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Selenium nanoparticles inhibit the growth of HeLa and MDA-MB-231 cells through induction of S phase arrest
Colloids and Surfaces B: Biointerfaces 94 (2012) 304–308
Contents lists available at SciVerse ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Selenium nanoparticles inhibit the growth of HeLa and MDA-MB-231 cells through induction of S phase arrest Haiying Luo a , Feifei Wang b,c , Yan Bai a,∗ , Tianfeng Chen a , Wenjie Zheng a,∗ a b c
College of Life Science and Technology, Jinan University, Guangzhou 510632, PR China State Key Laboratory of Biocontrol, Department of Biochemistry, College of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, PR China National Institutes for Food and Drug Control, Beijing 100050, PR China
a r t i c l e
i n f o
Article history: Received 28 October 2011 Received in revised form 4 February 2012 Accepted 6 February 2012 Available online 14 February 2012 Keywords: Selenium nanoparticles HeLa cells MDA-MB-231 cells Atomic force microscope Flow cytometric analysis
a b s t r a c t In vitro antiproliferative effects of selenium nanoparticles (nanoSe0 , 10–40 mol/L) on HeLa (human cervical carcinoma) cells and MDA-MB-231 (human breast carcinoma) cells were examined by optical microscopic inspection and MTT assay in the present study. The nanoSe0 effectively inhibited the growth of MDA-MB-231cells and HeLa cells in a dose-dependent manner. The morphology analysis with atomic force microscope showed that the HeLa cells treated with 10 mol/L nanoSe0 were rough and shrunken with truncated lamellipodia at terminal part of the cells. Flow cytometric analysis demonstrated that HeLa cells were arrested at S phase of the cell cycle after exposed to nanoSe0 (10 mol/L). Taken together, our results suggested that nanoSe0 may be more helpful in cancer chemoprevention as a potential anticancer drug. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Cancer chemoprevention is defined as the use of natural, synthetic, or biologic chemical agents to prevent the process of carcinogenesis before the development of malignancy [1]. Chemoprevention is a cost-effective approach to reduce cancer morbidity and mortality through inhibition of precancerous events before the clinical occurrence of the disease. Selenium is an essential micronutrient for human health and shows three levels of biological activity [2]. Selenium is an essential element in trace concentrations but a toxic element in elevated concentrations for humans, and selenium can be stored within organism as supplementary selenium source for maintaining the homeostatic functions in moderate concentrations. Selenium plays an important role in nourishment and medicine [3,4]. For example, selenium compounds exhibit potent chemopreventive activities against cancer [5–9] and selenium supplementation can reduce the cancer mortality and incidence, especially for several major cancers, including prostate, lung, colon, and liver cancers [10,11]. The role of selenium compounds as chemopreventive and chemotherapeutic agents has been supported by a large number of epidemiological, preclinical, and clinical studies [12,13]. Accumulated evidence has suggested that the dose and the chemical form (structure) are important
∗ Corresponding authors. Tel.: +86 2085220223. E-mail addresses: [email protected] (Y. Bai), [email protected] (W. Zheng). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2012.02.006
determinants of chemopreventive activities of selenium compounds [1,6,13]. Element selenium nanoparticles (nanoSe0 ) are attracting increasing attention due to their excellent biological activities and low toxicity [14–19]. Tan et al. indicated that adriamycin (ADM) and nanoSe0 were both able to inhibit Bel7402 cells proliferation in a dose-dependent manner and the combined treatment with ADM and nanoSe0 was more effective in inhibiting cancer cell growth than each of the two drugs alone [9]. Zhou et al. synthesized a novel Se-Fe3 O4 nanocomposites material to examine the magnetically enhanced cytotoxicity of nanoSe0 on human osteoblast-like MG-63 cells [20]. Our previous works reported that some cancer cells, such as A375 human melanoma cells, HepG2 hepatocellular carcinoma cells, MCF-7 breast adenocarcinoma cells, HeLa (human cervical carcinoma) cells, and human kidney HK-2 cells, treated with the nanoSe0 modified with sialic acid or Undaria pinnatifida polysaccharide resulted in dose-dependent cell apoptosis as indicated by DNA fragmentation and phosphatidylserine translocation [21,22]. Several mechanisms have been postulated to elucidate the anticancer activity of selenium, which include induction of cell apoptosis, inhibition of cell proliferation, modulation of redox state, detoxification of carcinogen, stimulation of the immune system, and inhibition of angiogenesis [23–25]. Among these potential mechanisms of anticancer activities of selenium, apoptosis receives most attention and has been postulated to be critical for cancer chemoprevention by selenocompounds [26]. However, so far very little information about the nanoSe0 could be obtained.
