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Available online at www.sciencedirect.com

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Citreoviridin inhibits cell proliferation and enhances apoptosis of human umbilical vein endothelial cells Haifeng Hou a,b,∗,1 , Ru Zhou a,1 , An Li c,d,1 , Cheng Li e , Qunwei Li a , Jianbao Liu a , Baofa Jiang b a

School of Public Health, Taishan Medical University, Taian 271000, China School of Public Health, Shandong University, Jinan 250012, China c College of Animal Science & Veterinary Medicine, Guangxi University, Nanning 530004, China d Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China e Ruijin Hospital, Shanghai Jiao Tong University, Shanghai 200025, China b

a r t i c l e

i n f o

a b s t r a c t

Article history:

In some areas of China, citreoviridin (CIT) is considered one of the risk factors for devel-

Received 15 November 2013

opment of cardiovascular disease (CVD). Apoptosis of endothelial cell may induce vascular

Received in revised form

endothelium injury and atherosclerosis, which result in CVD probably. In this study, we

16 February 2014

investigated the effect of CIT on apoptosis and proliferation of human umbilical vein

Accepted 20 February 2014

endothelial cells (HUVECs). The MTT assay was used to determinate HUVECs proliferation.

Available online 2 March 2014

Distribution of the cell cycle was analyzed by flow cytometry. The Annexin-V/PI staining was used to investigate cell apoptosis. Western blotting analysis was used to indicate

Keywords:

changes in the expression level of apoptosis-related proteins. The results indicated that

Citreoviridin

CIT inhibited HUVECs proliferation and the cells were arrested at G0/G1 phase, which is

Endothelial cells

associated with decreased levels of cyclinD1 and increased expression of p53 and p21. The

Apoptosis

apoptosis rate of HUVECs was improved by CIT. The expression of Bcl-2 were down-regulated

Proliferation

after CIT treatment, whereas the levels of Bax was significantly up-regulated. Furthermore,

Cell cycle

CIT-induced apoptosis was accompanied by the activation of caspase-3, -9. These findings

Caspase

demonstrate that CIT inhibits cell proliferation via DNA synthesis reduction and induces caspase-dependent apoptosis in HUVECs. CIT plays a pivotal role in the process of endothelial cell apoptosis, may thereby play an important role in the improvement of CVD in areas of China that have a high prevalence of CIT contamination. © 2014 Elsevier B.V. All rights reserved.

∗ Corresponding author at: Institute of Epidemiology, School of Public Health, Taishan Medical University, No. 2 YingSheng East Road, Taian 271000, China. Tel.: +86 15698122444; fax: +86 538 6222053. E-mail address: [email protected] (H. Hou). 1 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.etap.2014.02.016 1382-6689/© 2014 Elsevier B.V. All rights reserved.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 828–836

Fig. 1 – Structural formula of citreoviridin.

1.

Introduction

Atherosclerosis is the lesion primarily underlies cardiovascular disease (CVD), one of the most common causes of illness and death worldwide, which is the principal causes of death in China (Hou et al., 2013; Wang et al., 2011). Generally, the endothelial surface of the vessel lumen is a relatively non-adhesive and non-thrombogenic conduit for the transportation of cellular and macromolecular constituents of blood. Apoptosis and inflammation in endothelial cells are key factors involved during atherosclerosis (Rus et al., 1996; Traub and Berk, 1998). Apoptotic endothelial cell death may distroy the endothelial monolayer and thereby contribute to vascular injury. Citreoviridin (CIT) is one of mycotoxins derived from Penicillium strains, and the structural formula of CIT is showed in Fig. 1. CIT is thought to be the toxicological reason of acute cardiac beriberi that has prevailed in Japan and Keshan disease in China (Taatjes et al., 2008; Taylor et al., 2008; Hou et al., 2006; Nishie et al., 1988). A novel regular phenomenon was discovered that the contamination of CIT in foods was obvious in some areas in China where the prevalence of cardiovascular disease was serious. Previous research has demonstrated that the contamination levels of CIT (in grains) in target areas in China were between 4.9 ␮g/kg and 33.2 ␮g/kg (Li et al., 2009). Although studies have shown that CIT induce apoptosis in HUVECs (Hou et al., 2011), but the underlying mechanisms of its action are not completely understood. The objective of the current study is to investigate the direct effects of CIT in inducing apoptosis to vascular endothelial cells in vitro and to explore its underlying mechanisms. Our results showed that CIT inhibited cell proliferation and induce cell apoptosis. And the stimulated effect of CIT on apoptosis of HUVECs via caspase-dependent apoptotic pathway was found. As apoptosis of endothelial cells is also a key factor during atherosclerosis we suggest that CIT can regulate apoptosis in endothelial cells during the process of atherosclerosis. And our findings may have important implications and underscore the role of CIT as a novel risk factor for atherosclerosis and CVDs.

