplasmic vesiculation, condensation and apoptotic body; (C) UV + 5.68 mmol L 1 PCF: note that pretreatment of PCF preserves the cell integrity with little change in cytoplasmic and nuclear structures.
decrease in lipid peroxidation and ROS level were observed, which is correlated with anti-apoptotic function of PCF.
(49.3% of control), while PCF protected UV-irradiated thymocytes from losing mitochondrial transmembrane potential.
3.5. PCF inhibited UV-induced mitochondrial membrane potential reduction
4. Discussion
Mitochondrial changes are critical for the inductive and effect phase of apoptosis. The loss of mitochondrial transmembrane potential signifies metabolic cell death [27]. We have used rhodamine 123 as a molecular probe to assay the mitochondrial transmembrane potential in murine thymocytes exposed to UV. As shown in Table 2, UV dramatically reduced mitochondrial transmembrane potential
In this study, we characterized the effects of PCF on UVinduced apoptosis in murine thymocyte. Our results showed that: UV at a dose of 6.16 mJ cm 2 could markedly induce murine thymocyte apoptosis in vitro. UV-induced apoptosis was associated with increased MDA; intracellular ROS and reduced mitochondrial transmembrane potential. Adding exogenous PCF reversed UV-induced apoptosis, the effects of PCF was in a dose-dependent manner.
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Fig. 3. The effects of PCF on UV-induced thymocyte apoptosis. To quantitate apoptosis by flow cytometry, propidium iodide was used to stain the DNA and look for the sub-diploid, DNA fluorescence was analyzed by quantitative flow cytometry using CellQuest Software and being analyzed on FACS advantage flow cytometer. (A) Control, apoptotic rate was 13.8%; (B) UV alone, apoptotic rate was 65.2%; (C) UV + 5.68 mmol L 1 PCF, apoptotic rate was 14.6%; (D) UV + 2.84 mmol L 1 PCF, apoptotic rate was 33.4%; (E) UV + 1.42 mmol L 1 PCF, apoptotic rate was 51.6%.
Reactive oxygen species (ROS) are generated endogenously by all aerobic cells as byproducts of a number of metabolic reactions [28]. Oxidative stress results from an imbalance in the proxidant–antioxidant equilibrium during cell metabolism. While oxidative stress can damage cellular constituents and result in apoptosis, ultraviolet irradiation can induce the formation of ROS in biological systems. ROS can generate both cytotoxic effects on cells by oxidizing lipids and proteins, and genotoxic effects by (amongst other things) inducing base lesions [29]. UVB (280– 315 nm) irradiation can induce the formation of bulky DNA adducts such as cyclobutane pyrimidine dimers and
6–4 photoproducts, while exposure to UVA (315–400 nm) irradiation can result in alterations to single bases. However, much of the UV-induced DNA damage is caused indirectly via ROS [30,31]. In addition, ROS exacerbates formation of the highly reactive hydroxyl radical, which in turn can cause DNA strand breaks, damage protein residues, initiate lipid peroxidation and triggers apoptotic cellular death process. Therefore, drugs that exhibit free radical scavenging and iron chelating properties, may serve as potential candidates for protection of cell. Antioxidants may prevent apoptosis by means of suppression or scavenging ROS. As regards the role of PCF in thymocyte
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Fig. 4. Agarose gel electrophoresis of DNA fragmentation in thymocytes. After 2 h pretreatment with different concentrations of PCF (1.42 mmol L 1, 2.84 mmol L 1, 5.68 mmol L 1), cells were exposed to UV at a total dose of 6.16 mJ cm 2. Lane 1: DNA marker; Lane 2: cells irradiated with 6.16 mJ cm 2 UV only; Lane 3: control; Lanes 4–6: cells pretreated with 1.42 mmol L 1, 2.84 mmol L 1 and 5.68 mmol L 1 PCF, respectively. Table 1 Effects of PCF on UV-induced lipid peroxidation and intracellular ROS level Groups
MDA (lmol L (protein)
Control UV alone UV + 1.42 mmol L UV + 2.84 mmol L UV + 5.68 mmol L
4.18 ± 0.74 13.89 ± 0.87a 11.52 ± 1.11b 9.14 ± 0.53b,c 7.20 ± 0.59b,c,d
1 1 1
PCF PCF PCF
All values are means ± SD. a P < 0.01 vs. control. b P < 0.05 vs. UV alone. c P < 0.05 vs. UV + 1.42 mmol L d P < 0.05 vs. UV + 2.84 mmol L
1 1
1
g 1)
ROS (kU g 1) (protein) 336.1 ± 2.41 372.3 ± 5.40a 361.1 ± 1.82b 350.4 ± 1.77b,c 340.2 ± 5.06b,c,d
PCF. PCF.