H. Luo et al. / Colloids and Surfaces B: Biointerfaces 94 (2012) 304–308
In recent years, atomic force microscopy (AFM) is becoming one of the most important visualization technologies in cell biology. AFM can detect cellular topography, ultrastructure and change in cellular morphology after biochemistry reaction. Thus, AFM analysis could provide valuable information on cytoskeleton and morphology in the cell ultrastructure following treatment with drugs [27–29]. In this study, the effects of nanoSe0 on cell proliferation and growth in HeLa (human cervical carcinoma) cells and MDA-MB231 (human breast carcinoma) cells were examined using MTT colorimetry and flow cytometric analysis (FCM). Simultaneous experiments using optical microscopy and AFM were also performed for comparison purposes. 2. Materials and methods 2.1. Reagents and materials 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), parenzyme, acridine orange (AO), propidium iodide (PI), dimethyl sulfoxide (DMSO), penicillin and streptomycin were purchased from Sigma–Aldrich. Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum were purchased from Hyclone Laboratories Inc. Phosphate-buffer solution (PBS, pH 7.4) consisting of 138 mmol/L NaCl, 2.67 mmol/L KCl, 8.1 mmol/L Na2 HPO4 ·12H2 O and 1.47 mmol/L KH2 PO4 was used. Other chemicals were of analytical reagent grade. All solutions were prepared using ultrapure water. The resistivity of ultrapure water is 18.25 M cm. 2.2. Cell culture The HeLa cells and MDA-MB-231 cells were obtained from the cell lab of Sun Yat-sen University. Both cell lines were cultured in DMEM medium supplemented with fetal bovine serum (10%), penicillin (100 units/mL), and streptomycin (50 units/mL) at 37 ◦ C in CO2 incubator (95% relative humidity, 5% CO2 ). 2.3. Preparation of nanoSe0 The nanoSe0 sol (1 mmol/L) was prepared by mixing 5 mL SeO2 (2 mmol/L) and 5 mL vitamin C (Vc, 4 mmol/L) (1:2 in the molar ratio of SeO2 to Vc) and subsequently diluted to the required concentrations. The nanoSe0 was spherical with an average diameter of 133 nm, which was measured by using zetasizer nano (Nano ZS, Malvern, UK) and transmission electron microscopy technique (TEM, TECNAI-10, Philips, Germany). It was preferable to use nanoSe0 immediately after preparing. The amount of residual Vc in the nanoSe0 was detected by iodometric method combined with the automatic potentiometric titrator (TIM840, Radiometer Analytical, France). The concentration of the residual Vc in the 40 mol/L nanoSe0 was 7.6 mol/L. In the present study, the effect of 41.4 mol/L Vc as negative control group on the cell experiments was examined.
305
Infinite M200, Austria). Observation of cell morphological change was performed with an OLYMPUS Upright fluorescence microscope (Imager Z1, Carl Zeiss). 2.5. Flow cytometric analysis Well-cultivated HeLa cells were seeded in 60 mm tissue culture dishes and incubated over night at 37 ◦ C in a humidified atmosphere of 5% CO2 . The culture medium was aspirated and replaced with fresh medium containing the nanoSe0 diluted at final 10 mol/L. PBS served as blank control and 41.4 mol/L Vc served as negative control. After incubation for 24–48 h, the cell layer was trypsinized and washed once with cold PBS and incubated with cold 70% ethanol at 4 ◦ C for about 24 h. Cells were washed with cold PBS and resuspended in 250 L cold PBS. The samples were incubated with RNase and PI at 37 ◦ C for 30 min and analyzed by a FACSCalibur flow cytometer (FCM, Becton Dickinson & Co., Franklin Lakes, NJ). The samples were collected in FL2 channel (excitation at 488 nm and emission at 585 ± 21 nm) and the number of cells analyzed for each sample was 10,000. 2.6. Atomic force microscopy About 1 × 105 /mL of HeLa cells were seeded on a glass cover slip cultivated in fresh medium. Then the cover slips with cells were treated with nanoSe0 for 24 h. The HeLa cells in incubation solution (10 mL) were dropped onto cleaned coverslips and climbed for about 3–5 min. After cells were well adhered to coverslips, the cell layer was fixed with 2.5% glutaraldehyde for 15 min, then washed with pure water for three times. All the fixed samples were air dried. The prepared samples were imaged at room temperature using an atomic force microscope (AFM, Autoprobe CP Research, Veeco) in the tapping mode. The Si3 N4 tip was employed for cell scanning in this study. The tip cantilevers length was 100 m, the resonance frequency was around 100 kHz (Veeco, Santa Barbara, USA) and the force constant was 20–80 N/m. The scanning range of AFM scanner was 0.5 Hz, and all of the acquired images (512 × 512 pixels) were flattened with the provided software (Proscan Image Processing Software Version 2.1) to eliminate low-frequency background noise in scanning direction (flatten order: 0–1). AFM-based force spectroscopy was used for force detection. Force–distance curves were obtained by standard retraction. All force–distance curve experiments were performed at the same loading rate. 2.7. Statistical analysis Experiments were carried out at least in triplicate and results were expressed as mean ± S.D. Statistical analysis was performed by one-way analysis of variance (ANOVA). Difference with *P < 0.05 or **P < 0.01 was considered statistically significant. 3. Results and discussion
