- Email: [email protected]
Green synthesized zinc oxide nanoparticles regulates the apoptotic expression in bone cancer cells MG-63 cells
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Green synthesized zinc oxide nanoparticles regulates the apoptotic expression in bone cancer cells MG-63 cells
T
Jun Chenga,1, Xiaofeng Wangb,1, Lei Qiuc, Yunkai Lid, Najat Marraikie, Abdallah M. Elgorbane, ⁎ Li Xuef, a
Department of Orthopedics, Chongqing Three Gorges Central Hospital, Chongqing Province 404000, China Department of Neurosurgery, Weinan Central Hospital, Weinan, Shaanxi Province 714000, China c Department of Oncology, Zhucheng Hospital of Traditional Chinese Medicine, Zhucheng, Shandong Province 262200, China d Emergency Surgery the No.4 Hospital Jinan, Shangdong Province 250031, China e Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia f Department of Orthopaedics, the Third People's Hospital of Chengdu, Affiliated Hospital of Southwest Jiao Tong University Medical School, Chengdu, Sichuan Province 610031, China b
ARTICLE INFO
ABSTRACT
Keywords: Bone cancer Apoptosis Anticancer Rehmanniae Radix Zinc oxide nanoparticles ROS
Management of degenerative spine pathologies frequently leads to the need for bone growth. Rehmanniae Radix (RR), a Chinese herbal formulation was found to exhibit numerous therapeutic properties including its potent effect against cancer cell lines. However, the underlying mechanism through which the Zinc oxide nanoparticles (ZnONPs) synthesized from Rehmanniae Radix exerts its anti-cancer activity against osteosarcoma cell line MG63 needs to be explored. Therefore, the study was performed to evaluate the anticancer, cytotoxicity and apoptotic effectiveness of ZnONPs from RR against MG-63 cells. Characterization studies such UV–vis spectroscopy, FTIR, TEM and XRD analysis were performed. Cytotoxicity assay, mitochondrial membrane potential (MMP), morphological examination of cells and formation of reactive oxygen species (ROS), and apoptosis inducing ability of RR were evaluated by various procedures. Western blot analysis of apoptotic markers such as Bax, caspase-3 and caspase-9 were also performed. RR was found to inhibit growth of MG-63 cells at increasing dose. AO/EB staining confirmed the apoptotic efficacy of ZnONPs induced by RR in MG-63 cells. ZnONPs was also found to initiate increased generation of ROS and decreased MMP. Decreased MMP has resulted in increased levels of apoptotic proteins Bax, caspase-3 and caspase-9 and induction of apoptosis was substantiated by western blot analysis. The outcomes of the work propose that ZnONPs from RR exhibits strong anticancer action and inducing apoptosis on MG-63 cells via stimulating increased generation of ROS. Thus, ZnONPs from RR might be used as a hopeful drug target against several types of cancer cell lines.
1. Introduction Cancer is a highly complicated disease that booms in a heterogeneous environment that is uniquely adaptive [1]. Involvement of bones is the noteworthy complication in metastatic cancer that occurs in patients of breast, multiple myeloma, prostate, primary colon, lung and kidney tumours [2,3]. Most common primary malignant bone tumour includes Osteosarcoma (OS), Ewing sarcoma (ES) and chondrosarcoma (CS) which contributes to almost 70% of malignancies [4]. Metastasis of bone results in increased morbidity, joint pains, increased calcium levels, disability of joints, compression of spinal cord and other related pathological features that leads to major influence in quality of
life in patients [2]. Despite various clinical advances that has increased survival rate in patients, sarcomas are considered deadly till now and the spread of tumour to bone skeleton indicates the cancer is incurable and the treatment options available are also associated with undesirable side effects [4,5]. The therapies which are available clinically to treat cancer has progressively becoming outdated due to development in the fields of nanomedicine and multitargeted drug-delivery that invades and destroys cancer cells [6]. Nanomedicine belongs to the field of nanotechnology which utilizes engineered nanoparticles (NPs) to treat cancer. Nanomedicine has the potent capability to detect and treat cancer at its early stage [7]. Nanomedicine is highly reliable because of
Corresponding author. E-mail address: [email protected] (L. Xue). 1 Equal contribution. ⁎
https://doi.org/10.1016/j.jphotobiol.2019.111644 Received 5 September 2019; Received in revised form 26 September 2019; Accepted 4 October 2019 Available online 30 October 2019 1011-1344/ © 2019 Elsevier B.V. All rights reserved.
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
its potent targeting, high solubility, bioavailability and multipotent properties over other traditional method of cancer therapies available [8]. Among various nanoparticles available, zinc oxide nanoparticles (ZnONPs) have received improved attention at present for its potent antifungal, antibacterial and anticancer properties [9]. Zinc is an essential nutrient for adults and administration of ZnONPs in vivo are considered to be safe. At present, ZnONPs are mainly used in treating cancer [10] and it is found to exhibit cytotoxicity in various cancer cell lines [11–14]. ZnONPs make significant involvement in the development of treatment approaches in cancer drug delivery [15]. It also proves to be highly efficient, stable, low cost, soluble and non-toxic which makes it easy to reach the targeted sites of action [16,17]. The efficient action of ZnONPs results in weakening of mitochondria which produces increased ROS, lipid peroxides and damage to DNA [18–20]. When the ZnONPs interacts with the cancer cells, it disrupts the protein equilibrium and affects various important cellular processes including DNA damage, replication, apoptosis, activity of electron transport chain and cellular homeostasis thus increasing the cytotoxicity against cancer cells [21]. In recent years, various extracts from traditional Chinese medicine (TCM) are used clinically for management of cancer. In patients with advanced stage of cancer who are not eligible for other treatment options, TCM based medicines has proven to be beneficial in improving the general symptoms of cancer, augment immunity and maintain cancer metastasis. It also has beneficial effect in increasing the survival rate of cancer patients and ensure long-term survival among certain percentage of patients [22]. Rehmanniae Radix (RR) is the most commonly used herbal medicine in China to treat multiple diseases and it belongs to the family of Scrophulariaceae. It is prepared from washed root of Rehmannia Glutinosa and it is non-toxic and little bitter in taste [23–25]. Studies reported by Seo et al. (2008) [26] showed that RR exhibited no cytotoxic effect, inhibited the production of nitric oxide and displayed high DPPH radical scavenging ability. Lee et al. (2015) [27] reported the potent anti-apoptotic effect of catalpol, an active component present in RR and established the anticancer and antioxidant activities of RR. Other beneficial properties of RR includes inhibition of ROS production, antihypertensive, anti-inflammatory, antiarthritic, skin whitening effect, reduces wrinkles, improves cognitive function, depression, motility of the intestines, hypoglycaemia, immunity, reduces renal hypertension, inflammation and inhibits oxidation and recovers damaged liver cells [28–31]. RR administered HeLa cervical cancer cells displayed increased necrosis of cells, increased caspase activity and increased Fas expression which indicated the potent beneficial property of RR in treating cervical cancer [32]. Numerous potent therapeutic properties of RR has been reported from literature evidences enlightening its beneficial action and also its potent anticancer activity in certain forms of cancers. However, the effective action of RR against bone cancer cells is less investigated. Hence, the aim of the study is to explore the therapeutic action of ZnONPs from RR against bone cancer cell line MG-63 and to explore its anticancer activity in vitro.
2.2. Characterization of ZnONPs from RR The absorption spectrum of ZnONPs obtained from RR were obtained using UV–visible absorption spectrophotometer and the ZnONPs were characterized by Fourier transform infrared spectroscopy (FT-IR), Transmission Electron Microscopy (TEM) and X-ray diffraction (XRD) pattern for the nanoparticle was recorded using a X-ray diffractometer. 2.3. Fourier Transform Infrared Spectroscopy The lyophilized samples were mixed with dry potassium bromide (KBr pellet) and subjected to a pressure of about 5 × 10 Pa in an evacuated die to produce a clear transparent disc of diameter 2 cm and thickness 0.2 cm. IR spectra in frequency region 4000–400 cm−1, were recorded at room temperature on a Perkin Elmer Fourier transform spectrometer. 2.4. TEM The morphology of the synthesized ZnONPs was determined using TEM. The particle to be dispersed was diluted and it is stained using 2% phosphotungstic acid and it is placed in a copper grid. Then the particles were dried and morphology was studied. 2.5. Reagents and Chemicals Zinc acetate, Bovine Serum Albumin (BSA) and other chemicals were acquired from Sigma Chemical, USA. DMEM medium, Antibiotics and Fetal Bovine Serum were procured from Invitrogen (Groningen, The Netherlands). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Sigma,USA. 2.6. Maintenance of Cells Osteosarcoma cell line (MG-63) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), Antibiotics (mixture of 1% penicillin/streptomycin). Cells were incubated in T25 tissue culture flasks at 37 °C in a humidified atmosphere (5% CO2 & 95% air environment). ZnONPs from RR was dissolved in media and was resuspended in PBS. ZnONPs at the concentration of 30 μg/ml and 50 μg/ml were used for the study. The antibiotics and media required for the study was purchased from Sigma-Aldrich,USA. For passaging of cells, the cells were detached using trypsin/EDTA and they were reseeded. 2.7. Cell Cytotoxicity (MTT Assay) The cytotoxicity of ZnONPs from RR on the MG-63 cell line were determined by using conventional 3-(4, 5-dimethylthiazole-2-yl)-2, 5diphenyltetrazolium bromide (MTT) assay method. Briefly, cells were seeded in a 96-well plate (2 × 104 cells/well in 100 μl of complete medium) and then incubated. After 24 h, the ZnONPs at 30 and 50 μg/ ml concentrations were added on to the layer of cells. After incubation, 50 μl of MTT (1 mg/ml) was added to each well, and the cells were incubated in the dark at 37 °C for an additional 4 h. Thereafter, the medium was removed and the formazan crystals formed were dissolved in 200 μl of dimethyl sulphoxide, and within 15 min, absorbance was measured by a microplate reader at a wavelength of 540 nm. The percentage of reaction for each treatment was calculated assuming 100% reaction for untreated control cells.