Table 2 Effects of PCF on UV-induced loss of mitochondrial transmembrane potential Groups
Rhodamine 123 fluorescence intensity
Control UV alone UV + 1.42 mmol L UV + 2.84 mmol L UV + 5.68 mmol L
1473.96 ± 0.04 649.98 ± 0.08a 897.84 ± 0.09b 1123.46 ± 0.08b,c 1236.18 ± 0.10b,c,d
1 1 1
PCF PCF PCF
All values are means ± SD. a P < 0.01 vs. control. b P < 0.05 vs. UV alone. c P < 0.05 vs. UV + 1.42 mmol L d P < 0.05 vs. UV + 2.84 mmol L
1 1
PCF. PCF.
apoptosis, our results showed that PCF could powerfully protect thymocyte against UV-induced apoptosis. When PCF was pre-administered into the cultured media for
2 h, the formation of ROS were significantly decreased, apoptotic rate of thymocyte were decreased in a dosedependent manner after UV irradiation, suggesting that PCF contributes to protecting thymocytes from apoptosis by decreasing intracellular ROS. The amount of MDA reflects the level of lipid peroxidation [32]. The lipid peroxidation products alter the activities of numerous enzymes, which control the cellular intermediary metabolism, membrane-bound enzymes and ion transporters [33,34]. Exposure thymocyte to UV, which induced the formation of ROS, resulted in accumulation of lipid peroxidation. In this study, our data showed that the level of MDA in thymocytes was accumulated remarkably by UV irradiation. In the presence of PCF, the level of MDA in cells was significantly decreased, which suggested indirectly that the large amount of oxygen radicals was generated from UV irradiation, PCF could scavenge oxygen radicals and inhibit the reaction of lipid peroxidation. This effect was proportional to PCF concentration and may contribute to the anti-apoptotic mechanisms. Mitochondria play a pivotal role in the process of apoptosis from C. elegans to mammals. It has now become apparent that mitochondria act as integrators of pro-apoptotic signals, transducing these to the final execution machinery of apoptosis [35]. Mitochondria undergo the loss of mitochondrial membrane potential coincident with opening of the permeability transition pore under various conditions such as when cells are with overloaded with Ca2+ or oxidative stress, a state that leads to the release of apoptosis inducing factors [36]. One of the most popular methods of monitoring mitochondrial function is via the use of fluorescent potentiometric probes. Mitochondrial function in situ is monitored by measuring the accumulation of cationic fluorescent probes in response to the mitochondrial transmembrane potential in living cells. In this study, we have used the mitochondrial membrane potential indicator rhodamine 123 to assess the effects of PCF on the mitochondria membrane potential of UV-radiated murine thymocytes. UV-irradiation caused a dramatic reduction in rhodamine 123 staining; PCF was able to block the UV-induced loss of rhodamine 123 staining in a dosedependent manner. These results indicated that PCF could protect mitochondria of thymocytes against UVirradiation. Taken together, our results demonstrated that UV irradiation is a powerful inducer of apoptosis of murine thymocytes in vitro. PCF presented in the extracellular fluid could protect murine thymocyte against UV damage and reduce UV-induced apoptosis. These findings indicated that PCF might be a useful protective agent to modulate the function of murine thymocytes under UV irradiation. However, the protective function of PCF on thymocytes against UV irradiation may be more complex. One possibility is that PCF acts as a ROS scavenger that can protect cytoplasmic membrane from exogenic ROS damage. Further, PCF could enter cells and assist in the cellular antioxidant ability in case cells undergo oxidant stress.