2.4. MTT assay 3.1. Cell viability by MTT assay Cell viability was determined by measuring the ability of cells to converse MTT to a purple formazan dye. For our purpose, cells were seeded in 96-well tissue culture plates at 2.5 × 103 cells/well for 24 h, and then cells were incubated with medium containing different concentrations of nanoSe0 for 24 h. After incubation, 20 L/well of MTT solution (5 mg/mL PBS) was added and incubated for 4 h. The medium was aspirated and replaced with 150 L/well DMSO to dissolve the formazan salt formed. The color intensity of the formazan solution, which reflected the cell growth condition, was measured at 570 nm using a multimode microplate reader (Tecan
Cell viability was quantitatively evaluated by MTT assay. As shown in Fig. 1, nanoSe0 (10–40 mol/L) reduced the viability of MDA-MB-231 cells and HeLa cells. The viability of MDA-MB-231 cells was reduced to around 60% (Fig. 1a) and the viability of HeLa cells was dose-dependently reduced from around 73% to around 50% (Fig. 1b). In comparison, 20 mol/L SeO2 exhibited the intense cytotoxicity against HeLa cells and MDA-MB-231 cells. However, our previous works indicated that compare with SeO2 the nanoSe0 showed lower cytotoxicity toward normal cells [21]. Considering
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100
Cell viabitity (%)
80 60
a b **
**
** **
**
** **
**
**
**
40 20
20
(P µm os o iti l/L ve Se Bl con O2 (N ank trol ) eg ati ve co 10 nt µm ro l) ol /L na 20 no Se 0 µm ol /L na no 30 Se 0 µm ol /L na no 40 Se 0 µm ol /L na no Se 0
0
Fig. 1. Growth inhibition on MDA-MB-231 cells (a) and HeLa cells (b) after 24 h incubation by nanoSe0 and SeO2 . The data represent the mean values (±S.D.).
selectivity between cancer and normal cells, nanoSe0 may be superior to SeO2 in cancer chemoprevention. 3.2. Optical microscopic inspection The optical microscopic images of cells were displayed in Fig. 2. Obviously, the HeLa cells and MDA-MB-231 cells appeared
fan-shaped and square with smooth surface (Fig. 2a). A large number of cells became rounded and shrunken and the adherence cell number decreased. And the density of cells was evidently decreased with the treatment of 10–40 mol/L nanoSe0 (Fig. 2b–e), which was similar to that treated with 20 mol/L SeO2 served as a positive control (Fig. 2f). Furthermore, when the cancer cells were treated with high concentration of nanoSe0 (40 mol/L), most of the cells were gathered together and suspended in the culture medium. These results indicated that nanoSe0 could induce the change of cell morphology and inhibit the cancer cells growth in a dose-dependent manner. 3.3. Ultrastructure and quantitative roughness analysis of HeLa cells by AFM Visualization and quantitative roughness analysis of HeLa cells were investigated by AFM. The morphological features of HeLa cells and HeLa cells treated with Vc and nanoSe0 were shown in Fig. 3. The HeLa cells treated with or without 41.4 mol/L Vc were fan-shaped with complete membrane, regular shape and relatively smooth surface (Fig. 3a1–a3 and b1–b3), and had dense filopodia and lamellipodia at terminal part (Fig. 3a1 and b1). These ultrastructural parts may signify invasive property of the proliferating cancer cells. As shown in Fig. 3c, the surface morphology and ultrastructure of HeLa cells under the effect of nanoSe0 had changed significantly. The cells treated with 10 mol/L nanoSe0 displayed rough and shrunken cell morphology with truncated lamellipodia at terminal part of the cell, which may be an early injury response of HeLa cells to nanoSe0 . And also the surface roughness of the HeLa cells membranes increased after treated with 10 mol/L nanoSe0 (Table 1). These results indicated that nanoSe0 had intense
Fig. 2. Inverted microscopic images of HeLa cells and MDA-MB-231 cells incubated separately with only the culture medium as the control (a), 10 mol/L nanoSe0 (b), 20 mol/L nanoSe0 (c), 30 mol/L nanoSe0 (d), 40 mol/L nanoSe0 (e), 20 mol/L SeO2 (f) for 24 h. The magnification was 100×.