2. Materials and Methods 2.1. Synthesis of Zinc Oxide Nanoparticles from Radix Rehmanniae Zinc acetate solution (1 mM) was dissolved in water and it is stirred for about 1 h continuously. To this sodium hydroxide (20 ml) is added together with 20 ml of RR extract. After 1 h of incubation, the colour change of the solution was noticed. The same reaction mix was allowed in the stirrer for another 3 h and the solution turned yellow which indicated the existence of ZnONPs. Thus precipitate thus formed was separated and centrifuged at 60 °C for 15 min at 8000 rpm. Pellet thus collected following centrifugation can be dried in hot air oven and preserved for further studies.
%Viability =
2
Mean Absorbance of Sample × 100 Mean Absorbance of Negative control
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
2.8. Determination of Cell Morphology and Intracellular ROS The growth of the cells was monitored and the cells were detached from the flask and the floated suspension formed as clumps/aggregates were thus collected and visualized under microscope [33]. Control cells were also included for the morphological analysis. The ROS level of cells was measured using 2′7′-dichlorodihydrofluoresceindiacetate (DCFH-DA) (Applygen Co. Ltd., China) [34,35]. Cells at a concentration of 1 × 104 were seeded in six well plates and kept at overnight incubation. The following day, the medium is substituted with new medium containing 30 and 50 μg/ml of ZnONPs and kept at 37 °C for 24 h. The control was added with distilled water alone. After the growth period, the cells were trypsinized and detached from plates and stained for 30 min with DCFH-DA. Dye present in excess was detached by washing with 1 × PBS. Images of the cells were captured with fluorescence at 530 nm. 2.9. Determination of Mitochondrial Membrane Potential by Rhodamine123 (Rh-123) Staining Fig. 1. UV–Vis absorption spectra of ZnONPs.
MG-63 cells were seeded in six-well plate, and ZnONPs at different concentrations (30 and 50 μg/ml) were introduced and control cells contained distilled water and incubated for 24 h. The cells were then washed with PBS and fixed in methanol at room temperature for 10 min. The cells which are fixed were allowed to permeabilize with triton X-100 (0.2%) for 10 min in PBS solution and incubated for 30 min at room temperature with 5 μg/ml of Rh-123. The cells stained with Rh123(ThermoFisher Scientific, USA) were viewed under fluorescent microscope.
was performed. β-actin was loaded as positive control. 2.13. Statistical Analysis Values are expressed as Mean ± Standard deviation (SD). Using one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT) the statistical comparisons were performed. The results were considered statistically significant if the p values were < 0.05.
2.10. Acridine Orange (AO)/Ethidium Bromide (EB) Staining DMEM medium was added to each well of 96-well plate and cells were added to it at a density of 2 × 104/ml and incubated. Control cells were left untreated and other cells were incubated with ZnONPs at a concentration of 30 and 50 μg/ml. After culturing, trypsin was added into all the wells and cells are detached. Suspensions containing 25 μl were moved to glass slides. Double staining solution of 8 μl comprising of AO (100 μg/ml) and EB (100 μg/ml) were added to the suspensions and mixed gently for 20 mins. The mixture was placed in slide and covered using coverslip. The morphology of the cells undergone apoptosis was studied and the cells were counted using fluorescent microscope.
3. Results 3.1. UV–Visible Analysis Fig. 1 shows the UV–vis spectrum of ZnONPs prepared RR at room temperature. The characteristic absorption peak of ZnONPs was observed at 330 nm. The nanoparticles which are synthesized in the solution were determined by UV–vis spectrophotometer and it is the simplest technique available to identify the nanoparticles which are formed in the solution. The ZnONPs absorption spectrum ranges from 200 to 600 nm which clearly relates with the results of the present study.
2.11. Cell Adhesion Assay MG-63 osteosarcoma cells were seeded in a 24 well plate which is precoated with fibronectin (5 μg/ml). Then the cells were incubated at 37 °C for 2 h in cell specific media to get adhered and then washed with PBS 3 times to remove the cells which are not adhered. Then the remaining cells are fixed in formaldehyde for 15 min and washed and then stained for 1 h using Toluidene Blue. After incubation, the cells were viewed under the microscope to understand the adhesion capacity of MG-63 cells treated with or without ZnONPs.
3.2. Transmission Electron Microscopy Fig. 2 (a & b) show the representative images of ZnO nanoparticles. Rod shape ZnO nanoparticles were detected in TEM images of average size in the range of 10.0–12.0 nm which correlates with the size calculated by XRD. It shows that the particles are well crystallized. The diffraction rings on image matches with the peaks in XRD pattern which also proves the hexagonal wurtzite structure of ZnO nanoparticles.
2.12. Western Blot Analysis of Apoptotic Markers
3.3. XRD Analysis
The samples containing 30 micrograms of proteins were electrophoresed using 10% sodium dodecyl sulphate (SDS) polyacrylamide gel for analysis of apoptotic markers Bax, Caspase 3 and caspase 9. After gel electrophoresis, the gel was wet transferred on to membrane made of nitrocellulose. The membranes were then blocked with skimmed mik powder (5%) at room temperature for 1 h in TBST solution. After blocking the membranes were incubated overnight using primary antibody followed by addition of secondary antibody (Santa Cruz, US). The blots were then visualized by Chemidoc imaging and densitometry
The peaks are indexed as 31.84°, 34.52°, 36.38°, 47.64°, 56.7°, 62.92° (103), 63.06°, and 68.1° respectively. All diffraction peaks of sample correspond to the characteristic hexagonal wurtzite structure of zinc oxide nanoparticles (a = 0.315 nm and c = 0.529 nm). Average particle size of ZnO nanoparticles is found to be 10.0 nm using Scherrer equation. Diffraction pattern corresponding to impurities are found to be absent (Fig. 2c). Thus, this confirms the presence of ZnO nanoparticles synthesized which are synthesized of high purity. 3
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
Fig. 2. Transmission Electron Microscopic (TEM) images and X Ray Diffraction pattern of synthesized ZnONPs.
present in ZnONPs. The peaks at 1634 cm−1 and 1192 cm−1 represent the presence of CeN stretch of amino group respectively.
3.4. FTIR The synthesized ZnONPs from RR was lyophilized and subjected to FTIR spectral analysis in order to find out inter and intra molecular interactions present in the nanoparticle and to confirm the presence of RR in the nanoparticle. FTIR spectra of ZnONPs, in (Fig. 3) shows a strong peak at 3321 cm−1 indicating the NH stretch of amine group
3.5. Effect of ZnONPs from RR on the MG-63 Cells Survival The ability of ZnONPS from RR on osteosarcoma cancer cell was investigated by culturing MG-63 cells under different concentrations for
Fig. 3. Fourier transform infrared spectrum analysis of ZnONPs. 4
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
both live and dead cells (Fig. 5b). Cells which are supplemented with ZnONPs at a concentration of 50 μg/ml displayed reduced number of live cells (Fig. 5c). Cell injury resulting from oxidative stress holds a vital role in cancer cell progression. ROS generation in MG-63 were watched after incubating with ZnONPs and the levels of ROS generated were studied using DCFH-DA stain and the changes are viewed using fluorescent microscope (Fig. 5d–f). Cells which were administered with ZnONPs at a concentration of 30 and 50 μg/ml were analysed using DCFH-DA dye which exhibits green fluorescence (Fig. 5e and f). The observation of the study showed that the fluorescence strength of the cells was increased with increased concentration of ZnONPs and the ROS generation was also increased thus indicating that ZnONPs have brought apoptosis in MG-63 cells facilitated via ROS generation. 3.7. Effect of ZnONPs in Mitochondrial Membrane Potential
Fig. 4. Determination of cytotoxicity of ZnONPs synthesized from Radix Rehmanniae by MTT assay. 96-well plates were seeded with MG-63 cells at a density of 1 × 104 cells/well and treated with varying concentrations of ZnONPs synthesized from RR for 24 h. The results are denoted as means ± standard error (SE) of the mean and the error bar size reveals the sample variation of triplicates.