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5. Abbreviations DMSO dimethyl sulphoxidate HPLC high-pressure liquid chromatograph MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide MDA malondialdehyde PCF polypeptide from Chlamys farreri PBS phosphate-buffered saline ROS reactive oxygen species TEM transmission electron microscope UV ultraviolet
Acknowledgements We appreciate Ru-yong Yao and Bo-xiao Ding for their technical assistance. We also appreciate Professor Jian Feng for his writing assistance. This work was supported by National Nature Science Foundation of China (No. 30471458) and Technology Bureau of Qingdao (No. 2000-260). References [1] F.R. Abdulla, S.R. Feldman, P.M. Williford, D. Krowchuk, M. Kaur, Tanning and skin cancer, Pediatr. Dermatol. 22 (2005) 501– 512. [2] D.L. Miller, M.A. Weinstock, Non-melanoma skin cancer in the United States: incidence, J. Am. Acad. Dermatol. 30 (1994) 774–778. [3] Q. Zhan, K.A. Lord, I. Alamo, M.C. Hollander, F. Carrier, D. Ron, K.W. Kohn, B. Hoffman, D.A. Liebermann, A.J. Fornace, The GAD and MPD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth, Mol. Cell. Biol. 14 (1994) 2361–2371. [4] S.J. Kuerbitz, B.S. Plunkett, W.V. Walsh, M.B. Kastan, Wild-type p53 is a cell cycle checkpoint determinant following irradiation, Proc. Natl. Acad. Sci. USA 89 (1992) 7491–7495. [5] X. Wu, A.J. Levine, p53 and EGF-1 cooperate to mediate apoptosis, Proc. Natl. Acad. Sci. USA 91 (1994) 3602–3606. [6] Z. Ronai, S. Rutberg, Y.M. Yang, UV-responsive element (TGACAACA) from rat fibroblasts to human melanomas, Environ. Mol. Mutagen. 23 (1994) 157–163. [7] P. Herrlich, H. Ponta, H.J. Rahmsdorf, DNA damage-induced gene expression: signal transduction and relation to growth factor signaling, Rev. Physiol. Biochem. Pharmacol. 119 (1992) 187–223. [8] P. Herrlich, H.J. Rahmsdorf, Transcriptional and post-transcriptional responses to DNA-damaging agents, Curr. Opin. Cell Biol. 6 (1994) 425–431. [9] H. Schenk, M. Klein, W. Erdbrugger, W. Droge, K. Schulze-Osthoff, Distinct effects of thioredoxin and antioxidants on the activation of transcription factors NF-kappa B and AP-1, Proc. Natl. Acad. Sci. USA 91 (1994) 1672–1676. [10] M.S. Duthie, I. Kimber, M. Norval, The effects of ultraviolet radiation on the human immune system, Br. J. Dermatol. 140 (1999) 995–1009. [11] V.K. Shreedhar, M.W. Pride, Y. Su, Origin and characteristics of ultraviolet-B radiation induced suppressor T Lymphocytes, J. Immunol. 61 (1998) 1327–1335. [12] J. Fuchs, M.E. Huflejt, L.M. Rothfuss, D.S. Wilson, G. Carcamo, L. Packer, Acute effects of near ultraviolet and visible light on the cutaneous antioxidant defense system, Photochem. Photobiol. 50 (1989) 739–744.
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A polypeptide from Chlamys farreri abolishes UV-induced apoptosis in murine thymocytes in vitro Chun-Ling Yan
b
a,b
, Ru-Yong Yao c, Li-Yan Jing d, Yue-Jun Wang e, Wan-Shun Liu a, Chun-Bo Wang b,*
a Marine Life Sciences College, Ocean University of China, Qingdao 266003, China Department of Pharmacology, Medical College, Qingdao University, 38, Dongzhou Road, Qingdao 266021, China c Affiliated Hospital of Qingdao University, Qingdao 266003, China d Tai’an Public Health School, Taian 271000, China e Yellow Sea Fishery Research Institute, Qingdao 266071, China
Received 7 January 2006; received in revised form 10 February 2006; accepted 17 February 2006 Available online 4 May 2006
Abstract Previously we reported that a polypeptide from Chlamys farreri (PCF) was a potent photoprotective agent against ultraviolet (UV) irradiation in vitro. To understand the mechanism by which PCF protects cells from irradiation, we studied anti-apoptotic effects of PCF against UV irradiation on the murine thymocytes in vitro. MTT and flow cytometric analysis assays showed that 2 h pretreatment with PCF completely abolished UV induced cell death. TEM examination showed that PCF fully protected the ultrastructure of thymocytes exposed to UV irradiation. Lipid peroxidation and intracellular reactive oxygen species assays indicated that PCF efficiently blocked production of reactive oxygen intermediates induced by UV irradiation. Further, PCF protected UV-irradiated thymocytes from losing mitochondrial transmembrane potential and DNA fragmentation. Based on these observations we propose that PCF is a potent antiapoptotic factor, which protects cells from irradiation at multiple steps. 2006 Elsevier B.V. All rights reserved. Keywords: Polypeptide from Chlamys farreri; Ultraviolet; Apoptosis; Murine thymocytes
1. Introduction Skin cancer is the most common cancer in fair-skinned populations. The incidence of skin cancers is rising and thus poses a threat to public health [1,2]. Exposure to ultraviolet (UV) radiation such as sun or other commercial indoor tanning equipment plays a key role for development of the skin caner – both melanoma and non-melanoma. It is known that sunburns and excessive UV exposures cause cumulative damage which triggers a multitude of molecular changes, including defects in DNA repair [3], growth arrest
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Corresponding author. Tel.: +86 532 299 1202; fax: +86 532 299 1009. E-mail address: [email protected] (C.-B. Wang).