H. Luo et al. / Colloids and Surfaces B: Biointerfaces 94 (2012) 304–308
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Fig. 3. AFM images of HeLa cells induced for 24 h. (a) Control group; (b) cells treated with 41.4 mol/L Vc; (c) cells treated with 10 mol/L nanoSe0 . (a1) Error signal morphology (70 m × 70 m); (a2) topography; (a3) 3D image; (a4) error signal morphology (2 m × 2 m); (a5) ultrastructure topography of a4. (b1) Error signal morphology (80 m × 80 m); (b2) topography; (b3) 3D image; (b4) error signal morphology (2 m × 2 m); (b5) ultrastructure topography of b4. (c1) Error signal morphology (60 m × 60 m); (c2) topography; (c3) 3D image; (c4) error signal morphology (2 m × 2 m); (c5) ultrastructure topography of c4.
cytotoxic effect on HeLa cells. The AFM images and roughness analysis substantiated the cytotoxic effects of nanoSe0 on HeLa cells obtained by using optical microscopic inspection and MTT assay. 3.4. Effects of nanoSe0 on cell cycle distribution In agreement with the present findings, inhibition of proliferation in cancer cells treated with drugs could be the result of induction of apoptosis or cell cycle arrest or a combination of these two modes. When cancer cells are arrested in the S phase, the mitosis and proliferation of cancer cells are inhibited.
The content of DNA is very sensitive to reflect the cell metabolism and cell cycle progression. The proportion of cell cycle and content of DNA diploid were shown in Fig. 4 and Table 2. The results indicated that the HeLa cells treated with 10 mol/L nanoSe0 were arrested in the S phase. The percentage of DNA diploid in S phase was a cell proliferation index. Thus, nanoSe0 (10 mol/L) could significantly inhibit the proliferation and growth of HeLa cells. Compared with control group, Vc had no inhibitory effects on the growth and proliferation of HeLa cells. Taken together, our results suggested that nanoSe0 inhibited HeLa cell growth through induction of S phase arrest.
Fig. 4. Cell cycle distribution of HeLa cells treated with nanoSe0 for 48 h. (a) Control group; (b) cells treated with 41.4 mol/L Vc; (c) cells treated with 10 mol/L nanoSe0 .
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Table 1 Rough degrees of the HeLa cells membranes without or with 41.4 mol/L Vc and 10 mol/L nanoSe0 treatments. Treatments
Control Vc nanoSe0
10 m
2 m
Rms rough (nN)
Ave rough (nN)
Rms rough (nN)
Ave rough (nN)
92.0 144.1 266.1
74.8 109.7 227.8
28.7 34.7 66.9
22.5 29.1 53.4
Table 2 Effect of nanoSe0 on the HeLa cells in cell cycle pattern. Dip of HeLa cells (%)
Control group Treated with 41.4 mol/L Vc Treated with 10 mol/L nanoSe0
Cell cycle patterns Sub-G0
G0/G1
G2/M
S
0 0 1.41
53.38 54.56 33.16
12.12 11.59 12.40
34.50 33.54 54.44
All experiments were carried out in triplicate and the data represent the mean values (±S.D.).
4. Conclusions The results of optical microscopic inspection and MTT assay showed that the nanoSe0 (10–40 mol/L) could reduced the viability of HeLa cells and MDA-MB-231 cells in a dose-dependent manner. AFM analysis showed that the HeLa cells treated with nanoSe0 displayed rough and shrunken cell morphology with truncated lamellipodia at terminal part of the cell. FCM analysis showed that the HeLa cells treated with low doses of nanoSe0 were arrested in the S phase. These investigations demonstrated that nanoSe0 exhibited the intense inhibitory effects on the proliferation and growth of the cancer cells and may be a candidate for further evaluation as a chemopreventive and chemotherapeutic agent for human cancers. Acknowledgements This work was supported by Natural Science Foundation of China (21075053, 20901030), Planned Item of Science and Technology of Guangdong Province (2008A030201020) and 211 project grant of Jinan University. References [1] M. Suzuki, M. Endo, F. Shinohara, S. Echigo, H. Rikiishi, Differential apoptotic response of human cancer cells to organoselenium compounds, Cancer Chemother. Pharmacol. 66 (2010) 475–484. [2] J.S. Hamilton, Review of selenium toxicity in the aquatic food chain, Sci. Total Environ. 326 (2004) 1–31. [3] M.P. Rayman, The importance of selenium to human health, The Lancet 356 (2000) 233–241. [4] R. Sinha, K. Ei-Bayoumy, Apoptosis is a critical cellular event in cancer chemoprevention and chemotherapy by selenium compounds, Curr. Cancer Drug Targets 4 (2004) 13–28. [5] K.M. Carvalho, M.T. Gallardo-Williams, R.F. Benson, D. Mrtin, Chemical forms of selenium for cancer prevention, J. Agric. Food Chem. 51 (2003) 704–709.
