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Synergistic combination of PEGylated selenium nanoparticles and X-ray-induced radiotherapy for enhanced anticancer effect in human lung carcinoma
Biomedicine & Pharmacotherapy 107 (2018) 1135–1141
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Synergistic combination of PEGylated selenium nanoparticles and X-rayinduced radiotherapy for enhanced anticancer effect in human lung carcinoma Hejia Zhanga, Qingjia Sunb, Lingling Tongc, Yanru Haod, Tianyu Yue,
T
⁎
a
Ultrasonic Department, China-Japan Union Hospital of Jilin University, Changchun, Jilin, 130033, China Otorhinolaryngological Department, China-Japan Union Hospital of Jilin University, Changchun, Jilin, 130033, China c Department of Gynaecology and Obstetrics, China-Japan Union Hospital of Jilin University, Changchun, Jilin, 130033, China d Otorhinolaryngological Department, No. 2 Hospital of Jilin University, Changchun, Jilin, 130000, China e Department of Thyroid Surgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin, 130033, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lung cancer Selenium X-ray Apoptosis Nanoparticles Combination effect
In this study, PEGylated selenium nanoparticles (PSNP) was successfully prepared and combined with X-ray for effective anticancer efficacy in lung cancer cells. The particles were nanosized and observed in spherical shape. The combination of PSNP and X-ray effectively killed the cancer cells and decreased the cell viability in a concentration dependent manner. PSNP combined with X-ray showed a significantly higher apoptosis of cancer cells with around 23% of cells in late apoptosis stage. Consistently, Caspase-3 activity was significantly higher when exposed to X-ray than in the absence of X-ray. The caspase-3 activity has been doubled in the presence of X-ray and PSNPs were actively involved in the activation of effector caspase-3 and downstream target. Importantly, treatment with the combination of PSNP and X-ray showed predominant red fluorescence which is indicative of dead cells. The results clearly indicate the cytotoxic potential of PSNP + X-ray combination against lung cancer cells. Overall, novel strategy of combination of PSNP and X-ray could be an alternative and effective chemo-radiotherapy.
1. Introduction Non-small-cell lung cancer (NSCLC) is one of the leading cause of cancer-related death in US and other parts of world [1,2]. The seriousness of this cancer could be reflected by its low 5-year survival rate (15%). The poor prognosis of lung cancer was mainly attributed to the high metastasis, migration and cancer cell invasion [3,4]. Furthermore, heterogeneous nature of cancer and lack of successful therapy contributed to the high mortality rate. Compared to chemotherapy or surgery, radiotherapy is minimally invasive and has attracted the attention of researchers [5]. X-ray is a good way to induce the radiotherapy that can target even the deeper tumor sites. Radiotherapy is one of the important modalities for cancer treatment and according to an estimate 70% of patients with cancer are undergo radiation therapy [6,7]. Radiotherapy induces cancer cell death via direct and indirect mechanisms. In direct mechanism, gamma radiation from radiotherapy directly induces the DNA damage via excitation of target atoms. In the case of indirect mechanisms, gamma radiation produces free radicals via interacting with the nearby ⁎
molecules of DNA [8,9]. The interaction of gamma radiation with radiosensitizing materials further induces higher photoelectric effect and kills higher amount of cancer cells. In both the cases, radiotherapy induces a series of cellular event and damage the DNA and triggers the cell death [10,11]. Recently, several authors have demonstrated the potential of rare earth metals for application in radiotherapy [12]. For example, gold nanoparticles have been employed as an X-ray absorbing agent and exhibited excellent tumor killing efficacy in the presence of X-ray [13]. Therefore, nanomaterials that can induce the potential radiation and at the same time safe to healthy cell has generated lot curiosity among scientists. Selenium (Se) is one such compound which is reported to induce a significant chemotherapeutic effect in cancer cells [14]. The Se exhibits low toxicity to healthy cells while it induces significant toxicity to cancer cells. The Se exhibits the anticancer effect by inducing ROSmediated apoptosis mechanisms in the cancer cells. Studies have reported that selenocompounds could enhance the anticancer effect when exposed to X-ray via ROS-signaling pathways [15–17]. Therefore, we hypothesized that X-ray induction of Se will induce chemo- and
Corresponding author. E-mail address: [email protected] (T. Yu).
https://doi.org/10.1016/j.biopha.2018.08.074 Received 21 October 2017; Received in revised form 12 August 2018; Accepted 15 August 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 107 (2018) 1135–1141
H. Zhang et al.
again with PBS. The cellular uptake was then observed using confocal laser scanning microscopy (CLSM, Nikon A1+, Japan).
radiotherapy. It has been reported that the X-rays are very effective in inducing radio-sensitizing effect in combination with nanoparticle system than the metallic form itself [18–20]. Therefore, we have synthesized the selenium nanoparticle (SNP) in this study. Furthermore, we have coated SeNP with polyethylene glycol (PEG). The presence of PEG is expected to serve several functions. Firstly, PEG surface coating will enhance the colloidal stability of metallic nanoparticle which is generally not stable. Secondly, presence of PEG outer layer will prolong its blood circulation time after the intravenous administration [21–23]. Thus far, we have prepared PEG-coated selenium nanoparticle (PSNP) and combined with X-ray to have effective radiotherapy against lung cancers. The particles were prepared and characterized for size and morphology. The cell viability of PSNP was evaluated in the presence and absence of X-ray induction. The anticancer effect was further characterized in terms of apoptosis assay, flow cytometer, and caspase3 activity.