(CIT) was provided by Fermentek Company (Jerusalem, Israel) and dissolved with 100% DMSO (concentration of the stock solution was 100 mg/l). Carbobenzoxy-valyl alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) were from Promega corporation (Madison, WI, USA). 3[4,5-Dimethylthiazol-2-y-l]-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Genview (Houston, TX, USA). Propidium iodine (PI) was purchased from Sigma Chemical Co (St Louis, MO, USA). Annexin V-FITC apoptosis detection kit, caspase-3 activity assay kit and caspase-9 activity assay kit were purchased from KeyGEN Biotech (Nanjing, P. R. China). The ECL Western Blotting Detection Reagents was purchased from Thermo Scientific Pierce (Rockford, IL, USA). Mouse anti␤-Actin, Mouse anti-Bax and rabbit anti-Bcl-2 were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-p21, mouse anti-p53, rabbit anti-cyclinB1 and rabbit anticyclinD1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-caspase-3, mouse anti-caspase-8, mouse anti-caspase-9 and rabbit anti-PARP were purchased from Cell Signaling Technology (Danvers, MA, USA). The antiNF-␬B p65 primary antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DAPI and Alexa Fluor 568 labeled anti-rabbit IgG were provided by Invitrogen (Carlsbad, CA, USA).

2.2.

Materials and methods

2.1.

Reagents

Human umbilical vein endothelial cells (HUVECs) were provided by ATCC (Manassas, VA, USA). DMEM/F12 growth medium and fetal bovine serum (FBS) were purchased from GIBCO BRL (Grand Island, NY, USA). Citreoviridin

Cell cultures and treatments

HUVECs were cultured in DMEM medium supplemented with 10% FBS, penicillin-G (100 U/ml), streptomycin (100 ␮g/ml). The growth medium was changed every other day until the cells reached confluence. Cells of passage 4 and 6 were seeded in monolayers at 37 ◦ C in a humidified atmosphere (5% CO2 and 95% air). According to previous experiments and the reported contamination levels of CIT in grains obtained in Northeast China (Li et al., 2009), we cultured HUVECs in relevant medium, and divided the cells into 3 groups: low level CIT treatment group (addition of 0.1 mg/l CIT in the medium), high level CIT treatment group (addition of 0.2 mg/l CIT in the medium), HUVECs in untreated group (addition of the same volume of DMSO only). In order to analyze the recovery of CIT-induced apoptosis, we pre-treated the cells with the pan-caspase inhibitor, Z-VAD-FMK (50 ␮M) for 1 h, then induced the cells by CIT (0, 0.1 and 0.2 mg/l) for 24 h. The procedures of drug preparation and treatment were carried out in the dark. HUVECs were further cultured at 37 ◦ C for 24 h, 48 h or 72 h. Five replicates for each experiment were performed.

2.3.

2.