The alterations in membrane potential of mitochondria in MG-63 cells was assessed by means of Rh-123 stain. Control (untreated) cells showed increased green fluorescence which indicates higher mitochondrial membrane potential (Fig. 6a–c). Cells treated with ZnONPs at 30 μg/ml also showed higher mitochondrial membrane than those supplemented with 50 μg/ml (Fig. 6b and c). Cells subjected to 50 μg/ ml of ZnONPs showed low green fluorescence indicative of decrease in mitochondrial membrane potential with increased ROS generation (Fig. 6c). This clearly shows that ZnoNPs synthesized from RR indicates that cell death in MG-63 cells occurs through mitochondrial-mediated pathway.
24 h (5, 10, 20, 30, 40, 50, 60, 70 and 80 μg/ml) and the survival of the cells was determined by MTT assay (Fig. 4). MG-63 cells treated with ZnONPs from RR was found to inhibit cancer cell growth at increasing concentration. The inhibitory concentration (IC50 value) was calculated as xxx μg/ml. The results of the study indicated that the survival of cancer cell was decreased with increasing ZnONPs concentration. The findings of the study displayed that ZnONPs was more effective in minimizing the cancer cell growth and survival rate of MG-63 cells.
3.8. Apoptosis Induction in MG-63 Cells by ZnoNPs from RR Stained with AO/EB
3.6. Effect of ZnONPs from RR on Cell Morphology and ROS Generation
The cells which underwent apoptosis can be confirmed through AO/ EB stain. ZnoNPs treated cells at different concentrations of 30 and 50 μg/ml for 24 h, displayed typical changes in morphology distinctive of apoptosis such as fragmentation of nuclei or nuclear shrinkage, condensed chromatin fragments and shrinked cytoplasm. The results of the study are indicated in Fig. 7a–c. Control cells exhibited normal
Structural changes of MG-63 cells were detected by microscopy (Fig. 5a–c). The study showed that ZnONPS was effective in reducing the viability of cancer cells at various concentration (30 and 50 μg/ml). MG-63 cells treated with ZnONPs at 30 μg/ml showed occurrence of
Fig. 5. Morphological examination and generation of ROS in MG-63 cells treated with or without ZnONPs synthesized from RR. Control (a), Cells treated with ZnONPs at concentration of 30 and 50 μg/ml (b and c). MG-63 cells treated with 30 μg/ml of ZnONPs show a lesser number of viable cells (b). Cells treated with 50 μg/ml of ZnONPs display greater number of nonviable cells. (c). MG-63 cells stained with DCFH –DA showed increased intensity of fluorescence in 50 μg/ml than 30 μg/ml of ZnONPs synthesized. Fluorescence intensity was observed at least levels in control cells indicating a greater number of viable cells. 5
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
Fig. 6. Fluorescent microscopic images of mitochondrial membrane potential using Rh-123 in MG-63 cells treated with or without ZnONPs from RR. Control cells showed increased cellular uptake of Rh-123 dye which indicates the integrity of Δψm (a). MG-63 cells treated with ZnONPs at concentration of 30 μg/ml (b) and 50 μg/ml (c), showed reduced uptake of Rh-123 indicating the loss of Δψm.
4. Discussion
nuclear architecture (Fig. 7a). Cells supplied with 30 μg/ml of drug showed numerous early apoptotic cells which are observed as greenish yellow colour nuclei (Fig. 7b). Cells which are treated with ZnONPs at 50 μg/ml contained late apoptotic cells which appeared orange in colour (Fig. 7c). Treatment with ZnONPs showed marked increase in apoptosis and the number of viable cells was also decreased evidently.
The two major processes that underlie cancer cells development are formation of tumour and metastasis which results in increased proliferation of cells and reduced apoptosis rate. Thus, initiation of apoptosis in cancer cells might increase the treatment options for inhibiting cancer cell growth and metastasis [36]. Cell death by apoptosis is mediated through intrinsic or extrinsic apoptotic pathway and it is categorized by depolarization of mitochondrial membrane with loss of membrane potential [37]. Such imbalance in the cellular status results in increased production of ROS [38]. Increased intracellular ROS production results in significant increase in the rate of apoptosis in cancer cells [39]. Thus, the effective regimen to treat cancer is mainly through development of drugs which promotes ROS production and ensuing in apoptosis. Hence, with this background, the work was performed to explore the cytotoxic, anticancer and apoptotic effectiveness of ZnONPs synthesized from Chinese herbal formulation, Radix Rehmanniae in MG-63 osteosarcoma cell lines. The nanoparticle size plays a vital role in determining the property of synthesized nanomaterial. Thus, size of the particle is very essential to materialize the unique property of the nanomaterial and UV–vis spectroscopy is he most extensively used methodology to assess the optical property of synthesized nanoparticle. Thus, the sharp absorption spectrum of ZnONPs obtained at about 360 nm indicates the monodispersed property of nanoparticle present [40,41]. The ZnONPs metal‑oxygen frequencies which is observed in the present study is in accordance with earlier literature evidences [42,43]. FTIR spectra of ZnONPs corresponding to the spectra obtained in the present study correlates well with earlier investigation [44]. The findings of TEM studies shows that the ZnONPs appear in a well crystallized form and
3.9. Effect of ZnONPs from RR in Cell Adhesion Ability In order to assess the capacity of cancer cells to adhere, the cell adhesion assay was carried out. Fig. 8 represents the images of cell adhesion assay in MG-63 control and ZnONPs treated cells. Control cells depicted increased adhesion capacity of cancer cells following incubation in 24 well plate (Fig. 9a). Cancer cells which are treated with 30 μg/ml of ZnONPs displayed diminished adherence of cells than the control cells (Fig. 8b). MG-63 cells treated with 50 μg/ml of ZnONPs exhibited even more reduced adherence capacity than the cells treated with lower dose of ZnONPs (Fig. 8c). 3.10. Immunodetection of Apoptotic Markers Fig. 9 displays the influence of ZnONPs from RR on expression of apoptotic proteins in MG-63 cells. Results of the study revealed that there was a substantial increase in the protein levels of Bax, caspase-3 and caspase-9 in ZnONPs treated cells, when compared with control cells. Control cells displayed decreased rate of apoptosis. Cells administered with 50 μg/ml of ZnONPs exhibited higher rate of apoptosis than the cells supplemented with 30 μg/ml of ZnONPs.
Fig. 7. Detection of Apoptosis induced in MG-63 cells treated with or without ZnONPs from RR using AO/EB double staining. MG-63 cells treated with ZnONPs at varying concentrations 30 and 50 μg/ml (b and c). Untreated control cells showed uniform size and structure with green fluorescence (a). Cells treated with 30 μg/ml of ZnONPs exhibited early apoptotic cells and nuclear condensation which were observed in greenish yellow colour (b) & Cells treated with 50 μg/ml of ZnONPs showed greater number of late apoptotic cells and fragmented nucleus which were observed in yellowish orange colour (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 6
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
Fig. 8. Cell adhesion assay of MG-63 cells treated with or without ZnONPs synthesized from RR. MG-63 cells treated with ZnONPs at varying concentrations 30 and 50 μg/ml (b and c). Untreated control cells showed increased adherence of cells (a). Cells treated with 30 μg/ml of ZnONPs exhibited diminished cellular adhesion (b) & Cells treated with 50 μg/ml of ZnONPs showed greater reduction of cellular adhesion than the cells treated with 30 μg/ml (c).
various dyes available, Rh123 is the most commonly used dye for determining the membrane potential of mitochondria. The dye Rh123 will retain in the cells of mitochondria only when the cells contain intact membrane potential. The fluorescence will be reduced in cells which lacks membrane potential and results in outflow of stain from mitochondria and reduced fluorescence. Reduced fluorescence is indicative of diminished integrity of mitochondrial membrane [52]. Alterations in membrane potential of mitochondria indicates the initiation of apoptosis and the increased uptake of Rh-123 in control cells observed in the present study indicates intact mitochondria, whereas cells administered with 30 and 50 μg/ml of ZnONPs showed decreasing fluorescence at increasing doses indicative of decreased mitochondrial membrane potential. In the current study, the MG-63 cells supplemented with 30 and 50 μg/ml of ZnONPs from RR which undergone apoptosis was confirmed by AO/EB staining [53]. Apoptosis in MG-63 cells were determined by fluorescence microscopic findings for differentiating live and dead cells. Acridine orange has the ability to hold with all cells and it releases green fluorescence, whereas EB will interact with the cells that lacks cytoplasmic membrane and the nucleus will appear red in colour which is interpolating with DNA. Love cells exhibit green coloured nuclei and the cells which undergone early apoptosis, will emit bright green colour fluorescence. Condensed chromatin is viewed as green patches and the late apoptotic cells observed after treatment with ZnONPS are viewed as orange to red coloured cells. The areas which appear as opaque orange are indicative of depolarized mitochondria. All these morphological changes observed indicates that the cells which are treated with ZnONPs containing RR have underwent apoptosis. Apoptosis is tightly regulated by Bax (proapoptotic “gatecrashers” member) and Caspase-3 (executioner) [54]. Caspases play a critical role in apoptosis [55] in which Caspase-3 is one of the effector units found in apoptotic cells [56]. Hence, we assessed the protein levels of proapoptotic proteins to determine the efficacy of ZnONPs from RR in augmenting apoptosis in MG-63 cell line. The Bcl-2 family members mainly control the mitochondrial pathway of apoptosis. Bax and Bcl-2 are the important activators for release of cytochrome C and consequent activation of caspases [57]. Thus, in the present study, the molecular mechanism of ZnONPs in promoting apoptosis was evaluated and the protein expression of Bax, caspase-3 and caspase-9 was found to be augmented at increasing doses in cells treated with ZnONPs than the control cells. The increase in levels of Bax, caspase-3 and caspase-9 indicates that ZnONPs has induced apoptosis in MG-63 cell line through mediation of mitochondrial apoptotic pathway. Further, increased ROS production, and marked rise in apoptotic markers in cells treated with ZnONPs showed that cell death is mediated via mitochondrial apoptotic pathway [58,59].