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Contents lists available at SciVerse ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Selenium nanoparticles inhibit the growth of HeLa and MDA-MB-231 cells through induction of S phase arrest Haiying Luo a , Feifei Wang b,c , Yan Bai a,∗ , Tianfeng Chen a , Wenjie Zheng a,∗ a b c
College of Life Science and Technology, Jinan University, Guangzhou 510632, PR China State Key Laboratory of Biocontrol, Department of Biochemistry, College of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, PR China National Institutes for Food and Drug Control, Beijing 100050, PR China
a r t i c l e
i n f o
Article history: Received 28 October 2011 Received in revised form 4 February 2012 Accepted 6 February 2012 Available online 14 February 2012 Keywords: Selenium nanoparticles HeLa cells MDA-MB-231 cells Atomic force microscope Flow cytometric analysis
a b s t r a c t In vitro antiproliferative effects of selenium nanoparticles (nanoSe0 , 10–40 mol/L) on HeLa (human cervical carcinoma) cells and MDA-MB-231 (human breast carcinoma) cells were examined by optical microscopic inspection and MTT assay in the present study. The nanoSe0 effectively inhibited the growth of MDA-MB-231cells and HeLa cells in a dose-dependent manner. The morphology analysis with atomic force microscope showed that the HeLa cells treated with 10 mol/L nanoSe0 were rough and shrunken with truncated lamellipodia at terminal part of the cells. Flow cytometric analysis demonstrated that HeLa cells were arrested at S phase of the cell cycle after exposed to nanoSe0 (10 mol/L). Taken together, our results suggested that nanoSe0 may be more helpful in cancer chemoprevention as a potential anticancer drug. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Cancer chemoprevention is defined as the use of natural, synthetic, or biologic chemical agents to prevent the process of carcinogenesis before the development of malignancy [1]. Chemoprevention is a cost-effective approach to reduce cancer morbidity and mortality through inhibition of precancerous events before the clinical occurrence of the disease. Selenium is an essential micronutrient for human health and shows three levels of biological activity [2]. Selenium is an essential element in trace concentrations but a toxic element in elevated concentrations for humans, and selenium can be stored within organism as supplementary selenium source for maintaining the homeostatic functions in moderate concentrations. Selenium plays an important role in nourishment and medicine [3,4]. For example, selenium compounds exhibit potent chemopreventive activities against cancer [5–9] and selenium supplementation can reduce the cancer mortality and incidence, especially for several major cancers, including prostate, lung, colon, and liver cancers [10,11]. The role of selenium compounds as chemopreventive and chemotherapeutic agents has been supported by a large number of epidemiological, preclinical, and clinical studies [12,13]. Accumulated evidence has suggested that the dose and the chemical form (structure) are important
∗ Corresponding authors. Tel.: +86 2085220223. E-mail addresses: [email protected] (Y. Bai), [email protected] (W. Zheng). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2012.02.006
determinants of chemopreventive activities of selenium compounds [1,6,13]. Element selenium nanoparticles (nanoSe0 ) are attracting increasing attention due to their excellent biological activities and low toxicity [14–19]. Tan et al. indicated that adriamycin (ADM) and nanoSe0 were both able to inhibit Bel7402 cells proliferation in a dose-dependent manner and the combined treatment with ADM and nanoSe0 was more effective in inhibiting cancer cell growth than each of the two drugs alone [9]. Zhou et al. synthesized a novel Se-Fe3 O4 nanocomposites material to examine the magnetically enhanced cytotoxicity of nanoSe0 on human osteoblast-like MG-63 cells [20]. Our previous works reported that some cancer cells, such as A375 human melanoma cells, HepG2 hepatocellular carcinoma cells, MCF-7 breast adenocarcinoma cells, HeLa (human cervical carcinoma) cells, and human kidney HK-2 cells, treated with the nanoSe0 modified with sialic acid or Undaria pinnatifida polysaccharide resulted in dose-dependent cell apoptosis as indicated by DNA fragmentation and phosphatidylserine translocation [21,22]. Several mechanisms have been postulated to elucidate the anticancer activity of selenium, which include induction of cell apoptosis, inhibition of cell proliferation, modulation of redox state, detoxification of carcinogen, stimulation of the immune system, and inhibition of angiogenesis [23–25]. Among these potential mechanisms of anticancer activities of selenium, apoptosis receives most attention and has been postulated to be critical for cancer chemoprevention by selenocompounds [26]. However, so far very little information about the nanoSe0 could be obtained.