2.6. Cell culture and cytotoxicity analysis A549 cells were cultured in Roswell Park Memorial Institute (RPMI1640) medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin antibiotic mixture. The cells were maintained in the incubator under standard conditions of 5% CO2 and 37 °C. The cells were grown in culture medium and incubated in an incubator. The cytotoxicity assay was determined by MTT (3-(4,5Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Briefly, 10,000 cells were seeded in each well of 96-well plate and incubated for 24 h. The cells were treated with various concentrations of PSNP in the presence and absence of X-ray and incubated for 24 h. The cells were then treated with MTT solution and incubated for 3 h and then DMSO was added to it and kept for 15 min. The absorbance was measured at 570 nm by ELISA microplate reader (DYNEX, USA). Radiotherapy was administered in vitro using a Clinac IX 6 MeV beam linear accelerator (Varian Medical Systems, Palo Alto, CA, USA)
2. Materials and methods Selenium dioxide, PEG600, and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich, China. All other organic solvents ere used without any modifications and used as such.
2.7. Cell apoptosis The cell apoptosis was determined by Annexin-V/PI staining kit. Briefly, A549 cells at a seeding density of 3 × 105 cells/well was seeded in a 6-well plate and incubated for 24 h. The old media was replaced with new media containing PSNP in the presence and absence of X-ray and incubated for 24 h. The cells were then washed and scrapped and washed again. The cells were added with 50 μl of binding buffer and stained with Annexin V and PI with 2.5 μl each to all the samples. The cells were incubated for 15 min and then reconstituted with binding buffer for 1 ml. The cells were then studied in flow cytometer for 10,000 events. In this assay, normal viable cells are Annexin V and PI negative while Annexin V positive and PI negative indicates the early apoptosis. Annexin V positive indicates the late apoptosis.
2.1. Preparation of PEG-coated selenium nanoparticles 2 ml of selenium dioxide (125 M) was taken in a glass beaker and PEG600 (10:1 w/w) was added to the above solution and kept under magnetic stirring (2000 rpm) for 5 min at 25 ± 1 °C. 2 ml of ascorbic acid (5 M) was added and mixture was further stirred for 10 min. The pH was raised to pH 8. The PEG-coated Se nanoparticles were formed and which was centrifuged at 12,000 rpm for 15 min. The PSNP particles were washed twice with distilled water and stored at 4–8 °C. The concentration of Se was determined by ICP-AES analysis. The PSNP nanoparticles were diluted to 10 ml with Milli-Q water and analyzed by ICP-AES.
2.8. Caspase-3 activity analysis 2.2. Particle size analysis Caspase-3 activity has been evaluated by fluorometric method. Briefly, cells were seeded in 12-well plate and after 24 h incubation; cells were treated with PSNP and exposed with X-ray. The cells were then incubated for 24 h. The cells were harvested and lysed using lysis buffer and incubated for 1 h in ice. Caspase activity was determined by fluorescence intensity using caspase-3 substrates (Ac-DEVD-AMC) at an excitation wavelength of 380 nm and 460 nm as a emission wavelength.
The particle size of nanoparticle was evaluated by dynamic light scattering (DLS) method. The particles were suitably diluted and determined by Zetasizer (Malvern Instruments, UK). The experiment was performed at room temperature. 2.3. Stability analysis The stability of SNP and PSNP were determined in terms of particle size using Zetasizer. Briefly, formulations were diluted with Milli-Q water and particle size were measured at predetermined time points until 120 h of storage at ambient room temperature (25 °C).
2.9. Live/dead analysis A549 cells at a seeding density of 3 × 105 cells/well was seeded in a 6-well plate and incubated for 24 h. The old media was replaced with new media containing PSNP in the presence and absence of X-ray and incubated for 24 h. The cells were stained with Calcein AM and ethidium homodimer-1 as a live and dead cell staining agent. The cells were then observed through fluorescence microscope.
2.4. Transmission electron microscope (TEM) The morphology of particle was evaluated by transmission electron microscopy (TEM) using JEM 2010, JEOL, Japan at an accelerating voltage of 120 kV. The samples were suitably diluted and stained with 2% phosphotungistic acid and placed in a carbon-coated copper grid. The images were captured using AMT imaging system.