1011-1344/$ - see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2006.02.013
[4], apoptosis [5,6], changes of gene expressions [7–9], and reduced cellular immune responses [10,11]. Recent studies also show that a photodynamic action from UV irradiation can produce reactive oxygen intermediates that can indirectly affect a variety of cellular targets [12,13]. On the other hand, antioxidant reagents can block thymocyte apoptosis caused by gamma radiation [14,15]. In searching for potential blocker for such reactive oxygen intermediates, we isolated a marine antioxidant polypeptide from Chlamys farreri (a Chinese scallop), named PCF, a octal peptide (Mr = 879) consists of Pro, Asn, Ser, Thr, Arg, Hyl, Cys, and Gly. Previously, we reported that PCF had a potent antioxidant activity [16], and we have also showed that this peptide can protect hairless mouse skin from UV irradiation [17]. To understand the mechanism by which PCF protect skin from UV
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irradiation effects, in this study we investigated the potential role of PCF in improving immune system. Specifically we studied its anti-apoptotic role of PCF in murine thymocytes exposed to UV light. 2. Materials and methods 2.1. Agent PCF (purity >96%) was isolated from gonochoric Chinese scallop C. farreri that has been served as seafood for several 1000 years. The extraction yield of PCF was 1.14 g kg 1 C. farreri by our techniques. PCF was purified and analyzed by HPLC, dissolved in sterile deionized water, stored at 4 C.
2.4. Transmission electron microscopy At the end of the culture, cells from control, UV alone and UV + 5.68 mmol L 1 PCF were washed two times with PBS and fixed with 2.5% glutaraldehyde for 30 min at 4 C. Followed by washing 3 times with 0.1 M sodium cacodylate buffer, and post-fixed with 1% osmium tetroxide in 0.1 m sodium cacodylate buffer for 1 h. The cultures were then dehydrated with ethanol series and processed for embedding in epon resin (Poly/bed 812 Polysciences Int.) for 2 h. Complete polymerization is performed in a 60 C oven for 24 h. Thin sections are cut using a diamond knife (70 nm). The ultra thin sections were stained with uranium acetate and plumbum citrate then examined under a JEM-1200EX electron microscope [19] (JEOL Company, Japan).
2.2. Thymocyte culture and treatment 2.5. Measurement of apoptosis Thymocytes were collected after mincing the murine thymus and were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal calf serum, 100 U ml 1 penicillin, and 100 mg ml 1 streptomycin at 37 C in a humidified incubator containing 5% CO2. The cells were plated at a density of 1–5 · 106 cells ml 1 in 24-well plates. Cells were divided into five groups including: (1) Control (no exposure to UV light), (2) UV alone (model), (3) UV + 1.42 mmol L 1 PCF, (4) UV + 2.84 mmol L 1 PCF, and (5) UV + 5.68 mmol L 1 PCF. Before UV irradiation, PCF was added into the medium at a final concentration for 2 h followed by two times of PBS washing. Cells, with a very thin layer of PBS were irradiated for 10 s under UVA and UVB lamps (Beijing Normal University, China). The wavelength range of UVA lamp was 315–400 nm with a peak wavelength at 365 nm, and the wavelength range of UVB lamp was 280–315 nm with a peak wavelength at 297 nm. The total dosage irradiated to these cells, measured by an IL700 radiometer (International Light Inc.), was 6.16 mJ cm 2. After irradiation, the cells were incubated for additional 6 h, and processed for the following experiments. 2.3. MTT test for cell viability Cells were seeded in microtiter plates (96 wells) at a density of 2 · 106 cells per well. Two hours after UV irradiation, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) [18] was added into medium, and cells were incubated for additional 4 h. MTT reacted with dehydrogenase enzymes in metabolically active cells, which yielded a blue formazan product. The formazan product was dissolved in DMSO (Sigma) and the absorbance was determined with an enzyme-linked immunosorbent assay in a plate reader (Shanghai Medical Instrument, China) at 490 nm and the background readings were automatically subtracted. The results were expressed as percentage of the measurements obtained from untreated controls.