Fig. 9. Protein expression of Bax, caspase-3 and caspase-9 in MG-63 cells treated with or without ZnONPs. MG-63 cells treated with ZnONPs at varying concentrations 30 and 50 μg/ml and Each blot is a representative of three independent observations.
the images obtained by diffraction has the similar pattern observed in XRD analysis which confirms the hexagonal structure of ZnONPs [45]. Similar XRD analysis pattern for ZnONPs was reported in earlier investigations [46,47]. Viability of cancer cells displayed that ZnONPs from RR exhibited a strong chemo preventive effect and showed diminished cellular adhesion in MG-63 cell line along with some variations in morphology. Drugs which might interfere with the apoptotic signalling cascade demands increased attention in treating the cancer cells [48]. ZnONPs synthesized from RR exhibited less or no cytotoxic effect in MG-63 cancer cell lines which is correlated with the earlier evidence reported by Seo et al. (2008) [26]. Generation of ROS plays a vital role in the pathological processes which includes proliferation of cells, oxidative defense mechanism and accountable for killing of foreign organisms [49]. Cancer cells are highly prone to increased ROS production and promotes higher rate of apoptosis than the normal cell lines [50]. Decreased or altered intracellular antioxidant defense mechanism increased the production of ROS and extensively damages cellular macromolecules such as DNA, proteins and lipids. Thus any compound which increases the production of ROS could cause increased mitochondrial damage and finally results in increased rate of apoptosis [51]. Thus, the result of the study showed that ZnONPs synthesized from RR has the ability to increase ROS production and thereby increase the rate of apoptosis and promote cancer cell death. The apoptotic effect of RR observed in the study relates with the earlier literature evidence which also reported that RR administration to cancer cells has increased ROS production and apoptosis [28]. More studies were performed to examine the apoptosis induction in MG-63 cells by ZnONPs. Treatment targeting the cancer cells at present is mainly focussed on mitochondria to promote apoptosis. Among 7
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
5. Conclusion
[21] P.K. Mishra, H. Mishra, A. Ekielski, S. Talegaonkar, B. Vaidya, Zinc oxide nanoparticles: a promising nanomaterial for biomedical applications, Drug Discov. Today 22 (2017) 1825–1834. [22] F. Ghelfi, M. Tieri, S. Gori, F. Nicolis, M.C. Petrella, A. Filiberti, G. Apolone, L. Titta, Do cancer patients change their diet in the e-health information era? A review of the literature and a survey as a proposal for the Italian population, Food Res. Int. 104 (2018 Feb 1) 59–68. [23] C.S. Yook, Herbal medicine II, Gwangmyunguihagsa Publishers, Seoul, 1992, p. 141 Korean. [24] M.K. Shin, Clinical herb, Namsandang Publishers, Seoul, 1986, p. 218 Korean. [25] S.I. Lee, Clinical Application of Korean Medicine, Sungbosa Publishers, Seoul, 1986, pp. 199–201 Korean. [26] H.S. Seo, The experimental study on anti-inflammation and anti-oxidation of indigo naturalis and Rehmanniae Radix, J. Korean Orient. Med. Ophthalmol. Otolaryngol. Dermato. 21 (3) (2008) 104–110. [27] Y.H. Lee, J.W. Kim, S.K. Chae, J.H. Ahn, G.Y. Do, D.B. Koo, Anti-apoptotic effects of catalpol on preimplantaion porcine embryos, J. Emb. Trans. 30 (1) (2015) 23–31. [28] S.H. Kim, T.H. Yook, J.U. Kim, R. Radix, An effective treatment for patients with various inflammatory and metabolic diseases: results from a review of Korean publications, Aust. J. Pharm. 20 (2) (2017 Jun) 81–88. [29] J.D. Lim, H.Y. Kim, M.J. Kim, D.C. Yang, S.J. Yun, I.M. Chung, et al., Biological activities of resveratrol-3-O-ß-D-giucoside in transgenic Rehmanniae Radix, J. Plant. Biotechnol. 5 (1) (2005) 129. [30] C.H. Jin, Y.M. Lee, M.A. Kang, Y.D. Park, D.S. Choi, M.W. Byun, et al., Anti-inflammatory activities of ethylacetate extract of rehmannia glutinosa in LPS-induced RAW 264.7 cells, Food Sci. Biotechnol. 18 (4) (2009) 923–927. [31] Y.S. Ki, Y.J. Son, Y.J. Lee, Effects of Rehmanniae Radix on the rat liver damaged with CCl4, Kor. J. Herbol 15 (1) (2000) 45–51 Korean. [32] J.Y. Kim, O.H. Jo, C.M. Choi, H.B. Jo, Rhizoma Rehmanniae induced apoptosis in human cervical carcinoma HeLa cells, J. Orient. Obstet. Gynecol 19 (1) (2006) 69–80 Korean. [33] W. Gu, E. Yeo, N. McMillan, C. Yu, Silencing oncogene expression in cervical cancer stem-like cells inhibits their cell growth and self-renewal ability, Cancer Gene Ther. 18 (12) (2011 Dec) 897–905. [34] H. Huang, J. Chen, H. Lu, M. Zhou, Z. Chai, Y. Hu, Two mTOR inhibitors, rapamycin and Torin 1, differentially regulate iron-induced generation of mitochondrial ROS, Biometals. 30 (6) (2017 Dec 1) 975–980. [35] D.S. Yoon, M.H. Lee, D.S. Cha, Measurement of intracellular ROS in Caenorhabditis elegans using 2′, 7′-dichlorodihydrofluorescein diacetate, Bio. Protoc. 8 (6) (2018 Mar 20) e2774. [36] Jinhua Zhu, Meihui Liu, Yuanfen Liu, Yiting Zhang, Bing Yang, Wei Zhang, Zoledronic Acid Regulates Autophagy and Induces Apoptosis in Colon Cancer Cell Line CT26, Biomed. Res. Int. 2017 (2017) 6. Article ID 7203584. [37] M. Kurokawa, S. Kornbluth, Caspases and kinases in a death grip, Cell. 138 (5) (2009 Sep 4) 838–854. [38] C. Gorrini, I.S. Harris, T.W. Mak, Modulation of oxidative stress as an anticancer strategy, Nat. Rev. Drug Discov. 12 (12) (2013 Dec) 931–947. [39] D. Trachootham, J. Alexandre, P. Huang, Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov. 8 (7) (2009 Jul) 579–591. [40] Y.D. Jin, J.P. Yang, P.L. Heremans, et al., Single-layer organic light-emitting diode with 2.0% external quantum efficiency prepared by spin-coating, Chem. Phys. Lett. 320 (5–6) (2000) 387–392. [41] D.H. Zhang, Z.Y. Xue, Q.P. Wang, Formation of ZnO nanoparticles by the reaction of zinc metal with aliphatic alcohols, J. Phys. D 35 (21) (2002) 2837–2840. [42] C.N.R. Rao, Chemical Applications of Infrared Spectroscopy, Academic Press, New York and London, 1963. [43] Markova-Deneva, J. Uni. Chem. Technol. Metal. 45 (2010) 351–378. [44] V. Parthasarathi, G. Thilagavathi, International journal of pharmaceutical, Science 3 (2012) 1–7. [45] G. Voicu, O. Oprea, B.S. Vasile, E. Andronescu, Digest J. Nanomater. Biostruct. 8 (2013) 667–675. [46] B.Y. Chena, P. Liu, J.F. Liu, L. Wang, J. Ceram. Process. Res. 12 (2011) 420–425. [47] Swee-Yong Pung, Wen-Pie Lee, Azizan Aziz, Int. J. Inorg.Chem. 2012 (2012) 1–9. [48] P.N. Kelly, A. Strasser, The role of Bcl-2 and its pro-survival relatives in tumourigenesis and cancer therapy, Cell Death Differ. 18 (9) (2011 Sep) 1414–1424. [49] L. He, T. He, S. Farrar, L. Ji, T. Liu, X. Ma, Antioxidants maintain cellular redox homeostasis by elimination of reactive oxygen species, Cell. Physiol. Biochem. 44 (2) (2017) 532–553. [50] M.A. Gallego, C. Ballot, J. Kluza, N. Hajji, A. Martoriati, L. Castéra, C. Cuevas, P. Formstecher, B. Joseph, G. Kroemer, C. Bailly, P. Marchetti, Overcoming chemoresistance of non-small cell lung carcinoma through restoration of an AIF-dependent apoptotic pathway, Oncogene. 27 (14) (2008 Mar 27) 1981–1992. [51] K.C. Pramanik, S.R. Boreddy, S.K. Srivastava, Role of mitochondrial electron transport chain complexes in capsaicin mediated oxidative stress leading to apoptosis in pancreatic cancer cells, PLoS One 6 (5) (2011) e20151. [52] T. Sithara, K.B. Arun, H.P. Syama, T.R. Reshmitha, P. Nisha, Morin inhibits proliferation of SW480 colorectal cancer cells by inducing apoptosis mediated by reactive oxygen species formation and uncoupling of Warburg effect, Front. Pharmacol. 8 (2017 Sep 12) 640. [53] K. Liu, P.C. Liu, R. Liu, X. Wu, Dual AO/EB staining to detect apoptosis in osteosarcoma cells compared with flow cytometry, Med. Sci. Monit. Basic Res. 21 (2015 Feb 9) 15–20. [54] Z.N. Oltvai, C.L. Milliman, S.J. Korsmeyer, Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death, Cell. 74 (4) (1993 Aug 27) 609–619.