H. Luo et al. / Colloids and Surfaces B: Biointerfaces 94 (2012) 304–308
In recent years, atomic force microscopy (AFM) is becoming one of the most important visualization technologies in cell biology. AFM can detect cellular topography, ultrastructure and change in cellular morphology after biochemistry reaction. Thus, AFM analysis could provide valuable information on cytoskeleton and morphology in the cell ultrastructure following treatment with drugs [27–29]. In this study, the effects of nanoSe0 on cell proliferation and growth in HeLa (human cervical carcinoma) cells and MDA-MB231 (human breast carcinoma) cells were examined using MTT colorimetry and flow cytometric analysis (FCM). Simultaneous experiments using optical microscopy and AFM were also performed for comparison purposes. 2. Materials and methods 2.1. Reagents and materials 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), parenzyme, acridine orange (AO), propidium iodide (PI), dimethyl sulfoxide (DMSO), penicillin and streptomycin were purchased from Sigma–Aldrich. Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum were purchased from Hyclone Laboratories Inc. Phosphate-buffer solution (PBS, pH 7.4) consisting of 138 mmol/L NaCl, 2.67 mmol/L KCl, 8.1 mmol/L Na2 HPO4 ·12H2 O and 1.47 mmol/L KH2 PO4 was used. Other chemicals were of analytical reagent grade. All solutions were prepared using ultrapure water. The resistivity of ultrapure water is 18.25 M cm. 2.2. Cell culture The HeLa cells and MDA-MB-231 cells were obtained from the cell lab of Sun Yat-sen University. Both cell lines were cultured in DMEM medium supplemented with fetal bovine serum (10%), penicillin (100 units/mL), and streptomycin (50 units/mL) at 37 ◦ C in CO2 incubator (95% relative humidity, 5% CO2 ). 2.3. Preparation of nanoSe0 The nanoSe0 sol (1 mmol/L) was prepared by mixing 5 mL SeO2 (2 mmol/L) and 5 mL vitamin C (Vc, 4 mmol/L) (1:2 in the molar ratio of SeO2 to Vc) and subsequently diluted to the required concentrations. The nanoSe0 was spherical with an average diameter of 133 nm, which was measured by using zetasizer nano (Nano ZS, Malvern, UK) and transmission electron microscopy technique (TEM, TECNAI-10, Philips, Germany). It was preferable to use nanoSe0 immediately after preparing. The amount of residual Vc in the nanoSe0 was detected by iodometric method combined with the automatic potentiometric titrator (TIM840, Radiometer Analytical, France). The concentration of the residual Vc in the 40 mol/L nanoSe0 was 7.6 mol/L. In the present study, the effect of 41.4 mol/L Vc as negative control group on the cell experiments was examined.
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Infinite M200, Austria). Observation of cell morphological change was performed with an OLYMPUS Upright fluorescence microscope (Imager Z1, Carl Zeiss). 2.5. Flow cytometric analysis Well-cultivated HeLa cells were seeded in 60 mm tissue culture dishes and incubated over night at 37 ◦ C in a humidified atmosphere of 5% CO2 . The culture medium was aspirated and replaced with fresh medium containing the nanoSe0 diluted at final 10 mol/L. PBS served as blank control and 41.4 mol/L Vc served as negative control. After incubation for 24–48 h, the cell layer was trypsinized and washed once with cold PBS and incubated with cold 70% ethanol at 4 ◦ C for about 24 h. Cells were washed with cold PBS and resuspended in 250 L cold PBS. The samples were incubated with RNase and PI at 37 ◦ C for 30 min and analyzed by a FACSCalibur flow cytometer (FCM, Becton Dickinson & Co., Franklin Lakes, NJ). The samples were collected in FL2 channel (excitation at 488 nm and emission at 585 ± 21 nm) and the number of cells analyzed for each sample was 10,000. 2.6. Atomic force microscopy About 1 × 105 /mL of HeLa cells were seeded on a glass cover slip cultivated in fresh medium. Then the cover slips with cells were treated with nanoSe0 for 24 h. The HeLa cells in incubation solution (10 mL) were dropped onto cleaned coverslips and climbed for about 3–5 min. After cells were well adhered to coverslips, the cell layer was fixed with 2.5% glutaraldehyde for 15 min, then washed with pure water for three times. All the fixed samples were air dried. The prepared samples were imaged at room temperature using an atomic force microscope (AFM, Autoprobe CP Research, Veeco) in the tapping mode. The Si3 N4 tip was employed for cell scanning in this study. The tip cantilevers length was 100 m, the resonance frequency was around 100 kHz (Veeco, Santa Barbara, USA) and the force constant was 20–80 N/m. The scanning range of AFM scanner was 0.5 Hz, and all of the acquired images (512 × 512 pixels) were flattened with the provided software (Proscan Image Processing Software Version 2.1) to eliminate low-frequency background noise in scanning direction (flatten order: 0–1). AFM-based force spectroscopy was used for force detection. Force–distance curves were obtained by standard retraction. All force–distance curve experiments were performed at the same loading rate. 2.7. Statistical analysis Experiments were carried out at least in triplicate and results were expressed as mean ± S.D. Statistical analysis was performed by one-way analysis of variance (ANOVA). Difference with *P < 0.05 or **P < 0.01 was considered statistically significant. 3. Results and discussion