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MTT assay

The growth inhibitory effect of CIT on HUVECs was assessed by MTT. Cells were seeded at a density of 1 × 103 cells/well in 96-well plates. And cells were treated with CIT at 0.1 mg/l or 0.2 mg/l for 72 h. MTT assays were performed as instructed by the manufacturer every 24 h. The absorbance was read at 490 nm using a microplate reader (Sunrise RC, Tecan, Switzerland). Relative cell viability was expressed as the percentage of the control.

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2.4.

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Cell cycle and cell apoptosis assay

The DNA contents were analyzed by the propidium iodine staining method. The trypsin treated cells were harvested and fixed with 75% ethanol for 20 h at 4 ◦ C. Fixed cells were rinsed twice with PBS, and re-suspended in PBS solution lacking calcium and magnesium containing 10 ␮g/ml RNase A, incubated in 37 ◦ C for 30 min. Then PI was added (10 ␮g/ml) and incubated in the dark for 30 min. FACScan flow cytometry and CellQuest analysis software (BD, San Jose, CA) were used to analyze stained cells. Cell apoptosis assay was detected by Annexin V-FITC kits (KeyGen Biotech). The washed cells were resuspended in 500 ␮l of PBS and incubated with 10 ␮l PI and 10 ␮l of Annexin V-FITC for 15 min at room temperature in the dark. Flow cytometry method was used to detect cell apoptosis.

2.5.

Western blotting analysis

Western blotting analysis was carried out after treatment with different doses of CIT for 24 h. After washing twice with cold PBS, HUVECs were lysed at 4 ◦ C for 1 h in lysis buffer (150 mM NaCl, 20 mM Tris–HCl, pH 7.4, 0.1% SDS, 1.0% NP-40, 0.5% Na-deoxycholate, 0.2 mM PMSF, and protease inhibitor cocktails). Lysates were centrifuged at 12,000 × g for 20 min and the supernatants were used as total cell lysates. A quantity of 40 ␮g of total protein per lane was separated in SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% milk powder in 0.05% Tween-TBS, incubated with the relevant primary antibodies or the irrelevant isotypic control (␤-actin antibody), and the secondary antibody diluted in 1% BSA. Detection of the target proteins on the membranes was performed using the ECL Western Blotting Detection Reagents (Thermo Scientific Pierce, Rockford, IL, USA).

2.6.

Immunofluorescence staining of NF-B

HUVECs were seeded on sterilized glass coverslips in 6-well plates and treated with CIT and TNF-␣ after serum starvation. After washing twice with PBS, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature and their permeability was increased using 0.2% TritonX-100 for 5 min. The cells were washed twice with PBS and incubated with blocking buffer containing 3% goat serum in PBS for 1 h at 37 ◦ C. Then the cells were incubated with rabbit anti-NF-␬B p65 primary antibody (1:250 dilution) for 1 h at 37 ◦ C, followed by incubation with Alexa Fluor 568 labeled goat anti-rabbit IgG for 1 h (1:500 dilution). Cells were then labeled with DAPI (1 ␮g/ml) for 5 min and were washed three times with PBS. The images were captured with an Olympus BX51 fluorescence microscope (Olympus, Japan).

2.7.

Determination of activities of caspases in HUVEC

HUVECs were pre-treated with Z-VAD-FM (50 ␮M) for 1 h, then treated with CIT (0, 0.1 and 0.2 mg/l) for 24 h. Caspase-3, and -9 activities were measured using a colorimetric assay kit (Kaiji Biotechnology, Nanjing, China) according to the manufacturer’s instructions. Cells were collected and immediately used for the measurement of protein concentration

Fig. 2 – Effect of CIT on the viability of HUVECs. Cells were incubated with 0, 0.1 mg/l, 0.2 mg/l of CIT for 72 h, and the cell growth was determined by the MTT assay at every 24 h. All the data were expressed as fold change compared with the control group, and the graph displays the Mean ± SD of the five independent experiments (P < 0.05, compared in different concentration groups of CIT).

and caspase-3, and -9 activities. The protein concentration was determined by the Bradford assay. The enzymes activities were detected by a microplate reader (Sunrise RC, Tecan, Switzerland) at 405 nm. The activities of caspase enzymes are expressed as fold change compared with the control.