3. Results and discussion Non-small-cell lung cancer (NSCLC) is one of the leading causes of cancer-related death in US and other parts of world. Radiotherapy is one of the important modalities for cancer treatment; and according to an estimate 70% of patients of all cancer groups undergo radiation therapy [24]. X-ray is a good way to induce the radiotherapy that can target the deeper tumor sites as well. Studies have reported that selenocompounds could enhance the anticancer effect when exposed to Xray via ROS-signaling pathways [16]. Therefore, we hypothesized that X-ray induction of Se will induce chemo- and radiotherapy. We have
2.5. Cellular uptake analysis in A549 cells A549 cells (2 × 105) were seeded onto a 6-well plate and incubated for 24 h at 37 °C to promote cell adhesion. The cells were then incubated with PSNP (loaded with coumarin-6) for different time point, followed by washed with cold PBS. The cells were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, and washed 1136
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Fig. 1. Graphical presentation: Schematic presentation of preparation of PEG-coated selenium nanoparticles and its application to X-ray.
combined the PEG-coated selenium nanoparticle (PSNP) and X-ray to have effective radiotherapy in the treatment of lung cancers (Fig. 1). 3.1. Particle size and stability analysis The particle size of SNP and PSNP were determined by dynamic light scattering analysis. The mean particle size of SNP was observed to be ∼50 nm with uniform dispersion index (PDI∼0.1) (Fig. 2a). The particle size slightly increased to ∼80 nm upon PEG surface coating. The zeta potential of final nanoparticles was observed to be −21.2 ± 1.25 mV. Nevertheless, overall particle size was less than 100 nm indicating its suitability for cancer targeting application. The stability analysis of the two nanoparticle system was performed by dynamic light scattering (DLS) analysis. As shown (Fig. 2b), particle size of bare SNP increased due to the aggregation property of metal and resulted in large particle size within 1 h of preparation. On the other hand, PSNP was very stable and did not result in any increase in size. The nanosized nature of PSNP and presence of PEG on the surface of nanoparticles will enhance the blood circulation time of particles that might increase to the therapeutic efficacy due to enhanced accumulation in the tumor. Fig. 2. Particle size and stability analysis: (a) dynamic light scattering (DLS) analysis of PSNP using ZetaSizer (Malvern Instruments, UK); (b) stability analysis of SNP and PSNP. The experimental data are presented as mean ± SD (N = 6).
3.2. Morphology of particles The morphology of particle was evaluated by transmission electron microscope (TEM). Representative images of SNP and PSNP are presented in Fig. 3. It is clearly seen that a spherical shaped nanoparticles
Fig. 3. Morphology analysis: Transmission electron microscope (TEM) image of SNP and PSNP. The particles were counterstained with phosphotungistic acid and observed under TEM microscope. 1137
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Fig. 4. Cytotoxicity analysis: (a) in vitro cell viability of A549 cancer cells upon treated with PSNP with and without X-ray irradiation; (b) in vitro cell viability of IMR-90 normal fibroblast cells after treated with PSNP. The cytotoxicity assay was performed by MTT assay protocol and presented as concentration vs cell viability. The experimental data are presented as mean ± SD (N = 6). *p < 0.05 and **P < 0.01 is the statistical difference between the groups.
Fig. 5. Cellular uptake analysis: The cellular uptake of PSNP in A549 cancer cells. The cellular uptake of nanoparticle was evaluated by fluorescence microscope in a concentration dependent manner. ***p < 0.0001 is the statistical difference between cellular uptake between 4 h and 0.5 h. Image J software was used to calculate the fluorescence intensity.
of A549 cells were ∼48%, ∼20% and ∼5% for the exposure of 20 μg/ ml, 60 μg/ml and 100 μg/ml, respectively. The synergistic effect of PSNP + X-ray is responsible for the enhanced cell killing effect. The possible mechanism for enhanced cell killing effect might be cell apoptosis and cell cycle arrest. Based on the extensive literature survey, we expected that the enhanced cell killing effect might be due to the photoelectric absorption and secondary electron caused by X-ray irradiation that could produce reactive oxygen species (ROS) [25]. It is well known that ROS is an important parameter controlling the cancer cell progression and fate during radio- and chemotherapy. Theoretically, photoelectric absorption of radiosensitive objects depends on its atomic radius. Therefore, selenium with an atomic number of Z = 34 could be a potential candidate for photoelectric effect [26,27]. We have performed cytotoxicity analysis in cancer cell and healthy cells. As shown in Fig. 4b; SeNP showed potent cytotoxicity effect in
(SNP) was formed and present in a size range of 50–60 nm and uniformly distributed in the TEM grid. The morphology of PSNP did not change after PEG assembly and spherical outfit was observed although the size was slightly increased 80–90 nm. 3.3. Anticancer effect of PSNP under the influence of X-ray The radio-sensitizing effect of selenium nanoparticles under the influence of X-ray was evaluated by MTT assay. As shown in Fig. 4a; treatment of PSNP reduced the cell viability of A549 cells in a concentration dependent manner. For example, cell viability of A549 cells were ∼75%, ∼50% and ∼30% for the exposure of 20 μg/ml, 60 μg/ml and 100 μg/ml, respectively. As expected, combination of PSNP + Xray induced a remarkable cell killing effect and showed significantly higher anticancer effect in lung cancer cells. For example, cell viability 1138
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Fig. 6. Apoptosis assay: in vitro apoptosis analysis of cancer cells after treatment with PSNP with and without X-ray irradiation. Apoptosis was determined by Annexin-V/PI double staining assay. The number cell event was fixed at 10,000 cells per sample. The experiment was performed in triplicate (n = 3). **p < 0.01 is the statistical difference between PSNP + X-ray and PSNP.