Flow cytometric analysis was performed for quantification of cell death by apoptosis. Due to DNA degradation and subsequent leakage from cells, apoptotic cells can be detected via their diminished staining with DNA-specific fluorochromes such as propidium iodide (PI) [20]. Thymocytes (1 · 106 cells ml 1) were harvested and then cells were washed twice with cold PBS and subsequently stained with propidium iodide (Becton Dickinson Company, San Diego, CA, USA) before being analyzed on FACS Advantage flow cytometer. DNA fluorescence was analyzed by quantitative flow cytometry using CellQuest Software. Analyzing hypodiploid areas identified the percentage of apoptotic cells, 10,000 cells in each group were analyzed. Apoptosis was also quantified by measuring DNA fragmentation, which is one of the later steps during the apoptotic program [21] and by agarose gel electrophoresis as previously described [22]. Briefly, 1 · 106 ml 1 cells were harvested and centrifuged at 1000g for 10 min. Cell pellets were suspended in Tris–HCl 10 mmol L 1 (PH 7.4), edetic acid 10 mmol L 1, 0.5% Triton X-100 and proteinase K 40 lg L 1 (Merck,USA) at 37 C for 2 h. The lysate was extracted with 0.5% NaCl 5 M and 50% 2-propanol and incubated overnight at 20C, and centrifuged at 7000g for 20 min. The supernatant was precipitated with 70% ethanol and centrifuged. The pellets was dried and suspended in Tris–HCl 10 mmol L 1 (PH 7.4), edetic acid 1 mmol L 1, and RNase A 40 lg L 1 (sigma, USA) at 37 C for 60 min, electrophoresis was performed in 1.5% agarose gels in Tris–borate buffer at 50 V cm 1 for 30 min and stained with 0.1% ethidium bromide, then visualized under 305 nm ultraviolet (UV) illumination, and photographed. 2.6. Lipid peroxidation and intracellular reactive oxygen species (ROS) assays Malondialdehyde (MDA), a terminal product of lipid peroxidation, was measured to estimate the extent of lipid
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peroxidation in cells. The measurements of MDA and ROS formation were performed following the instruction of reagent kits (Nanjing Institute of Jiancheng Biological Engineering, China) [23,24].
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mental concentration of PCF (1.42 mmol L 1, 1 1 2.84 mmol L , and 5.68 mmol L ) for 2 h, significantly increases the cell survival rate at all tested concentrations. Particularly, at the concentration of 5.68 mmol L 1, PCF completely abolish the effect of UV on cell death.
2.7. Detection of mitochondrial transmembrane potential The cells were washed two times with PBS, and then resuspended in PBS at a concentration of 1 · 109 cells L 1. Five hundred microliters of cell solution were transferred into a 5 ml culture tube and rhodamine 123 solution (Sigma, USA) was added with a final concentration of 1 lmol L 1. Cells were then incubated for 30 min at 37 C. At the end, cells were washed two times with PBS, and analyzed using Becton Dickinson FACS Vantage flow cytometer for rhodamine 123 fluorescence that indicates the level of mitochondrial transmembrane potential. For each sample, 10,000 events were collected and measured [25].
In further examination of morphological changes in irradiated thymocytes, we observed apoptotic characteristics in these cells: cytoplasmic vesiculation, condensation and margination of the nuclear chromatin (Fig. 2B) compared to the control (Fig. 2A). The ultrastructure of the cell pretreated with PCF appeared close to normal (Fig. 2C), suggesting that PCF can preserve cellular morphology against UV irradiation. 3.3. PCF effectively slows down apoptosis induced by UV irradiation
2.8. Statistical analysis All data were expressed as mean ± SD one-way analysis of variance (ANOVA) was performed. The dose–response data for PCF were analyzed with Student–Newman–Keuls’ test. Differences were considered significant if P-value <0.05. 3. Results 3.1. PCF improves survival rate of UV-radiated thymocytes The short-term MTT assay is a simple and reproducible method used for prediction of the cell survival rate [26]. With this chemosensitivity technique we showed that UV irradiation reduced cell survival rate of thymocytes 60% (Fig. 1). Pretreatment of thymocytes with PCF, with incre-
Cell viability (percentage of the control)
3.2. PCF protects ultrastructure of thymocyte exposed to UV irradiation
120
bcd 100
bc 80
a 60
In the irradiated cells undergoing apoptosis, DNA is degraded to low molecular weight DNA fragment with subsequent leakage from the cell leading to reduction of DNA content. Flow cytometry, a technique for analyzing DNA content, is valuable for identification of apoptotic cells. In this technique, apoptotic cells stained with a DNA specific fluorochrome PI will exhibit a special DNA peak, called the sub-G1 peak or apoptotic peak. In the control group the apoptotic cells count for 13.8% of total cell numbers (Fig. 3A). The apoptotic cells were sharply increased to 5-fold (65.2%, Fig. 3B) after UV-irradiation. Pretreatment of PCF significantly slowed down cell apoptosis lead by UV-irradiation to 14.6% at the concentration of 5.68 mmol L 1 (Fig. 3C), 33.4% at the concentration of 2.84 mmol L 1 (Fig. 3D) and 51.6% at the concentration of 1.42 mmol L 1 (Fig. 3E) separately. One of the important hallmarks of apoptosis is the DNA fragmentation of 180–200 bp in length, which appears in the typical ‘‘DNA laddering’’ pattern on DNA electrophoresis gel. As shown in Fig. 4, DNA ladders appeared in the cells exposed to UV. Two-hour pretreatment of PCF at the concentration of 5.68 mmol L 1 completely prevent appearances of DNA ladders (DNA fragmentation).