2.8.

Statistical analysis

All the experiments were repeated five times. The data were expressed as the Mean ± SD. Statistical evaluation of the data was performed using a one-way analysis of variance followed by Dunnett’s test for comparisons among more than two groups. P < 0.05 was considered statistical significant.

3.

Results

3.1.

Effect of CIT on the HUVECs proliferation

To determine whether CIT can inhibit the HUVECs proliferation, the cells were treated with CIT (0.1 mg/l or 0.2 mg/l) for 72 h, and the proliferation rate was determined by the MTT assay at every 24 h. As shown in Fig. 2, CIT inhibited the growth of HUVECs in a dose- and time-dependent manner (P < 0.05). The effect of CIT on the cell cycle distribution was analyzed by flow cytometry. As shown in Fig. 3, CIT treatment results in the accumulation of HUVECs in the G0/G1 phase, and cell numbers in S-phase had a significant decrease. The results suggest that CIT inhibits HUVECs growth by blocking the G0/G1 to S phase transition in the cell cycle in a dose-dependent manner. To understand the G0/G1 arrest signaling pathway, we analyzed the expression of G0/G1 checkpoint regulators in HUVECs by western blot analysis. As shown in Fig. 4, the expression of p53 and p21 was obviously increased after HUVECs exposed to CIT for 24 h, whereas the expression of

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Fig. 3 – CIT induces cell cycle arrest in HUVECs. Cells were treated with 0, 0.1 mg/l, 0.2 mg/l of CIT for 24 h and then stained with PI. The DNA content was analyzed by means of flow cytometry. Five replicates for each experiment were performed. The figure expresses a dose-dependent increased G0/G1 phase, and decreased S-phase significantly (P < 0.05, compared in 3 groups).

cyclinD1 were marked decreased compared to that of control group. Our data demonstrated that down-regulation of cyclinD1 and up-regulation of p53 and p21 might contribute to the CIT-inhibited G0/G1 to S phase transition.

3.2. CIT induces HUVECs apoptosis in time- and dose-dependent manner We determined whether CIT can cause apoptosis of HUVECs in different doses or time, asymmetry and permeability of the cell membrane by Annexin V-FITC and PI staining. HUVECs were incubated with 0, 0.1 mg/l, 0.2 mg/l of CIT for 24 or 48 h, and then analyzed. Flow cytometry analysis results indicate that CIT induces apoptosis in HUVECs (Fig. 5). At a concentration of 0.2 mg/l CIT, the effect of apoptosis in HUVECs at 24 h is equivalent to the effect of 0.1 mg/l of CIT at 48 h. The results suggest that CIT can damage HUVECs by inducing apoptosis of the cells.

3.3. CIT treatment modifies the expression of Bcl-2 in HUVECs Bcl-2 family proteins have either pro- or anti-apoptotic activities and modulate the mitochondrial apoptosis signal pathway. The balance between anti-apoptotic and proapoptotic is critical in determining the susceptibility of cells to death signals. Western blot analysis indicates that the treatment of HUVECs with CIT results in the dose-dependent decreasing levels of anti-apoptotic Bcl-2 protein. The expression of the pro-apoptotic Bax protein was up-regulated significantly after CIT treatment (Fig. 6).

CIT-induced apoptosis is mediated via activation 3.4. of caspases in HUVECs To identify the possible pathway of HUVECs apoptosis induced by CIT, we examined the cleavage of caspases in HUVECS treated with CIT for 24 h. Western blot analysis showed the

Fig. 4 – Effects of CIT on expression of p21, p53, cyclinB1 and cyclinD1 in HUVECs. (a) Western blotting analysis showing p21, p53 cyclinB1 and cyclinD1 in response to 0, 0.1 mg/l, 0.2 mg/l of CIT; (b) All the data were expressed as fold change compared with the control group. The levels of p21, p53 cyclinB1 and cyclinD1 are expressed as the means ± SD for five independent experiments. *P < 0.05 compared with the control group, **P < 0.01 compared with the control group.