2.4. MTT assay 3.1. Cell viability by MTT assay Cell viability was determined by measuring the ability of cells to converse MTT to a purple formazan dye. For our purpose, cells were seeded in 96-well tissue culture plates at 2.5 × 103 cells/well for 24 h, and then cells were incubated with medium containing different concentrations of nanoSe0 for 24 h. After incubation, 20 L/well of MTT solution (5 mg/mL PBS) was added and incubated for 4 h. The medium was aspirated and replaced with 150 L/well DMSO to dissolve the formazan salt formed. The color intensity of the formazan solution, which reflected the cell growth condition, was measured at 570 nm using a multimode microplate reader (Tecan
Cell viability was quantitatively evaluated by MTT assay. As shown in Fig. 1, nanoSe0 (10–40 mol/L) reduced the viability of MDA-MB-231 cells and HeLa cells. The viability of MDA-MB-231 cells was reduced to around 60% (Fig. 1a) and the viability of HeLa cells was dose-dependently reduced from around 73% to around 50% (Fig. 1b). In comparison, 20 mol/L SeO2 exhibited the intense cytotoxicity against HeLa cells and MDA-MB-231 cells. However, our previous works indicated that compare with SeO2 the nanoSe0 showed lower cytotoxicity toward normal cells [21]. Considering
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100
Cell viabitity (%)
80 60
a b **
**
** **
**
** **
**
**
**
40 20
20
(P µm os o iti l/L ve Se Bl con O2 (N ank trol ) eg ati ve co 10 nt µm ro l) ol /L na 20 no Se 0 µm ol /L na no 30 Se 0 µm ol /L na no 40 Se 0 µm ol /L na no Se 0
0
Fig. 1. Growth inhibition on MDA-MB-231 cells (a) and HeLa cells (b) after 24 h incubation by nanoSe0 and SeO2 . The data represent the mean values (±S.D.).
selectivity between cancer and normal cells, nanoSe0 may be superior to SeO2 in cancer chemoprevention. 3.2. Optical microscopic inspection The optical microscopic images of cells were displayed in Fig. 2. Obviously, the HeLa cells and MDA-MB-231 cells appeared
fan-shaped and square with smooth surface (Fig. 2a). A large number of cells became rounded and shrunken and the adherence cell number decreased. And the density of cells was evidently decreased with the treatment of 10–40 mol/L nanoSe0 (Fig. 2b–e), which was similar to that treated with 20 mol/L SeO2 served as a positive control (Fig. 2f). Furthermore, when the cancer cells were treated with high concentration of nanoSe0 (40 mol/L), most of the cells were gathered together and suspended in the culture medium. These results indicated that nanoSe0 could induce the change of cell morphology and inhibit the cancer cells growth in a dose-dependent manner. 3.3. Ultrastructure and quantitative roughness analysis of HeLa cells by AFM Visualization and quantitative roughness analysis of HeLa cells were investigated by AFM. The morphological features of HeLa cells and HeLa cells treated with Vc and nanoSe0 were shown in Fig. 3. The HeLa cells treated with or without 41.4 mol/L Vc were fan-shaped with complete membrane, regular shape and relatively smooth surface (Fig. 3a1–a3 and b1–b3), and had dense filopodia and lamellipodia at terminal part (Fig. 3a1 and b1). These ultrastructural parts may signify invasive property of the proliferating cancer cells. As shown in Fig. 3c, the surface morphology and ultrastructure of HeLa cells under the effect of nanoSe0 had changed significantly. The cells treated with 10 mol/L nanoSe0 displayed rough and shrunken cell morphology with truncated lamellipodia at terminal part of the cell, which may be an early injury response of HeLa cells to nanoSe0 . And also the surface roughness of the HeLa cells membranes increased after treated with 10 mol/L nanoSe0 (Table 1). These results indicated that nanoSe0 had intense
Fig. 2. Inverted microscopic images of HeLa cells and MDA-MB-231 cells incubated separately with only the culture medium as the control (a), 10 mol/L nanoSe0 (b), 20 mol/L nanoSe0 (c), 30 mol/L nanoSe0 (d), 40 mol/L nanoSe0 (e), 20 mol/L SeO2 (f) for 24 h. The magnification was 100×.