40
3.4. PCF efficiently blocked production of ROS induced by UV irradiation
20 0 control
UV alone
UV+1.42mM PCF
UV+2.84mM PCF
UV+5.68mM PCF
Fig. 1. Protective effect of PCF on UV-irradiated thymocytes. Thymocytes were pretreated with increasing concentrations of PCF (1.42 mmol L 1, 2.84 mmol L 1, 5.68 mmol L 1) before UV irradiation (6.16 mJ cm 2). Cell viability was assayed with MTT and expressed as percentage of control. All values are means ± SD: a, P < 0.05 vs. control; b, P < 0.01 vs. UV alone; c, P < 0.01 vs. UV + 1.42 mmol L 1 PCF; d, P < 0.01 vs. UV + 2.84 mmol L 1 PCF.
UV irradiation can also activate reactive oxygen intermediates that subsequently induce apoptosis. To test if PCF could slow down production of lipid peroxidation in UV-irradiated cells, we compared levels of MDA and ROS in control, and UV irradiated cells in the absence or presence of PCF with different dosages. Table 1 showed that UV significantly increases the levels of both MDA and ROS, while in pretreatment of PCF cells, a dose-dependent
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Fig. 2. Effect of PCF on the ultrastructure of UV-irradiated thymocytes. (A) Untreated control cells; (B) UV alone: cells showed apoptotic features, such as cytoplasmic vesiculation, condensation and apoptotic body; (C) UV + 5.68 mmol L 1 PCF: note that pretreatment of PCF preserves the cell integrity with little change in cytoplasmic and nuclear structures.
decrease in lipid peroxidation and ROS level were observed, which is correlated with anti-apoptotic function of PCF.
(49.3% of control), while PCF protected UV-irradiated thymocytes from losing mitochondrial transmembrane potential.
3.5. PCF inhibited UV-induced mitochondrial membrane potential reduction
4. Discussion
Mitochondrial changes are critical for the inductive and effect phase of apoptosis. The loss of mitochondrial transmembrane potential signifies metabolic cell death [27]. We have used rhodamine 123 as a molecular probe to assay the mitochondrial transmembrane potential in murine thymocytes exposed to UV. As shown in Table 2, UV dramatically reduced mitochondrial transmembrane potential
In this study, we characterized the effects of PCF on UVinduced apoptosis in murine thymocyte. Our results showed that: UV at a dose of 6.16 mJ cm 2 could markedly induce murine thymocyte apoptosis in vitro. UV-induced apoptosis was associated with increased MDA; intracellular ROS and reduced mitochondrial transmembrane potential. Adding exogenous PCF reversed UV-induced apoptosis, the effects of PCF was in a dose-dependent manner.
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Fig. 3. The effects of PCF on UV-induced thymocyte apoptosis. To quantitate apoptosis by flow cytometry, propidium iodide was used to stain the DNA and look for the sub-diploid, DNA fluorescence was analyzed by quantitative flow cytometry using CellQuest Software and being analyzed on FACS advantage flow cytometer. (A) Control, apoptotic rate was 13.8%; (B) UV alone, apoptotic rate was 65.2%; (C) UV + 5.68 mmol L 1 PCF, apoptotic rate was 14.6%; (D) UV + 2.84 mmol L 1 PCF, apoptotic rate was 33.4%; (E) UV + 1.42 mmol L 1 PCF, apoptotic rate was 51.6%.