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Fig. 5 – CIT induces the apoptosis of HUVECs. Cells were treated with 0, 0.1 mg/l, 0.2 mg/l of CIT for 24 or 48 h, then analyzed for apoptosis by flow cytometry with Annexin-V-FITC/PI dual staining. The figure shows a dose-dependent increase in apoptotic cells to CIT treatment (P < 0.05, compared in different concentration groups of CIT). Results were expressed as percentage of apoptotic cells that include the cells in early and later apoptosis.

cleavage of apical pro-caspase-9 and -3 into the characteristic active fragments was already evident after treatment with CIT (Fig. 7). We also examined the effect of CIT on PARP, and the results indicated that CIT induced the cleavage of PARP in a dose-dependent manner. Because no obvious cleavage of casepase-8 occurs, we conclude that CIT plays a critical role in the intrinsic pathway of apoptosis. In order to determine whether NF-␬B nuclear translocation affects HUVECs apoptosis induced by CIT, we detected localization of NF-␬B p65 with immunofluorescence staining assay. As shown in Fig. 8, no alteration of NF-␬B localization is found in response to CIT treatment.

3.5. Z-VAD-FMK inhibits CIT-induced apoptosis and activities of caspases in HUVECs. To completely understand the role of caspases in CIT induced HUVECs apoptosis, a cell permeable pan-caspase inhibitor, Z-VAD-FMK, was used to investigate the effects of caspases blockade. HUVECs were pre-treated with 50 ␮M of Z-VAD-FMK for 1 h, followed treated with CIT (0, 0.1 and 0.2 mg/l) for 24 h, then rates of apoptosis and activities of caspase-3 and caspase-9 were determined. The results show that Z-VAD-FMK significantly inhibited apoptosis rates of CIT treated HUVECs (Fig. 9). And activities of caspase-3 and caspase-9 were blocked by Z-VAD-FMK (Fig. 10).

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Fig. 6 – CIT treatment modifies the expression of Bcl-2 and Bax in HUVECs. (a) Expressions of Bcl-2 and Bax were determined by western blot analysis. (b) Each bar corresponds to the Mean ± SD for five independent experiments. *P < 0.05 compared with the control group, **P < 0.01 compared with the control group.

4.

Discussion

CIT was reported as a stimulant for apoptosis in HUVECs, and played the pathogenic role in cardiovascular disease in CIT polluted areas of China (Hou et al., 2013; Li et al., 2009). But the mechanism of apoptosis induced by CIT is not clearly mentioned. The present study reports the mechanism of CIT in inhibiting cell proliferation and inducing cell apoptosis in HUVECs. Apoptosis is regulated via the action of several oncogenes and subsequently oncoproteins that display inhibiting or promoting action(Wang et al., 2012). Bcl-2, a gene located at chromosome, encodes a 26-kD protein that blocks programmed cell death without affecting cellular proliferation (Adams and Cory, 1998; Hockenbery et al., 1990). The bax protein is a member of the bcl-2 family which promotes

apoptosis (Fei et al., 2012; Oltvai et al., 1993). Bax resides in the cytosol and translocates to mitochondria upon induction of apoptosis (Hsu et al., 1997; Wolter et al., 1997). Bax has been shown to induce cytochrome C release and activate caspase in in vivo and in vitro(Jürgensmeier et al., 1998; Rossé et al., 1998). The ratio of bax to bcl-2 determines the susceptibility of a cell to apoptosis (Yang and Korsmeyer, 1996). In many systems, members of the bcl-2 family modulate apoptosis, with the bax/bcl-2 ratio serving to as a rheostat to determine cell susceptibility to apoptosis (Korsmeyer, 1999). In this study, upregulation in the levels of Bax was observed when HUVECs were treated with CIT. The expression of Bcl-2 protein, however, was simultaneously decreased after CIT treatment. Caspases, a family of cysteine proteases, are synthesized as inactive pro-enzymes which are processed to active form in cells undergoing apoptosis. Upon induction of apoptosis, cytochrome C is released from mitochondria associates with