assay is based on the translocation of phosphatidylserine to the outer membrane which is an indication of cell apoptosis. As shown in Fig. 6; 99% of cells were viable in untreated group. On the contrary, treatment of A549 cells with PSNP exhibited 20% of early and late apoptosis (total) indicating the potential of Se to induce the apoptosis and cell death eventually. Consistently, combination of PSNP and X-ray showed a significantly higher apoptosis of cancer cells with around 23% of cells in late apoptosis stage and 35% of cells in necrosis phase indicating the superior anticancer efficacy of x-ray therapy. Specifically, co-treatment of cancer cells with PSNP and X-ray significantly and synergistically enhanced the cells growth inhibition through induction of cell apoptosis, as evidenced by DNA fragmentation and activation of caspase-3. Taken together, results clearly highlighted the importance of combination of X-ray with selenium particles. Fig. 7. Caspase-3 activity: Caspase-3 activity of A549 cells upon treated with PSNP with and without X-ray irradiation. The experiment was performed in triplicate (n = 3). **P < 0.01 is the statistical difference between the groups.
3.6. Caspase-3 activity Caspase-3 has been considered as a primary signaling component of the apoptosis cascade and this protein responsible for the cleavage of other caspases and proteins, resulting in a programmed cell death. The downstream activation of signaling pathways depends on the activity of caspase-3. Therefore, we have attempted to evaluate the caspase-3 activity following the PSNP exposure to the lung cancer cells. As shown in Fig. 7, PSNP showed significantly higher caspase-3 activity compared to that of control. As expected, caspase-3 activity was significantly higher when exposed to X-ray than in the absence of X-ray. It can be clearly seen that the caspase-3 activity has been doubled in the presence of Xray. Therefore, results showed that PSNP was actively involved in the activation of effector caspase-3 and downstream target that might significantly inhibit the cancer cell proliferation, revealing apoptosis as main mechanism of death. These results indicate that the structural sensitivity of PSNP to X-ray stimulation was the fatal factor that led to the generation of significant amounts of ROS that led to the apoptosis of cancer cells.
cancer cells whereas it showed relatively less toxic effect in normal healthy cells. For example, A549 cells showed ∼60% cell viability compared to ∼35% cell viability for normal healthy cells indicating the potential application in cancer cell killing. 3.4. Cellular uptake analysis The anticancer property of the nanoparticle depends on its ability to internalize the cancer cells (Fig. 5). Therefore, we have studied the cellular uptake in the A549 cancer cells. As shown, remarkable green fluorescence was observed right from 30 min of incubation. The intensity of green fluorescence kept increasing with the increase in the incubation time of the nanoparticles to the cancer cells. 3.5. Flow cytometer-based apoptosis assay Annexin V/PI double staining apoptosis assay was performed to evaluate the apoptosis potential of PSNP. The principle of Annexin V/PI 1139
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Fig. 8. Qualitative cytotoxic effect: Live/dead assay of cancer cells upon treated with PSNP with and without X-ray irradiation. The images were captured from fluorescence microscope. The cancer cells were stained with staining agents corresponding to live and dead cells. **p < 0.001 is the statistical difference between PSNP + X-ray and PSNP.
3.7. Live/dead assay of A549 cancer cells
References
Live/dead assay was performed to evaluate the cellular viability. The cells were treated with PSNP with and without the X-ray exposure (Fig. 8). The presence of calcein in the live cell is indicated by the bright green fluorescence while ethidium homodimer-1 enters the dead cells with damaged surface membrane. After 24 h treatment, untreated cells showed predominant live cells as indicated by green fluorescence. On the contrary, PSNP showed higher number of dead cells as red and green fluorescence were mixed. Importantly, PSNP after treatment with X-ray showed predominant red fluorescence which is indicative of dead cells. The results clearly indicate the cytotoxic potential of PSNP + Xray combination in lung cancer cells.
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4. Conclusion In summary, PEGylated selenium nanoparticles (PSNP) was successfully prepared and combined with X-ray for effective anticancer efficacy in lung cancer cells. The particles were nanosized and observed in spherical shape. The combination of SeNP and X-ray effectively killed the cancer cells and decreased the cell viability in a concentration dependent manner. PSNP combined with X-ray showed a significantly higher apoptosis of cancer cells with around 23% of cells in late apoptosis stage. Consistently, Caspase-3 activity was significantly higher when exposed to X-ray than in the absence of X-ray. The results clearly showed that the caspase-3 activity has been doubled in the presence of X-ray and the fact that PSNP were actively involved in the activation of effector caspase-3 and downstream target. Importantly, PSNP after treatment with X-ray showed predominant red fluorescence which is indicative of dead cells. The results clearly indicate the cytotoxic potential of PSNP + X-ray combination in lung cancer cells. Overall, novel strategy of combination of selenium and X-ray could be an alternative and effective chemo-radiotherapy.