Our study has enlightened that MG-63 cells that are supplemented with ZnONPs at varying concentrations have brought increased synthesis of ROS. Increased ROS generation has led to altered mitochondrial membrane potential and thus resulted in apoptosis. Thus, ZnONPs have the efficacy to attenuate the progress and survival of cancer cells via mediating mitochondrial pathway of apoptosis. Induction of apoptosis as well as prevention of metastasis collectively might have resulted in the potent anticancer activity of ZnONPs from RR in MG-63 cells. Considering all these evidences, it suggests that ZnONPs synthesized from RR can be used as an potent therapeutic regime in cancer treatment. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgment The authors extend their appreciation to The Researchers Supporting Project number (RSP-2019/56) King Saud University, Riyadh, Saudi Arabia. References [1] T.S. Park, V.S. Donnenberg, A.D. Donnenberg, E.T. Zambidis, L. Zimmerlin, Dynamic interactions between cancer stem cells and their stromal partners, Curr. Pathobiol. Rep. 2 (2014) 41–52. [2] G.D. Roodman, Mechanisms of bone metastasis, N. Engl. J. Med. 350 (2004) 1655–1664 (CrossRefMedlineGoogle Scholar). [3] R.E. Coleman, Metastatic bone disease: clinical features, pathophysiology and treatment strategies, Cancer Treat. Rev. 27 (2001) 165–176. [4] V.O. Lewis, What’s new in musculoskeletal oncology, J. Bone Joint Surg. Am. 91 (2009) 1546–1556. [5] J. Fornetti, A.L. Welm, S.A. Stewart, Understanding the bone in cancer metastasis, J. Bone Miner. Res. 33 (2018) 2099–2113. [6] R. Gowda, N.R. Jones, S. Banerjee, G.P. Robertson, Use of nanotechnology to develop multi-drug inhibitors for cancer therapy, J Nanomed Nanotechnol. 4 (6) (2013). [7] R. Wang, P.S. Billone, W.M. Mullett, Nanomedicine in action: an overview of cancer nanomedicine on the market and in clinical trials, J. Nanomater. 2013 (2013) 12. [8] S.E. McNeil, Nanoparticle therapeutics: a personal perspective, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 1 (3) (2009) 264–271. [9] T. Ates, C. Tatar, F. Yakuphanoglu, Preparation of semiconductor ZnO powders by solegel method: humidity sensors, Sens. Actuator A 190 (2013) 150e160. [10] M.J. Akhtar, M. Ahamed, M.A. Majeed Khan Sudhir Kumar, Javed Ahmad, S.A. Alrokayan, Zinc oxide nanoparticles selectively induce apoptosis in human cancer cells through reactive oxygen species, Int. J. Nano Med. 7 (2012) 845e857. [11] R. Wahab, M.A. Siddiqui, Q. Saquib, S. Dwivedi, J. Ahmad, J. Musarrat, A.A. AlKhedhairy, H.S. Shin, ZnO nanoparticles induced oxidative stress and apoptosis in HepG2 and MCF-7 cancer cells and their antibacterial activity, Colloids Surf. B: Biointerfaces 117 (2014) 267e276. [12] I. Pujalte, B. Passagne, M. Brouillaud, E. Treguer, C. Durand, Ohayon-Courtes, Beatrice L’Azou, cytotoxicity and oxidative stress induced by different metallic nanoparticles on human kidney cells, Part. Fibre Toxicol. 3 (2011) 8e10. [13] R. Guan, T. Kang, F. Lu, Z. Zhang, H. Shen, M. Liu, Cytotoxicity, oxidative stress, and genotoxicity in human hepatocyte and embryonic kidney cells exposed to ZnO nanoparticles, Nanoscale Res. Lett. 7 (2012) 602e610. [14] T. Kang, R. Guan, X. Chen, Y. Song, H. Jiang, J. Zhao, In vitro toxicity of differentsized ZnO nanoparticles in Caco-2 cells, Nanoscale Res. Lett. 8 (2013) 496e501. [15] M. Liong, J. Lu, M. Kovochich, T. Xia, S.G. Ruehm, A.E. Nel, et al., Multifunctional inorganic nanoparticles for imaging, targeting, and drug delivery, ACS Nano 2 (2008) 889–896. [16] D. Selvakumari, R. Deepa, V. Mahalakshmi, P. Subhashini, N. Lakshminarayan, Anticancer activity of ZnO nanoparticles on MCF7 (breast cancer cell) and A549 (cancer cell). ARPN, J. Eng. Appl. Sci. 10 (2015) 5418–5421. [17] P. Amornpitoksuk, S. Suwanboon, S. Sangkanu, A. Sukhoom, J. Wudtipan, K. Srijan, S. Kaewtaro, Synthesis, photocatalytic and antibacterial activities of ZnO particles modified by diblock copolymer, Powder Technol. 212 (2011) 432–438. [18] P. Yousefpour, F. Atyabi, E.V. Farahani, R. Sakhtianchi, R. Dinarvand, Polyanionic carbohydrate doxorubicin-dextran nanocomplex as a delivery system for anticancer drugs: in vitro analysis and evaluations, Int. J. Nanomedicine 6 (2011) 1487–1496. [19] S. Ibsen, E. Zahavy, W. Wrasdilo, M. Berns, M. Chan, S. Esener, et al., A novel doxorubicin prodrug with controllable photolysis activation for cancer chemotherapy, Pharm. Res. 27 (2010) 1848–1860. [20] A. Chahal, A.K. Saini, A. Kumar, R.V. Saini, Natural antioxidants as defense against cancer, Asian J. Pharm. Res. 11 (2018) 38–44.
8
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al. [55] D.S. Chelur, M. Chalfie, Targeted cell killing by reconstituted caspases, Proc. Natl. Acad. Sci. U. S. A. 104 (7) (2007 Feb 13) 2283–2288 Epub 2007 Feb 5. Erratum in: Proc Natl Acad Sci U S A. 2008 Jan 15;105(2):824. [56] M. Masumura, R. Hata, I. Nishimura, T. Uetsuki, T. Sawada, K. Yoshikawa, Caspase3 activation and inflammatory responses in rat hippocampus inoculated with a recombinant adenovirus expressing the Alzheimer amyloid precursor protein, Brain Res. Mol. Brain Res. 80 (2) (2000 Sep 15) 219–227. [57] C.C. Su, J.Y.F. Chen, Z.H. Din, J.H. Su, Z.Y. Yang, Y.J. Chen, R.Y. Wang, Y.J. Wu, 13-acetoxysarcocrassolide induces apoptosis on human gastric carcinoma cells
through mitochondria-related apoptotic pathways: P38/JNK activation and PI3K/ AKT suppression, Mar. Drugs 12 (2014) 5295–5315. [58] K. Schulze-Osthoff, A.C. Bakker, B. Vanhaesebroek, R. Beyaert, W.A. Jacob, W. Fiers, Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions: evidence for the involvement of mitochondrial radical generation, J. Biol. Chem. 267 (1992) 5317–5323. [59] T.M. Buttke, P.A. Sandstrom, Oxidative stress as a mediator of apoptosis, Immunol. Today 15 (1994) 7–10.