Fig. 7 – Effects of CIT on caspase-3, -8, -9 and PARP activities in HUVECs. (a) Western blotting analysis showing cleavage of caspase-3, -9 and PARP in response to 0, 0.1 mg/l, 0.2 mg/l of CIT while No cleavage of caspase-8 is found. (b) All the data were expressed as fold change compared with the control group. The levels of cleaved caspases and PARP are expressed as the means ± SD for five independent experiments. *P < 0.05 compared with the control group, **P < 0.01 compared with the control group.

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Fig. 8 – Nuclear translocation of NF-␬B p65 in HUVECs. Cells were cultured on coverslips and treated with 0, 0.1, 0.2 mg/l of CIT for 24 h. Then the cells were fixed, their permeability was increased, and labeling was performed with an anti-human NF-␬B p65 antibody followed by a Alexa Fluor 568-labeled secondary antibody. The nucleus was stained with 4 ,6-diamidino-2-phenylindole (DAPI). The results were observed using fluorescent microscopy. Slides were viewed with an Olympus BX51 fluorescence microscope (Olympus Japan) using a Splan 40PL lens at 40×/0.70.

pro-caspase-9 and Apaf-1, and then it processes pro-caspase9 into a large subunit (37 kDa/17 kDa) and a small subunit (10 kDa) (Allan and Clarke, 2009). Cleaved caspase-9 further processes caspase-3, one of the key executioners of apoptosis, and PARP resulting in the downstream related protein

Fig. 10 – Effect of CIT on caspases activities in HUVECs. Cells were pre-treated with 50 ␮M of Z-VAD-FMK for 1 h, followed with CIT (0, 0.1 and 0.2 mg/l) for 24 h, and caspase-3, -9 activities were determined. All the data were expressed as fold change compared with the control group, and the graph displays the Mean ± SD of the five independent experiments. *P < 0.05, **P < 0.01.

activation and eventually cell death (Droga-Mazovec et al., 2008; Yun et al., 2009). In present study, we found the increased cleavage of caspase-3, -9, PARP in HUVECs treated with CIT. As no obvious casepase-8 activation and NF-␬B nuclear translocation were triggered, we conclude that CIT plays a critical role in the intrinsic pathway of apoptosis. CIT induces HUVECs apoptosis by activation of the intrinsic-mediated cell apoptosis pathway, characterized by elevated levels of caspase-9, followed by caspase-3. Vascular endothelial cells form the inner lining of blood vessels and play an important role in regulating vascular function. Since the vascular endothelium is involved in various physiological processes, endothelial cell apoptosis may constitute an initial

Fig. 9 – Z-VAD-FMK inhibits CIT-induced apoptosis of HUVECs. Cells were pre-treated with 50 ␮M of Z-VAD-FMK for 1 h, followed treated with CIT (0, 0.1 and 0.2 mg/l) for 24 h, then cell apoptosis rates were determined. The effect of Z-VAD-FMK on the recovery of CIT induced apoptosis is significant. Results were expressed as percentage of apoptotic cells that include the cells in early and later apoptosis. *P < 0.05, **P < 0.01.