Reactive oxygen species (ROS) are generated endogenously by all aerobic cells as byproducts of a number of metabolic reactions [28]. Oxidative stress results from an imbalance in the proxidant–antioxidant equilibrium during cell metabolism. While oxidative stress can damage cellular constituents and result in apoptosis, ultraviolet irradiation can induce the formation of ROS in biological systems. ROS can generate both cytotoxic effects on cells by oxidizing lipids and proteins, and genotoxic effects by (amongst other things) inducing base lesions [29]. UVB (280– 315 nm) irradiation can induce the formation of bulky DNA adducts such as cyclobutane pyrimidine dimers and
6–4 photoproducts, while exposure to UVA (315–400 nm) irradiation can result in alterations to single bases. However, much of the UV-induced DNA damage is caused indirectly via ROS [30,31]. In addition, ROS exacerbates formation of the highly reactive hydroxyl radical, which in turn can cause DNA strand breaks, damage protein residues, initiate lipid peroxidation and triggers apoptotic cellular death process. Therefore, drugs that exhibit free radical scavenging and iron chelating properties, may serve as potential candidates for protection of cell. Antioxidants may prevent apoptosis by means of suppression or scavenging ROS. As regards the role of PCF in thymocyte
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Fig. 4. Agarose gel electrophoresis of DNA fragmentation in thymocytes. After 2 h pretreatment with different concentrations of PCF (1.42 mmol L 1, 2.84 mmol L 1, 5.68 mmol L 1), cells were exposed to UV at a total dose of 6.16 mJ cm 2. Lane 1: DNA marker; Lane 2: cells irradiated with 6.16 mJ cm 2 UV only; Lane 3: control; Lanes 4–6: cells pretreated with 1.42 mmol L 1, 2.84 mmol L 1 and 5.68 mmol L 1 PCF, respectively. Table 1 Effects of PCF on UV-induced lipid peroxidation and intracellular ROS level Groups
MDA (lmol L (protein)
Control UV alone UV + 1.42 mmol L UV + 2.84 mmol L UV + 5.68 mmol L
4.18 ± 0.74 13.89 ± 0.87a 11.52 ± 1.11b 9.14 ± 0.53b,c 7.20 ± 0.59b,c,d
1 1 1
PCF PCF PCF
All values are means ± SD. a P < 0.01 vs. control. b P < 0.05 vs. UV alone. c P < 0.05 vs. UV + 1.42 mmol L d P < 0.05 vs. UV + 2.84 mmol L
1 1
1
g 1)
ROS (kU g 1) (protein) 336.1 ± 2.41 372.3 ± 5.40a 361.1 ± 1.82b 350.4 ± 1.77b,c 340.2 ± 5.06b,c,d
PCF. PCF.
Table 2 Effects of PCF on UV-induced loss of mitochondrial transmembrane potential Groups
Rhodamine 123 fluorescence intensity
Control UV alone UV + 1.42 mmol L UV + 2.84 mmol L UV + 5.68 mmol L
1473.96 ± 0.04 649.98 ± 0.08a 897.84 ± 0.09b 1123.46 ± 0.08b,c 1236.18 ± 0.10b,c,d
1 1 1
PCF PCF PCF
All values are means ± SD. a P < 0.01 vs. control. b P < 0.05 vs. UV alone. c P < 0.05 vs. UV + 1.42 mmol L d P < 0.05 vs. UV + 2.84 mmol L
1 1
PCF. PCF.
apoptosis, our results showed that PCF could powerfully protect thymocyte against UV-induced apoptosis. When PCF was pre-administered into the cultured media for
2 h, the formation of ROS were significantly decreased, apoptotic rate of thymocyte were decreased in a dosedependent manner after UV irradiation, suggesting that PCF contributes to protecting thymocytes from apoptosis by decreasing intracellular ROS. The amount of MDA reflects the level of lipid peroxidation [32]. The lipid peroxidation products alter the activities of numerous enzymes, which control the cellular intermediary metabolism, membrane-bound enzymes and ion transporters [33,34]. Exposure thymocyte to UV, which induced the formation of ROS, resulted in accumulation of lipid peroxidation. In this study, our data showed that the level of MDA in thymocytes was accumulated remarkably by UV irradiation. In the presence of PCF, the level of MDA in cells was significantly decreased, which suggested indirectly that the large amount of oxygen radicals was generated from UV irradiation, PCF could scavenge oxygen radicals and inhibit the reaction of lipid peroxidation. This effect was proportional to PCF concentration and may contribute to the anti-apoptotic mechanisms. Mitochondria play a pivotal role in the process of apoptosis from C. elegans to mammals. It has now become apparent that mitochondria act as integrators of pro-apoptotic signals, transducing these to the final execution machinery of apoptosis [35]. Mitochondria undergo the loss of mitochondrial membrane potential coincident with opening of the permeability transition pore under various conditions such as when cells are with overloaded with Ca2+ or oxidative stress, a state that leads to the release of apoptosis inducing factors [36]. One of the most popular methods of monitoring mitochondrial function is via the use of fluorescent potentiometric probes. Mitochondrial function in situ is monitored by measuring the accumulation of cationic fluorescent probes in response to the mitochondrial transmembrane potential in living cells. In this study, we have used the mitochondrial membrane potential indicator rhodamine 123 to assess the effects of PCF on the mitochondria membrane potential of UV-radiated murine thymocytes. UV-irradiation caused a dramatic reduction in rhodamine 123 staining; PCF was able to block the UV-induced loss of rhodamine 123 staining in a dosedependent manner. These results indicated that PCF could protect mitochondria of thymocytes against UVirradiation. Taken together, our results demonstrated that UV irradiation is a powerful inducer of apoptosis of murine thymocytes in vitro. PCF presented in the extracellular fluid could protect murine thymocyte against UV damage and reduce UV-induced apoptosis. These findings indicated that PCF might be a useful protective agent to modulate the function of murine thymocytes under UV irradiation. However, the protective function of PCF on thymocytes against UV irradiation may be more complex. One possibility is that PCF acts as a ROS scavenger that can protect cytoplasmic membrane from exogenic ROS damage. Further, PCF could enter cells and assist in the cellular antioxidant ability in case cells undergo oxidant stress.