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Fig. 3. AFM images of HeLa cells induced for 24 h. (a) Control group; (b) cells treated with 41.4 mol/L Vc; (c) cells treated with 10 mol/L nanoSe0 . (a1) Error signal morphology (70 m × 70 m); (a2) topography; (a3) 3D image; (a4) error signal morphology (2 m × 2 m); (a5) ultrastructure topography of a4. (b1) Error signal morphology (80 m × 80 m); (b2) topography; (b3) 3D image; (b4) error signal morphology (2 m × 2 m); (b5) ultrastructure topography of b4. (c1) Error signal morphology (60 m × 60 m); (c2) topography; (c3) 3D image; (c4) error signal morphology (2 m × 2 m); (c5) ultrastructure topography of c4.
cytotoxic effect on HeLa cells. The AFM images and roughness analysis substantiated the cytotoxic effects of nanoSe0 on HeLa cells obtained by using optical microscopic inspection and MTT assay. 3.4. Effects of nanoSe0 on cell cycle distribution In agreement with the present findings, inhibition of proliferation in cancer cells treated with drugs could be the result of induction of apoptosis or cell cycle arrest or a combination of these two modes. When cancer cells are arrested in the S phase, the mitosis and proliferation of cancer cells are inhibited.
The content of DNA is very sensitive to reflect the cell metabolism and cell cycle progression. The proportion of cell cycle and content of DNA diploid were shown in Fig. 4 and Table 2. The results indicated that the HeLa cells treated with 10 mol/L nanoSe0 were arrested in the S phase. The percentage of DNA diploid in S phase was a cell proliferation index. Thus, nanoSe0 (10 mol/L) could significantly inhibit the proliferation and growth of HeLa cells. Compared with control group, Vc had no inhibitory effects on the growth and proliferation of HeLa cells. Taken together, our results suggested that nanoSe0 inhibited HeLa cell growth through induction of S phase arrest.
Fig. 4. Cell cycle distribution of HeLa cells treated with nanoSe0 for 48 h. (a) Control group; (b) cells treated with 41.4 mol/L Vc; (c) cells treated with 10 mol/L nanoSe0 .
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Table 1 Rough degrees of the HeLa cells membranes without or with 41.4 mol/L Vc and 10 mol/L nanoSe0 treatments. Treatments
Control Vc nanoSe0
10 m
2 m
Rms rough (nN)
Ave rough (nN)
Rms rough (nN)
Ave rough (nN)
92.0 144.1 266.1
74.8 109.7 227.8
28.7 34.7 66.9
22.5 29.1 53.4
Table 2 Effect of nanoSe0 on the HeLa cells in cell cycle pattern. Dip of HeLa cells (%)
Control group Treated with 41.4 mol/L Vc Treated with 10 mol/L nanoSe0
Cell cycle patterns Sub-G0
G0/G1
G2/M
S
0 0 1.41
53.38 54.56 33.16
12.12 11.59 12.40
34.50 33.54 54.44
All experiments were carried out in triplicate and the data represent the mean values (±S.D.).
4. Conclusions The results of optical microscopic inspection and MTT assay showed that the nanoSe0 (10–40 mol/L) could reduced the viability of HeLa cells and MDA-MB-231 cells in a dose-dependent manner. AFM analysis showed that the HeLa cells treated with nanoSe0 displayed rough and shrunken cell morphology with truncated lamellipodia at terminal part of the cell. FCM analysis showed that the HeLa cells treated with low doses of nanoSe0 were arrested in the S phase. These investigations demonstrated that nanoSe0 exhibited the intense inhibitory effects on the proliferation and growth of the cancer cells and may be a candidate for further evaluation as a chemopreventive and chemotherapeutic agent for human cancers. Acknowledgements This work was supported by Natural Science Foundation of China (21075053, 20901030), Planned Item of Science and Technology of Guangdong Province (2008A030201020) and 211 project grant of Jinan University. References [1] M. Suzuki, M. Endo, F. Shinohara, S. Echigo, H. Rikiishi, Differential apoptotic response of human cancer cells to organoselenium compounds, Cancer Chemother. Pharmacol. 66 (2010) 475–484. [2] J.S. Hamilton, Review of selenium toxicity in the aquatic food chain, Sci. Total Environ. 326 (2004) 1–31. [3] M.P. Rayman, The importance of selenium to human health, The Lancet 356 (2000) 233–241. [4] R. Sinha, K. Ei-Bayoumy, Apoptosis is a critical cellular event in cancer chemoprevention and chemotherapy by selenium compounds, Curr. Cancer Drug Targets 4 (2004) 13–28. [5] K.M. Carvalho, M.T. Gallardo-Williams, R.F. Benson, D. Mrtin, Chemical forms of selenium for cancer prevention, J. Agric. Food Chem. 51 (2003) 704–709.
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