Acknowledgement The work was supported from the Research Grant of China-Japan Union Hospital of Jilin University, Changchun, Jilin, China. 1140
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Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Synergistic combination of PEGylated selenium nanoparticles and X-rayinduced radiotherapy for enhanced anticancer effect in human lung carcinoma Hejia Zhanga, Qingjia Sunb, Lingling Tongc, Yanru Haod, Tianyu Yue,
T
⁎
a
Ultrasonic Department, China-Japan Union Hospital of Jilin University, Changchun, Jilin, 130033, China Otorhinolaryngological Department, China-Japan Union Hospital of Jilin University, Changchun, Jilin, 130033, China c Department of Gynaecology and Obstetrics, China-Japan Union Hospital of Jilin University, Changchun, Jilin, 130033, China d Otorhinolaryngological Department, No. 2 Hospital of Jilin University, Changchun, Jilin, 130000, China e Department of Thyroid Surgery, China-Japan Union Hospital of Jilin University, Changchun, Jilin, 130033, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lung cancer Selenium X-ray Apoptosis Nanoparticles Combination effect
In this study, PEGylated selenium nanoparticles (PSNP) was successfully prepared and combined with X-ray for effective anticancer efficacy in lung cancer cells. The particles were nanosized and observed in spherical shape. The combination of PSNP and X-ray effectively killed the cancer cells and decreased the cell viability in a concentration dependent manner. PSNP combined with X-ray showed a significantly higher apoptosis of cancer cells with around 23% of cells in late apoptosis stage. Consistently, Caspase-3 activity was significantly higher when exposed to X-ray than in the absence of X-ray. The caspase-3 activity has been doubled in the presence of X-ray and PSNPs were actively involved in the activation of effector caspase-3 and downstream target. Importantly, treatment with the combination of PSNP and X-ray showed predominant red fluorescence which is indicative of dead cells. The results clearly indicate the cytotoxic potential of PSNP + X-ray combination against lung cancer cells. Overall, novel strategy of combination of PSNP and X-ray could be an alternative and effective chemo-radiotherapy.
1. Introduction Non-small-cell lung cancer (NSCLC) is one of the leading cause of cancer-related death in US and other parts of world [1,2]. The seriousness of this cancer could be reflected by its low 5-year survival rate (15%). The poor prognosis of lung cancer was mainly attributed to the high metastasis, migration and cancer cell invasion [3,4]. Furthermore, heterogeneous nature of cancer and lack of successful therapy contributed to the high mortality rate. Compared to chemotherapy or surgery, radiotherapy is minimally invasive and has attracted the attention of researchers [5]. X-ray is a good way to induce the radiotherapy that can target even the deeper tumor sites. Radiotherapy is one of the important modalities for cancer treatment and according to an estimate 70% of patients with cancer are undergo radiation therapy [6,7]. Radiotherapy induces cancer cell death via direct and indirect mechanisms. In direct mechanism, gamma radiation from radiotherapy directly induces the DNA damage via excitation of target atoms. In the case of indirect mechanisms, gamma radiation produces free radicals via interacting with the nearby ⁎
molecules of DNA [8,9]. The interaction of gamma radiation with radiosensitizing materials further induces higher photoelectric effect and kills higher amount of cancer cells. In both the cases, radiotherapy induces a series of cellular event and damage the DNA and triggers the cell death [10,11]. Recently, several authors have demonstrated the potential of rare earth metals for application in radiotherapy [12]. For example, gold nanoparticles have been employed as an X-ray absorbing agent and exhibited excellent tumor killing efficacy in the presence of X-ray [13]. Therefore, nanomaterials that can induce the potential radiation and at the same time safe to healthy cell has generated lot curiosity among scientists. Selenium (Se) is one such compound which is reported to induce a significant chemotherapeutic effect in cancer cells [14]. The Se exhibits low toxicity to healthy cells while it induces significant toxicity to cancer cells. The Se exhibits the anticancer effect by inducing ROSmediated apoptosis mechanisms in the cancer cells. Studies have reported that selenocompounds could enhance the anticancer effect when exposed to X-ray via ROS-signaling pathways [15–17]. Therefore, we hypothesized that X-ray induction of Se will induce chemo- and
Corresponding author. E-mail address: [email protected] (T. Yu).
https://doi.org/10.1016/j.biopha.2018.08.074 Received 21 October 2017; Received in revised form 12 August 2018; Accepted 15 August 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
Biomedicine & Pharmacotherapy 107 (2018) 1135–1141
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again with PBS. The cellular uptake was then observed using confocal laser scanning microscopy (CLSM, Nikon A1+, Japan).
radiotherapy. It has been reported that the X-rays are very effective in inducing radio-sensitizing effect in combination with nanoparticle system than the metallic form itself [18–20]. Therefore, we have synthesized the selenium nanoparticle (SNP) in this study. Furthermore, we have coated SeNP with polyethylene glycol (PEG). The presence of PEG is expected to serve several functions. Firstly, PEG surface coating will enhance the colloidal stability of metallic nanoparticle which is generally not stable. Secondly, presence of PEG outer layer will prolong its blood circulation time after the intravenous administration [21–23]. Thus far, we have prepared PEG-coated selenium nanoparticle (PSNP) and combined with X-ray to have effective radiotherapy against lung cancers. The particles were prepared and characterized for size and morphology. The cell viability of PSNP was evaluated in the presence and absence of X-ray induction. The anticancer effect was further characterized in terms of apoptosis assay, flow cytometer, and caspase3 activity.