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step in a variety of pathological situations such as atherosclerosis and hypertension. As the results show, cell growth suppression was caused by CIT. Exposure of HUVECs to CIT resulted in the enrichment of G0/G1 fraction in a dose-dependent manner, which was accompanied by a decrease in S phase. The effect of CIT on cell cycle distribution was significant. Many kinases and kinase inhibitors in the cell cycle are involved in mediating the G1/S transition, and cyclinD1 is important at various stages of the cell cycle (Lapenna and Giordano, 2009; Satyanarayana and Kaldis, 2009). The results in our experiments indicated that cyclinD1 was down-regulated by CIT treatment. Moreover, p21, which plays a crucial role in the regulation of G1/S transition by a p53-independent pathway, was also triggered by CIT in HUVECs. However, the levels of cyclinB1 were not altered in CIT-treated cells. Other research in lung cancer cells showed that CIT was an ATP-synthase inhibitor and induced the unfolded protein response (UPR) associated with phosphorylation the translation initiation factor 2␣ (eIF2␣), triggering cell growth inhibition (Chang et al., 2012). This conclusion is likely due to different cell line used, and the treatment dose of CIT. Moreover, CIT is not stable in light, when exposed in candescent, fluorescent or sun light for brief period of time, there was a significant amount of other substance. In this study, all procedures of drug preparation and treatment were strictly carried out in the dark. Our previous study showed that CIT increased TNF-␣-induced activation of endothelial cells in a higher dose of 1 mg/l, because CIT was used in natural illumination of culturing room (Hou et al., 2013). In conclusion, our results suggest that CIT mediated cell apoptosis via caspase-dependent apoptotic pathway. CIT inhibits HUVECs growth through induction of G0/G1 phase arrest in a dose-dependent manner. We hypothesize that CIT may play an important role in the improvement of atherosclerosis and cardiovascular disease in areas of China that have a high prevalence of CIT contamination and cardiovascular disease.

Conflict of interest statement None declared.

Acknowledgments This study was supported by the National Natural Science Foundation of China (Grants: NSFC, no. 81202170), Shandong Provincial Natural Science Foundation, China (no. ZR20010HQ006) and the Project of Shandong Province Higher Educational Science and Technology Program (no. J10LF17).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.etap. 2014.02.016.