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5. Abbreviations DMSO dimethyl sulphoxidate HPLC high-pressure liquid chromatograph MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide MDA malondialdehyde PCF polypeptide from Chlamys farreri PBS phosphate-buffered saline ROS reactive oxygen species TEM transmission electron microscope UV ultraviolet
Acknowledgements We appreciate Ru-yong Yao and Bo-xiao Ding for their technical assistance. We also appreciate Professor Jian Feng for his writing assistance. This work was supported by National Nature Science Foundation of China (No. 30471458) and Technology Bureau of Qingdao (No. 2000-260). References [1] F.R. Abdulla, S.R. Feldman, P.M. Williford, D. Krowchuk, M. Kaur, Tanning and skin cancer, Pediatr. Dermatol. 22 (2005) 501– 512. [2] D.L. Miller, M.A. Weinstock, Non-melanoma skin cancer in the United States: incidence, J. Am. Acad. Dermatol. 30 (1994) 774–778. [3] Q. Zhan, K.A. Lord, I. Alamo, M.C. Hollander, F. Carrier, D. Ron, K.W. Kohn, B. Hoffman, D.A. Liebermann, A.J. Fornace, The GAD and MPD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth, Mol. Cell. Biol. 14 (1994) 2361–2371. [4] S.J. Kuerbitz, B.S. Plunkett, W.V. Walsh, M.B. Kastan, Wild-type p53 is a cell cycle checkpoint determinant following irradiation, Proc. Natl. Acad. Sci. USA 89 (1992) 7491–7495. [5] X. Wu, A.J. Levine, p53 and EGF-1 cooperate to mediate apoptosis, Proc. Natl. Acad. Sci. USA 91 (1994) 3602–3606. [6] Z. Ronai, S. Rutberg, Y.M. Yang, UV-responsive element (TGACAACA) from rat fibroblasts to human melanomas, Environ. Mol. Mutagen. 23 (1994) 157–163. [7] P. Herrlich, H. Ponta, H.J. Rahmsdorf, DNA damage-induced gene expression: signal transduction and relation to growth factor signaling, Rev. Physiol. Biochem. Pharmacol. 119 (1992) 187–223. [8] P. Herrlich, H.J. Rahmsdorf, Transcriptional and post-transcriptional responses to DNA-damaging agents, Curr. Opin. Cell Biol. 6 (1994) 425–431. [9] H. Schenk, M. Klein, W. Erdbrugger, W. Droge, K. Schulze-Osthoff, Distinct effects of thioredoxin and antioxidants on the activation of transcription factors NF-kappa B and AP-1, Proc. Natl. Acad. Sci. USA 91 (1994) 1672–1676. [10] M.S. Duthie, I. Kimber, M. Norval, The effects of ultraviolet radiation on the human immune system, Br. J. Dermatol. 140 (1999) 995–1009. [11] V.K. Shreedhar, M.W. Pride, Y. Su, Origin and characteristics of ultraviolet-B radiation induced suppressor T Lymphocytes, J. Immunol. 61 (1998) 1327–1335. [12] J. Fuchs, M.E. Huflejt, L.M. Rothfuss, D.S. Wilson, G. Carcamo, L. Packer, Acute effects of near ultraviolet and visible light on the cutaneous antioxidant defense system, Photochem. Photobiol. 50 (1989) 739–744.
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