2.6. Cell culture and cytotoxicity analysis A549 cells were cultured in Roswell Park Memorial Institute (RPMI1640) medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin antibiotic mixture. The cells were maintained in the incubator under standard conditions of 5% CO2 and 37 °C. The cells were grown in culture medium and incubated in an incubator. The cytotoxicity assay was determined by MTT (3-(4,5Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Briefly, 10,000 cells were seeded in each well of 96-well plate and incubated for 24 h. The cells were treated with various concentrations of PSNP in the presence and absence of X-ray and incubated for 24 h. The cells were then treated with MTT solution and incubated for 3 h and then DMSO was added to it and kept for 15 min. The absorbance was measured at 570 nm by ELISA microplate reader (DYNEX, USA). Radiotherapy was administered in vitro using a Clinac IX 6 MeV beam linear accelerator (Varian Medical Systems, Palo Alto, CA, USA)
2. Materials and methods Selenium dioxide, PEG600, and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich, China. All other organic solvents ere used without any modifications and used as such.
2.7. Cell apoptosis The cell apoptosis was determined by Annexin-V/PI staining kit. Briefly, A549 cells at a seeding density of 3 × 105 cells/well was seeded in a 6-well plate and incubated for 24 h. The old media was replaced with new media containing PSNP in the presence and absence of X-ray and incubated for 24 h. The cells were then washed and scrapped and washed again. The cells were added with 50 μl of binding buffer and stained with Annexin V and PI with 2.5 μl each to all the samples. The cells were incubated for 15 min and then reconstituted with binding buffer for 1 ml. The cells were then studied in flow cytometer for 10,000 events. In this assay, normal viable cells are Annexin V and PI negative while Annexin V positive and PI negative indicates the early apoptosis. Annexin V positive indicates the late apoptosis.
2.1. Preparation of PEG-coated selenium nanoparticles 2 ml of selenium dioxide (125 M) was taken in a glass beaker and PEG600 (10:1 w/w) was added to the above solution and kept under magnetic stirring (2000 rpm) for 5 min at 25 ± 1 °C. 2 ml of ascorbic acid (5 M) was added and mixture was further stirred for 10 min. The pH was raised to pH 8. The PEG-coated Se nanoparticles were formed and which was centrifuged at 12,000 rpm for 15 min. The PSNP particles were washed twice with distilled water and stored at 4–8 °C. The concentration of Se was determined by ICP-AES analysis. The PSNP nanoparticles were diluted to 10 ml with Milli-Q water and analyzed by ICP-AES.
2.8. Caspase-3 activity analysis 2.2. Particle size analysis Caspase-3 activity has been evaluated by fluorometric method. Briefly, cells were seeded in 12-well plate and after 24 h incubation; cells were treated with PSNP and exposed with X-ray. The cells were then incubated for 24 h. The cells were harvested and lysed using lysis buffer and incubated for 1 h in ice. Caspase activity was determined by fluorescence intensity using caspase-3 substrates (Ac-DEVD-AMC) at an excitation wavelength of 380 nm and 460 nm as a emission wavelength.
The particle size of nanoparticle was evaluated by dynamic light scattering (DLS) method. The particles were suitably diluted and determined by Zetasizer (Malvern Instruments, UK). The experiment was performed at room temperature. 2.3. Stability analysis The stability of SNP and PSNP were determined in terms of particle size using Zetasizer. Briefly, formulations were diluted with Milli-Q water and particle size were measured at predetermined time points until 120 h of storage at ambient room temperature (25 °C).
2.9. Live/dead analysis A549 cells at a seeding density of 3 × 105 cells/well was seeded in a 6-well plate and incubated for 24 h. The old media was replaced with new media containing PSNP in the presence and absence of X-ray and incubated for 24 h. The cells were stained with Calcein AM and ethidium homodimer-1 as a live and dead cell staining agent. The cells were then observed through fluorescence microscope.
2.4. Transmission electron microscope (TEM) The morphology of particle was evaluated by transmission electron microscopy (TEM) using JEM 2010, JEOL, Japan at an accelerating voltage of 120 kV. The samples were suitably diluted and stained with 2% phosphotungistic acid and placed in a carbon-coated copper grid. The images were captured using AMT imaging system.