9
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Green synthesized zinc oxide nanoparticles regulates the apoptotic expression in bone cancer cells MG-63 cells
T
Jun Chenga,1, Xiaofeng Wangb,1, Lei Qiuc, Yunkai Lid, Najat Marraikie, Abdallah M. Elgorbane, ⁎ Li Xuef, a
Department of Orthopedics, Chongqing Three Gorges Central Hospital, Chongqing Province 404000, China Department of Neurosurgery, Weinan Central Hospital, Weinan, Shaanxi Province 714000, China c Department of Oncology, Zhucheng Hospital of Traditional Chinese Medicine, Zhucheng, Shandong Province 262200, China d Emergency Surgery the No.4 Hospital Jinan, Shangdong Province 250031, China e Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia f Department of Orthopaedics, the Third People's Hospital of Chengdu, Affiliated Hospital of Southwest Jiao Tong University Medical School, Chengdu, Sichuan Province 610031, China b
ARTICLE INFO
ABSTRACT
Keywords: Bone cancer Apoptosis Anticancer Rehmanniae Radix Zinc oxide nanoparticles ROS
Management of degenerative spine pathologies frequently leads to the need for bone growth. Rehmanniae Radix (RR), a Chinese herbal formulation was found to exhibit numerous therapeutic properties including its potent effect against cancer cell lines. However, the underlying mechanism through which the Zinc oxide nanoparticles (ZnONPs) synthesized from Rehmanniae Radix exerts its anti-cancer activity against osteosarcoma cell line MG63 needs to be explored. Therefore, the study was performed to evaluate the anticancer, cytotoxicity and apoptotic effectiveness of ZnONPs from RR against MG-63 cells. Characterization studies such UV–vis spectroscopy, FTIR, TEM and XRD analysis were performed. Cytotoxicity assay, mitochondrial membrane potential (MMP), morphological examination of cells and formation of reactive oxygen species (ROS), and apoptosis inducing ability of RR were evaluated by various procedures. Western blot analysis of apoptotic markers such as Bax, caspase-3 and caspase-9 were also performed. RR was found to inhibit growth of MG-63 cells at increasing dose. AO/EB staining confirmed the apoptotic efficacy of ZnONPs induced by RR in MG-63 cells. ZnONPs was also found to initiate increased generation of ROS and decreased MMP. Decreased MMP has resulted in increased levels of apoptotic proteins Bax, caspase-3 and caspase-9 and induction of apoptosis was substantiated by western blot analysis. The outcomes of the work propose that ZnONPs from RR exhibits strong anticancer action and inducing apoptosis on MG-63 cells via stimulating increased generation of ROS. Thus, ZnONPs from RR might be used as a hopeful drug target against several types of cancer cell lines.
1. Introduction Cancer is a highly complicated disease that booms in a heterogeneous environment that is uniquely adaptive [1]. Involvement of bones is the noteworthy complication in metastatic cancer that occurs in patients of breast, multiple myeloma, prostate, primary colon, lung and kidney tumours [2,3]. Most common primary malignant bone tumour includes Osteosarcoma (OS), Ewing sarcoma (ES) and chondrosarcoma (CS) which contributes to almost 70% of malignancies [4]. Metastasis of bone results in increased morbidity, joint pains, increased calcium levels, disability of joints, compression of spinal cord and other related pathological features that leads to major influence in quality of
life in patients [2]. Despite various clinical advances that has increased survival rate in patients, sarcomas are considered deadly till now and the spread of tumour to bone skeleton indicates the cancer is incurable and the treatment options available are also associated with undesirable side effects [4,5]. The therapies which are available clinically to treat cancer has progressively becoming outdated due to development in the fields of nanomedicine and multitargeted drug-delivery that invades and destroys cancer cells [6]. Nanomedicine belongs to the field of nanotechnology which utilizes engineered nanoparticles (NPs) to treat cancer. Nanomedicine has the potent capability to detect and treat cancer at its early stage [7]. Nanomedicine is highly reliable because of
Corresponding author. E-mail address: [email protected] (L. Xue). 1 Equal contribution. ⁎
https://doi.org/10.1016/j.jphotobiol.2019.111644 Received 5 September 2019; Received in revised form 26 September 2019; Accepted 4 October 2019 Available online 30 October 2019 1011-1344/ © 2019 Elsevier B.V. All rights reserved.
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
its potent targeting, high solubility, bioavailability and multipotent properties over other traditional method of cancer therapies available [8]. Among various nanoparticles available, zinc oxide nanoparticles (ZnONPs) have received improved attention at present for its potent antifungal, antibacterial and anticancer properties [9]. Zinc is an essential nutrient for adults and administration of ZnONPs in vivo are considered to be safe. At present, ZnONPs are mainly used in treating cancer [10] and it is found to exhibit cytotoxicity in various cancer cell lines [11–14]. ZnONPs make significant involvement in the development of treatment approaches in cancer drug delivery [15]. It also proves to be highly efficient, stable, low cost, soluble and non-toxic which makes it easy to reach the targeted sites of action [16,17]. The efficient action of ZnONPs results in weakening of mitochondria which produces increased ROS, lipid peroxides and damage to DNA [18–20]. When the ZnONPs interacts with the cancer cells, it disrupts the protein equilibrium and affects various important cellular processes including DNA damage, replication, apoptosis, activity of electron transport chain and cellular homeostasis thus increasing the cytotoxicity against cancer cells [21]. In recent years, various extracts from traditional Chinese medicine (TCM) are used clinically for management of cancer. In patients with advanced stage of cancer who are not eligible for other treatment options, TCM based medicines has proven to be beneficial in improving the general symptoms of cancer, augment immunity and maintain cancer metastasis. It also has beneficial effect in increasing the survival rate of cancer patients and ensure long-term survival among certain percentage of patients [22]. Rehmanniae Radix (RR) is the most commonly used herbal medicine in China to treat multiple diseases and it belongs to the family of Scrophulariaceae. It is prepared from washed root of Rehmannia Glutinosa and it is non-toxic and little bitter in taste [23–25]. Studies reported by Seo et al. (2008) [26] showed that RR exhibited no cytotoxic effect, inhibited the production of nitric oxide and displayed high DPPH radical scavenging ability. Lee et al. (2015) [27] reported the potent anti-apoptotic effect of catalpol, an active component present in RR and established the anticancer and antioxidant activities of RR. Other beneficial properties of RR includes inhibition of ROS production, antihypertensive, anti-inflammatory, antiarthritic, skin whitening effect, reduces wrinkles, improves cognitive function, depression, motility of the intestines, hypoglycaemia, immunity, reduces renal hypertension, inflammation and inhibits oxidation and recovers damaged liver cells [28–31]. RR administered HeLa cervical cancer cells displayed increased necrosis of cells, increased caspase activity and increased Fas expression which indicated the potent beneficial property of RR in treating cervical cancer [32]. Numerous potent therapeutic properties of RR has been reported from literature evidences enlightening its beneficial action and also its potent anticancer activity in certain forms of cancers. However, the effective action of RR against bone cancer cells is less investigated. Hence, the aim of the study is to explore the therapeutic action of ZnONPs from RR against bone cancer cell line MG-63 and to explore its anticancer activity in vitro.
2.2. Characterization of ZnONPs from RR The absorption spectrum of ZnONPs obtained from RR were obtained using UV–visible absorption spectrophotometer and the ZnONPs were characterized by Fourier transform infrared spectroscopy (FT-IR), Transmission Electron Microscopy (TEM) and X-ray diffraction (XRD) pattern for the nanoparticle was recorded using a X-ray diffractometer. 2.3. Fourier Transform Infrared Spectroscopy The lyophilized samples were mixed with dry potassium bromide (KBr pellet) and subjected to a pressure of about 5 × 10 Pa in an evacuated die to produce a clear transparent disc of diameter 2 cm and thickness 0.2 cm. IR spectra in frequency region 4000–400 cm−1, were recorded at room temperature on a Perkin Elmer Fourier transform spectrometer. 2.4. TEM The morphology of the synthesized ZnONPs was determined using TEM. The particle to be dispersed was diluted and it is stained using 2% phosphotungstic acid and it is placed in a copper grid. Then the particles were dried and morphology was studied. 2.5. Reagents and Chemicals Zinc acetate, Bovine Serum Albumin (BSA) and other chemicals were acquired from Sigma Chemical, USA. DMEM medium, Antibiotics and Fetal Bovine Serum were procured from Invitrogen (Groningen, The Netherlands). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from Sigma,USA. 2.6. Maintenance of Cells Osteosarcoma cell line (MG-63) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), Antibiotics (mixture of 1% penicillin/streptomycin). Cells were incubated in T25 tissue culture flasks at 37 °C in a humidified atmosphere (5% CO2 & 95% air environment). ZnONPs from RR was dissolved in media and was resuspended in PBS. ZnONPs at the concentration of 30 μg/ml and 50 μg/ml were used for the study. The antibiotics and media required for the study was purchased from Sigma-Aldrich,USA. For passaging of cells, the cells were detached using trypsin/EDTA and they were reseeded. 2.7. Cell Cytotoxicity (MTT Assay) The cytotoxicity of ZnONPs from RR on the MG-63 cell line were determined by using conventional 3-(4, 5-dimethylthiazole-2-yl)-2, 5diphenyltetrazolium bromide (MTT) assay method. Briefly, cells were seeded in a 96-well plate (2 × 104 cells/well in 100 μl of complete medium) and then incubated. After 24 h, the ZnONPs at 30 and 50 μg/ ml concentrations were added on to the layer of cells. After incubation, 50 μl of MTT (1 mg/ml) was added to each well, and the cells were incubated in the dark at 37 °C for an additional 4 h. Thereafter, the medium was removed and the formazan crystals formed were dissolved in 200 μl of dimethyl sulphoxide, and within 15 min, absorbance was measured by a microplate reader at a wavelength of 540 nm. The percentage of reaction for each treatment was calculated assuming 100% reaction for untreated control cells.