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references

Adams, J.M., Cory, S., 1998. The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322–1326. Allan, L.A., Clarke, P.R., 2009. Apoptosis and autophagy: regulation of caspase-9 by phosphorylation. FEBS J. 276, 6063–6073. Chang, H.Y., Huang, H.C., Huang, T.C., Yang, P.C., Wang, Y.C., Juan, H.F., 2012. Ectopic ATP synthase blockade suppresses lung adenocarcinoma growth by activating the unfolded protein response. Cancer Res. 72, 4696–4706. Droga-Mazovec, G., Bojic, L., Petelin, A., Ivanova, S., Romih, R., Repnik, U., Salvesen, G.S., Stoka, V., Turk, V., Turk, B., 2008. Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues. J. Biol. Chem. 283, 19140–19150. Fei, H.R., Chen, H.L., Xiao, T., Chen, G., Wang, F.Z., 2012. Caudatin induces cell cycle arrest and caspase-dependent apoptosis in HepG2 cell. Mol. Biol. Rep. 39, 131–138. Hockenbery, D., Nunez, G., Milligan, C., Schreiber, R.D., Korsmeyer, S.J., 1990. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348, 333–336. Hou, H., Li, Q., Qi, Y., 2006. Effect on relevant of Penicillium citreoviridin toxin production. Chin. J. Public Health 22, 811–812. Hou, H., Li, C., Li, Q., Li, D., Ye, W., Jiao, P., 2011. Effect of apoptosis and DNA damage on human umbilical vein endothelial cells is induced by citreoviridin. Chin. Prev. Med. 12, 304–306. Hou, H., Zhou, R., Jia, Q., Li, Q., Kang, L., Jiao, P., Li, D., Jiang, B., 2013. Citreoviridin enhances tumor necrosis factor-␣-induced adhesion of human umbilical vein endothelial cells. Toxicol. Ind. Health, http://dx.doi.org/10.1177/0748233713483194. Hsu, Y.T., Wolter, K.G., Youle, R.J., 1997. Cytosol-to-membrane redistribution of Bax and Bcl-X(L) during apoptosis. Proc. Natl. Acad. Sci. U. S. A. 94, 3668–3672. Jürgensmeier, J.M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D., Reed, J.C., 1998. Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl. Acad. Sci. U. S. A. 95, 4997–5002. Korsmeyer, S.J., 1999. BCL-2 gene family and the regulation of programmed cell death. Cancer Res. 59, 1693–1700. Lapenna, S., Giordano, A., 2009. Cell cycle kinases as therapeutic targets for cancer. Nat. Rev. Drug Discov. 8, 547–566. Li, D., Lv, H., Duan, L., Guo, Y., Chen, Z., Lu, W., 2009. Disease monitoring report of Keshan disease area of Jilin Province in 2008. Chin. J. Ctrl. Endem. Dis. 24, 226–233. Nishie, K., Cole, R.J., Dorner, J.W., 1988. Toxicity of citreoviridin. Res. Commun. Chem. Pathol. Pharmacol. 59, 31–52. Oltvai, Z., Milliman, C., Korsmeyer, S.J., 1993. Bcl-2 heterodimerizes in vivo with a conversed homolog Bax, that accelerates programmed cell death. Cell 74, 609–619. Rossé, T., Olivier, R., Monney, L., Rager, M., Conus, S., Fellay, I., Jansen, B., Borner, C., 1998. Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c. Nature 391, 496–499. Rus, H.G., Vlaicu, R., Niculesch, F., 1996. Interleukine-6 and interleukine-8 protein and gene expression in human arterial atherosclerotic wall. Atherosclerosis 127, 263–271. Satyanarayana, A., Kaldis, P., 2009. Mammalian cell-cycle regulation: several Cdks, numerous cyclins and diverse compensatory mechanisms. Oncogene 28, 2925–2939. Taatjes, D.J., Sobel, B.E., Budd, R.C., 2008. Morphological and cytochemical determination of cell death by apoptosis. Histochem. Cell Biol. 129, 33–43. Taylor, R.C., Cullen, S.P., Martin, S.J., 2008. Apoptosis: controlled demolition at the cellular level. Nat. Rev. Mol. Cell Biol. 9, 231–241.

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Traub, O., Berk, B.C., 1998. Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscl. Throm. Vas. Biol. 18, 677–685. Wang, N., Han, Y., Tao, J., Huang, M., You, Y., Zhang, H., Liu, S., Zhang, X., Yan, C., 2011. Overexpression of CREG attenuates atherosclerotic endothelium apoptosis via VEGF/PI3K/AKT pathway. Atherosclerosis 218, 543–551. Wang, F.Z., Fei, H.R., Li, X.Q., Shi, R., Wang de, C., 2012. Perifosine as potential anti-cancer agent inhibits proliferation, migration, and tube formation of human umbilical vein endothelial cells. Mol. Cell. Biochem. 368, 1–8.

Wolter, K.G., Hsu, Y.T., Smith, C.L., Nechushtan, A., Xi, X.G., Youle, R.J., 1997. Movement of Bax from the cytosol to mitochondria during apoptosis. J. Cell Biol. 139, 1281–1292. Yang, E., Korsmeyer, S.J., 1996. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 88, 386–401. Yun, S.I., Yoon, H.Y., Chung, Y.S., 2009. Glycogen synthase kinase-3beta regulates etoposide-induced apoptosis via Bcl-2 mediated caspase-3 activation in C3H10T1/2 cells. Apoptosis 14, 771–777.

Citreoviridin inhibits cell proliferation and enhances apoptosis of human umbilical vein endothelial cells.

In some areas of China, citreoviridin (CIT) is considered one of the risk factors for development of cardiovascular disease (CVD). Apoptosis of endoth...
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