3. Results and discussion Non-small-cell lung cancer (NSCLC) is one of the leading causes of cancer-related death in US and other parts of world. Radiotherapy is one of the important modalities for cancer treatment; and according to an estimate 70% of patients of all cancer groups undergo radiation therapy [24]. X-ray is a good way to induce the radiotherapy that can target the deeper tumor sites as well. Studies have reported that selenocompounds could enhance the anticancer effect when exposed to Xray via ROS-signaling pathways [16]. Therefore, we hypothesized that X-ray induction of Se will induce chemo- and radiotherapy. We have
2.5. Cellular uptake analysis in A549 cells A549 cells (2 × 105) were seeded onto a 6-well plate and incubated for 24 h at 37 °C to promote cell adhesion. The cells were then incubated with PSNP (loaded with coumarin-6) for different time point, followed by washed with cold PBS. The cells were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, and washed 1136
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Fig. 1. Graphical presentation: Schematic presentation of preparation of PEG-coated selenium nanoparticles and its application to X-ray.
combined the PEG-coated selenium nanoparticle (PSNP) and X-ray to have effective radiotherapy in the treatment of lung cancers (Fig. 1). 3.1. Particle size and stability analysis The particle size of SNP and PSNP were determined by dynamic light scattering analysis. The mean particle size of SNP was observed to be ∼50 nm with uniform dispersion index (PDI∼0.1) (Fig. 2a). The particle size slightly increased to ∼80 nm upon PEG surface coating. The zeta potential of final nanoparticles was observed to be −21.2 ± 1.25 mV. Nevertheless, overall particle size was less than 100 nm indicating its suitability for cancer targeting application. The stability analysis of the two nanoparticle system was performed by dynamic light scattering (DLS) analysis. As shown (Fig. 2b), particle size of bare SNP increased due to the aggregation property of metal and resulted in large particle size within 1 h of preparation. On the other hand, PSNP was very stable and did not result in any increase in size. The nanosized nature of PSNP and presence of PEG on the surface of nanoparticles will enhance the blood circulation time of particles that might increase to the therapeutic efficacy due to enhanced accumulation in the tumor. Fig. 2. Particle size and stability analysis: (a) dynamic light scattering (DLS) analysis of PSNP using ZetaSizer (Malvern Instruments, UK); (b) stability analysis of SNP and PSNP. The experimental data are presented as mean ± SD (N = 6).
3.2. Morphology of particles The morphology of particle was evaluated by transmission electron microscope (TEM). Representative images of SNP and PSNP are presented in Fig. 3. It is clearly seen that a spherical shaped nanoparticles
Fig. 3. Morphology analysis: Transmission electron microscope (TEM) image of SNP and PSNP. The particles were counterstained with phosphotungistic acid and observed under TEM microscope. 1137
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Fig. 4. Cytotoxicity analysis: (a) in vitro cell viability of A549 cancer cells upon treated with PSNP with and without X-ray irradiation; (b) in vitro cell viability of IMR-90 normal fibroblast cells after treated with PSNP. The cytotoxicity assay was performed by MTT assay protocol and presented as concentration vs cell viability. The experimental data are presented as mean ± SD (N = 6). *p < 0.05 and **P < 0.01 is the statistical difference between the groups.
Fig. 5. Cellular uptake analysis: The cellular uptake of PSNP in A549 cancer cells. The cellular uptake of nanoparticle was evaluated by fluorescence microscope in a concentration dependent manner. ***p < 0.0001 is the statistical difference between cellular uptake between 4 h and 0.5 h. Image J software was used to calculate the fluorescence intensity.
of A549 cells were ∼48%, ∼20% and ∼5% for the exposure of 20 μg/ ml, 60 μg/ml and 100 μg/ml, respectively. The synergistic effect of PSNP + X-ray is responsible for the enhanced cell killing effect. The possible mechanism for enhanced cell killing effect might be cell apoptosis and cell cycle arrest. Based on the extensive literature survey, we expected that the enhanced cell killing effect might be due to the photoelectric absorption and secondary electron caused by X-ray irradiation that could produce reactive oxygen species (ROS) [25]. It is well known that ROS is an important parameter controlling the cancer cell progression and fate during radio- and chemotherapy. Theoretically, photoelectric absorption of radiosensitive objects depends on its atomic radius. Therefore, selenium with an atomic number of Z = 34 could be a potential candidate for photoelectric effect [26,27]. We have performed cytotoxicity analysis in cancer cell and healthy cells. As shown in Fig. 4b; SeNP showed potent cytotoxicity effect in
(SNP) was formed and present in a size range of 50–60 nm and uniformly distributed in the TEM grid. The morphology of PSNP did not change after PEG assembly and spherical outfit was observed although the size was slightly increased 80–90 nm. 3.3. Anticancer effect of PSNP under the influence of X-ray The radio-sensitizing effect of selenium nanoparticles under the influence of X-ray was evaluated by MTT assay. As shown in Fig. 4a; treatment of PSNP reduced the cell viability of A549 cells in a concentration dependent manner. For example, cell viability of A549 cells were ∼75%, ∼50% and ∼30% for the exposure of 20 μg/ml, 60 μg/ml and 100 μg/ml, respectively. As expected, combination of PSNP + Xray induced a remarkable cell killing effect and showed significantly higher anticancer effect in lung cancer cells. For example, cell viability 1138
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Fig. 6. Apoptosis assay: in vitro apoptosis analysis of cancer cells after treatment with PSNP with and without X-ray irradiation. Apoptosis was determined by Annexin-V/PI double staining assay. The number cell event was fixed at 10,000 cells per sample. The experiment was performed in triplicate (n = 3). **p < 0.01 is the statistical difference between PSNP + X-ray and PSNP.