2. Materials and Methods 2.1. Synthesis of Zinc Oxide Nanoparticles from Radix Rehmanniae Zinc acetate solution (1 mM) was dissolved in water and it is stirred for about 1 h continuously. To this sodium hydroxide (20 ml) is added together with 20 ml of RR extract. After 1 h of incubation, the colour change of the solution was noticed. The same reaction mix was allowed in the stirrer for another 3 h and the solution turned yellow which indicated the existence of ZnONPs. Thus precipitate thus formed was separated and centrifuged at 60 °C for 15 min at 8000 rpm. Pellet thus collected following centrifugation can be dried in hot air oven and preserved for further studies.
%Viability =
2
Mean Absorbance of Sample × 100 Mean Absorbance of Negative control
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
2.8. Determination of Cell Morphology and Intracellular ROS The growth of the cells was monitored and the cells were detached from the flask and the floated suspension formed as clumps/aggregates were thus collected and visualized under microscope [33]. Control cells were also included for the morphological analysis. The ROS level of cells was measured using 2′7′-dichlorodihydrofluoresceindiacetate (DCFH-DA) (Applygen Co. Ltd., China) [34,35]. Cells at a concentration of 1 × 104 were seeded in six well plates and kept at overnight incubation. The following day, the medium is substituted with new medium containing 30 and 50 μg/ml of ZnONPs and kept at 37 °C for 24 h. The control was added with distilled water alone. After the growth period, the cells were trypsinized and detached from plates and stained for 30 min with DCFH-DA. Dye present in excess was detached by washing with 1 × PBS. Images of the cells were captured with fluorescence at 530 nm. 2.9. Determination of Mitochondrial Membrane Potential by Rhodamine123 (Rh-123) Staining Fig. 1. UV–Vis absorption spectra of ZnONPs.
MG-63 cells were seeded in six-well plate, and ZnONPs at different concentrations (30 and 50 μg/ml) were introduced and control cells contained distilled water and incubated for 24 h. The cells were then washed with PBS and fixed in methanol at room temperature for 10 min. The cells which are fixed were allowed to permeabilize with triton X-100 (0.2%) for 10 min in PBS solution and incubated for 30 min at room temperature with 5 μg/ml of Rh-123. The cells stained with Rh123(ThermoFisher Scientific, USA) were viewed under fluorescent microscope.
was performed. β-actin was loaded as positive control. 2.13. Statistical Analysis Values are expressed as Mean ± Standard deviation (SD). Using one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test (DMRT) the statistical comparisons were performed. The results were considered statistically significant if the p values were < 0.05.
2.10. Acridine Orange (AO)/Ethidium Bromide (EB) Staining DMEM medium was added to each well of 96-well plate and cells were added to it at a density of 2 × 104/ml and incubated. Control cells were left untreated and other cells were incubated with ZnONPs at a concentration of 30 and 50 μg/ml. After culturing, trypsin was added into all the wells and cells are detached. Suspensions containing 25 μl were moved to glass slides. Double staining solution of 8 μl comprising of AO (100 μg/ml) and EB (100 μg/ml) were added to the suspensions and mixed gently for 20 mins. The mixture was placed in slide and covered using coverslip. The morphology of the cells undergone apoptosis was studied and the cells were counted using fluorescent microscope.
3. Results 3.1. UV–Visible Analysis Fig. 1 shows the UV–vis spectrum of ZnONPs prepared RR at room temperature. The characteristic absorption peak of ZnONPs was observed at 330 nm. The nanoparticles which are synthesized in the solution were determined by UV–vis spectrophotometer and it is the simplest technique available to identify the nanoparticles which are formed in the solution. The ZnONPs absorption spectrum ranges from 200 to 600 nm which clearly relates with the results of the present study.
2.11. Cell Adhesion Assay MG-63 osteosarcoma cells were seeded in a 24 well plate which is precoated with fibronectin (5 μg/ml). Then the cells were incubated at 37 °C for 2 h in cell specific media to get adhered and then washed with PBS 3 times to remove the cells which are not adhered. Then the remaining cells are fixed in formaldehyde for 15 min and washed and then stained for 1 h using Toluidene Blue. After incubation, the cells were viewed under the microscope to understand the adhesion capacity of MG-63 cells treated with or without ZnONPs.
3.2. Transmission Electron Microscopy Fig. 2 (a & b) show the representative images of ZnO nanoparticles. Rod shape ZnO nanoparticles were detected in TEM images of average size in the range of 10.0–12.0 nm which correlates with the size calculated by XRD. It shows that the particles are well crystallized. The diffraction rings on image matches with the peaks in XRD pattern which also proves the hexagonal wurtzite structure of ZnO nanoparticles.
2.12. Western Blot Analysis of Apoptotic Markers
3.3. XRD Analysis
The samples containing 30 micrograms of proteins were electrophoresed using 10% sodium dodecyl sulphate (SDS) polyacrylamide gel for analysis of apoptotic markers Bax, Caspase 3 and caspase 9. After gel electrophoresis, the gel was wet transferred on to membrane made of nitrocellulose. The membranes were then blocked with skimmed mik powder (5%) at room temperature for 1 h in TBST solution. After blocking the membranes were incubated overnight using primary antibody followed by addition of secondary antibody (Santa Cruz, US). The blots were then visualized by Chemidoc imaging and densitometry
The peaks are indexed as 31.84°, 34.52°, 36.38°, 47.64°, 56.7°, 62.92° (103), 63.06°, and 68.1° respectively. All diffraction peaks of sample correspond to the characteristic hexagonal wurtzite structure of zinc oxide nanoparticles (a = 0.315 nm and c = 0.529 nm). Average particle size of ZnO nanoparticles is found to be 10.0 nm using Scherrer equation. Diffraction pattern corresponding to impurities are found to be absent (Fig. 2c). Thus, this confirms the presence of ZnO nanoparticles synthesized which are synthesized of high purity. 3
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
Fig. 2. Transmission Electron Microscopic (TEM) images and X Ray Diffraction pattern of synthesized ZnONPs.
present in ZnONPs. The peaks at 1634 cm−1 and 1192 cm−1 represent the presence of CeN stretch of amino group respectively.
3.4. FTIR The synthesized ZnONPs from RR was lyophilized and subjected to FTIR spectral analysis in order to find out inter and intra molecular interactions present in the nanoparticle and to confirm the presence of RR in the nanoparticle. FTIR spectra of ZnONPs, in (Fig. 3) shows a strong peak at 3321 cm−1 indicating the NH stretch of amine group
3.5. Effect of ZnONPs from RR on the MG-63 Cells Survival The ability of ZnONPS from RR on osteosarcoma cancer cell was investigated by culturing MG-63 cells under different concentrations for
Fig. 3. Fourier transform infrared spectrum analysis of ZnONPs. 4
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
both live and dead cells (Fig. 5b). Cells which are supplemented with ZnONPs at a concentration of 50 μg/ml displayed reduced number of live cells (Fig. 5c). Cell injury resulting from oxidative stress holds a vital role in cancer cell progression. ROS generation in MG-63 were watched after incubating with ZnONPs and the levels of ROS generated were studied using DCFH-DA stain and the changes are viewed using fluorescent microscope (Fig. 5d–f). Cells which were administered with ZnONPs at a concentration of 30 and 50 μg/ml were analysed using DCFH-DA dye which exhibits green fluorescence (Fig. 5e and f). The observation of the study showed that the fluorescence strength of the cells was increased with increased concentration of ZnONPs and the ROS generation was also increased thus indicating that ZnONPs have brought apoptosis in MG-63 cells facilitated via ROS generation. 3.7. Effect of ZnONPs in Mitochondrial Membrane Potential
Fig. 4. Determination of cytotoxicity of ZnONPs synthesized from Radix Rehmanniae by MTT assay. 96-well plates were seeded with MG-63 cells at a density of 1 × 104 cells/well and treated with varying concentrations of ZnONPs synthesized from RR for 24 h. The results are denoted as means ± standard error (SE) of the mean and the error bar size reveals the sample variation of triplicates.