assay is based on the translocation of phosphatidylserine to the outer membrane which is an indication of cell apoptosis. As shown in Fig. 6; 99% of cells were viable in untreated group. On the contrary, treatment of A549 cells with PSNP exhibited 20% of early and late apoptosis (total) indicating the potential of Se to induce the apoptosis and cell death eventually. Consistently, combination of PSNP and X-ray showed a significantly higher apoptosis of cancer cells with around 23% of cells in late apoptosis stage and 35% of cells in necrosis phase indicating the superior anticancer efficacy of x-ray therapy. Specifically, co-treatment of cancer cells with PSNP and X-ray significantly and synergistically enhanced the cells growth inhibition through induction of cell apoptosis, as evidenced by DNA fragmentation and activation of caspase-3. Taken together, results clearly highlighted the importance of combination of X-ray with selenium particles. Fig. 7. Caspase-3 activity: Caspase-3 activity of A549 cells upon treated with PSNP with and without X-ray irradiation. The experiment was performed in triplicate (n = 3). **P < 0.01 is the statistical difference between the groups.
3.6. Caspase-3 activity Caspase-3 has been considered as a primary signaling component of the apoptosis cascade and this protein responsible for the cleavage of other caspases and proteins, resulting in a programmed cell death. The downstream activation of signaling pathways depends on the activity of caspase-3. Therefore, we have attempted to evaluate the caspase-3 activity following the PSNP exposure to the lung cancer cells. As shown in Fig. 7, PSNP showed significantly higher caspase-3 activity compared to that of control. As expected, caspase-3 activity was significantly higher when exposed to X-ray than in the absence of X-ray. It can be clearly seen that the caspase-3 activity has been doubled in the presence of Xray. Therefore, results showed that PSNP was actively involved in the activation of effector caspase-3 and downstream target that might significantly inhibit the cancer cell proliferation, revealing apoptosis as main mechanism of death. These results indicate that the structural sensitivity of PSNP to X-ray stimulation was the fatal factor that led to the generation of significant amounts of ROS that led to the apoptosis of cancer cells.
cancer cells whereas it showed relatively less toxic effect in normal healthy cells. For example, A549 cells showed ∼60% cell viability compared to ∼35% cell viability for normal healthy cells indicating the potential application in cancer cell killing. 3.4. Cellular uptake analysis The anticancer property of the nanoparticle depends on its ability to internalize the cancer cells (Fig. 5). Therefore, we have studied the cellular uptake in the A549 cancer cells. As shown, remarkable green fluorescence was observed right from 30 min of incubation. The intensity of green fluorescence kept increasing with the increase in the incubation time of the nanoparticles to the cancer cells. 3.5. Flow cytometer-based apoptosis assay Annexin V/PI double staining apoptosis assay was performed to evaluate the apoptosis potential of PSNP. The principle of Annexin V/PI 1139
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Fig. 8. Qualitative cytotoxic effect: Live/dead assay of cancer cells upon treated with PSNP with and without X-ray irradiation. The images were captured from fluorescence microscope. The cancer cells were stained with staining agents corresponding to live and dead cells. **p < 0.001 is the statistical difference between PSNP + X-ray and PSNP.
3.7. Live/dead assay of A549 cancer cells
References
Live/dead assay was performed to evaluate the cellular viability. The cells were treated with PSNP with and without the X-ray exposure (Fig. 8). The presence of calcein in the live cell is indicated by the bright green fluorescence while ethidium homodimer-1 enters the dead cells with damaged surface membrane. After 24 h treatment, untreated cells showed predominant live cells as indicated by green fluorescence. On the contrary, PSNP showed higher number of dead cells as red and green fluorescence were mixed. Importantly, PSNP after treatment with X-ray showed predominant red fluorescence which is indicative of dead cells. The results clearly indicate the cytotoxic potential of PSNP + Xray combination in lung cancer cells.
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4. Conclusion In summary, PEGylated selenium nanoparticles (PSNP) was successfully prepared and combined with X-ray for effective anticancer efficacy in lung cancer cells. The particles were nanosized and observed in spherical shape. The combination of SeNP and X-ray effectively killed the cancer cells and decreased the cell viability in a concentration dependent manner. PSNP combined with X-ray showed a significantly higher apoptosis of cancer cells with around 23% of cells in late apoptosis stage. Consistently, Caspase-3 activity was significantly higher when exposed to X-ray than in the absence of X-ray. The results clearly showed that the caspase-3 activity has been doubled in the presence of X-ray and the fact that PSNP were actively involved in the activation of effector caspase-3 and downstream target. Importantly, PSNP after treatment with X-ray showed predominant red fluorescence which is indicative of dead cells. The results clearly indicate the cytotoxic potential of PSNP + X-ray combination in lung cancer cells. Overall, novel strategy of combination of selenium and X-ray could be an alternative and effective chemo-radiotherapy.
Acknowledgement The work was supported from the Research Grant of China-Japan Union Hospital of Jilin University, Changchun, Jilin, China. 1140
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