The alterations in membrane potential of mitochondria in MG-63 cells was assessed by means of Rh-123 stain. Control (untreated) cells showed increased green fluorescence which indicates higher mitochondrial membrane potential (Fig. 6a–c). Cells treated with ZnONPs at 30 μg/ml also showed higher mitochondrial membrane than those supplemented with 50 μg/ml (Fig. 6b and c). Cells subjected to 50 μg/ ml of ZnONPs showed low green fluorescence indicative of decrease in mitochondrial membrane potential with increased ROS generation (Fig. 6c). This clearly shows that ZnoNPs synthesized from RR indicates that cell death in MG-63 cells occurs through mitochondrial-mediated pathway.
24 h (5, 10, 20, 30, 40, 50, 60, 70 and 80 μg/ml) and the survival of the cells was determined by MTT assay (Fig. 4). MG-63 cells treated with ZnONPs from RR was found to inhibit cancer cell growth at increasing concentration. The inhibitory concentration (IC50 value) was calculated as xxx μg/ml. The results of the study indicated that the survival of cancer cell was decreased with increasing ZnONPs concentration. The findings of the study displayed that ZnONPs was more effective in minimizing the cancer cell growth and survival rate of MG-63 cells.
3.8. Apoptosis Induction in MG-63 Cells by ZnoNPs from RR Stained with AO/EB
3.6. Effect of ZnONPs from RR on Cell Morphology and ROS Generation
The cells which underwent apoptosis can be confirmed through AO/ EB stain. ZnoNPs treated cells at different concentrations of 30 and 50 μg/ml for 24 h, displayed typical changes in morphology distinctive of apoptosis such as fragmentation of nuclei or nuclear shrinkage, condensed chromatin fragments and shrinked cytoplasm. The results of the study are indicated in Fig. 7a–c. Control cells exhibited normal
Structural changes of MG-63 cells were detected by microscopy (Fig. 5a–c). The study showed that ZnONPS was effective in reducing the viability of cancer cells at various concentration (30 and 50 μg/ml). MG-63 cells treated with ZnONPs at 30 μg/ml showed occurrence of
Fig. 5. Morphological examination and generation of ROS in MG-63 cells treated with or without ZnONPs synthesized from RR. Control (a), Cells treated with ZnONPs at concentration of 30 and 50 μg/ml (b and c). MG-63 cells treated with 30 μg/ml of ZnONPs show a lesser number of viable cells (b). Cells treated with 50 μg/ml of ZnONPs display greater number of nonviable cells. (c). MG-63 cells stained with DCFH –DA showed increased intensity of fluorescence in 50 μg/ml than 30 μg/ml of ZnONPs synthesized. Fluorescence intensity was observed at least levels in control cells indicating a greater number of viable cells. 5
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
Fig. 6. Fluorescent microscopic images of mitochondrial membrane potential using Rh-123 in MG-63 cells treated with or without ZnONPs from RR. Control cells showed increased cellular uptake of Rh-123 dye which indicates the integrity of Δψm (a). MG-63 cells treated with ZnONPs at concentration of 30 μg/ml (b) and 50 μg/ml (c), showed reduced uptake of Rh-123 indicating the loss of Δψm.
4. Discussion
nuclear architecture (Fig. 7a). Cells supplied with 30 μg/ml of drug showed numerous early apoptotic cells which are observed as greenish yellow colour nuclei (Fig. 7b). Cells which are treated with ZnONPs at 50 μg/ml contained late apoptotic cells which appeared orange in colour (Fig. 7c). Treatment with ZnONPs showed marked increase in apoptosis and the number of viable cells was also decreased evidently.
The two major processes that underlie cancer cells development are formation of tumour and metastasis which results in increased proliferation of cells and reduced apoptosis rate. Thus, initiation of apoptosis in cancer cells might increase the treatment options for inhibiting cancer cell growth and metastasis [36]. Cell death by apoptosis is mediated through intrinsic or extrinsic apoptotic pathway and it is categorized by depolarization of mitochondrial membrane with loss of membrane potential [37]. Such imbalance in the cellular status results in increased production of ROS [38]. Increased intracellular ROS production results in significant increase in the rate of apoptosis in cancer cells [39]. Thus, the effective regimen to treat cancer is mainly through development of drugs which promotes ROS production and ensuing in apoptosis. Hence, with this background, the work was performed to explore the cytotoxic, anticancer and apoptotic effectiveness of ZnONPs synthesized from Chinese herbal formulation, Radix Rehmanniae in MG-63 osteosarcoma cell lines. The nanoparticle size plays a vital role in determining the property of synthesized nanomaterial. Thus, size of the particle is very essential to materialize the unique property of the nanomaterial and UV–vis spectroscopy is he most extensively used methodology to assess the optical property of synthesized nanoparticle. Thus, the sharp absorption spectrum of ZnONPs obtained at about 360 nm indicates the monodispersed property of nanoparticle present [40,41]. The ZnONPs metal‑oxygen frequencies which is observed in the present study is in accordance with earlier literature evidences [42,43]. FTIR spectra of ZnONPs corresponding to the spectra obtained in the present study correlates well with earlier investigation [44]. The findings of TEM studies shows that the ZnONPs appear in a well crystallized form and
3.9. Effect of ZnONPs from RR in Cell Adhesion Ability In order to assess the capacity of cancer cells to adhere, the cell adhesion assay was carried out. Fig. 8 represents the images of cell adhesion assay in MG-63 control and ZnONPs treated cells. Control cells depicted increased adhesion capacity of cancer cells following incubation in 24 well plate (Fig. 9a). Cancer cells which are treated with 30 μg/ml of ZnONPs displayed diminished adherence of cells than the control cells (Fig. 8b). MG-63 cells treated with 50 μg/ml of ZnONPs exhibited even more reduced adherence capacity than the cells treated with lower dose of ZnONPs (Fig. 8c). 3.10. Immunodetection of Apoptotic Markers Fig. 9 displays the influence of ZnONPs from RR on expression of apoptotic proteins in MG-63 cells. Results of the study revealed that there was a substantial increase in the protein levels of Bax, caspase-3 and caspase-9 in ZnONPs treated cells, when compared with control cells. Control cells displayed decreased rate of apoptosis. Cells administered with 50 μg/ml of ZnONPs exhibited higher rate of apoptosis than the cells supplemented with 30 μg/ml of ZnONPs.
Fig. 7. Detection of Apoptosis induced in MG-63 cells treated with or without ZnONPs from RR using AO/EB double staining. MG-63 cells treated with ZnONPs at varying concentrations 30 and 50 μg/ml (b and c). Untreated control cells showed uniform size and structure with green fluorescence (a). Cells treated with 30 μg/ml of ZnONPs exhibited early apoptotic cells and nuclear condensation which were observed in greenish yellow colour (b) & Cells treated with 50 μg/ml of ZnONPs showed greater number of late apoptotic cells and fragmented nucleus which were observed in yellowish orange colour (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 6
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
Fig. 8. Cell adhesion assay of MG-63 cells treated with or without ZnONPs synthesized from RR. MG-63 cells treated with ZnONPs at varying concentrations 30 and 50 μg/ml (b and c). Untreated control cells showed increased adherence of cells (a). Cells treated with 30 μg/ml of ZnONPs exhibited diminished cellular adhesion (b) & Cells treated with 50 μg/ml of ZnONPs showed greater reduction of cellular adhesion than the cells treated with 30 μg/ml (c).
various dyes available, Rh123 is the most commonly used dye for determining the membrane potential of mitochondria. The dye Rh123 will retain in the cells of mitochondria only when the cells contain intact membrane potential. The fluorescence will be reduced in cells which lacks membrane potential and results in outflow of stain from mitochondria and reduced fluorescence. Reduced fluorescence is indicative of diminished integrity of mitochondrial membrane [52]. Alterations in membrane potential of mitochondria indicates the initiation of apoptosis and the increased uptake of Rh-123 in control cells observed in the present study indicates intact mitochondria, whereas cells administered with 30 and 50 μg/ml of ZnONPs showed decreasing fluorescence at increasing doses indicative of decreased mitochondrial membrane potential. In the current study, the MG-63 cells supplemented with 30 and 50 μg/ml of ZnONPs from RR which undergone apoptosis was confirmed by AO/EB staining [53]. Apoptosis in MG-63 cells were determined by fluorescence microscopic findings for differentiating live and dead cells. Acridine orange has the ability to hold with all cells and it releases green fluorescence, whereas EB will interact with the cells that lacks cytoplasmic membrane and the nucleus will appear red in colour which is interpolating with DNA. Love cells exhibit green coloured nuclei and the cells which undergone early apoptosis, will emit bright green colour fluorescence. Condensed chromatin is viewed as green patches and the late apoptotic cells observed after treatment with ZnONPS are viewed as orange to red coloured cells. The areas which appear as opaque orange are indicative of depolarized mitochondria. All these morphological changes observed indicates that the cells which are treated with ZnONPs containing RR have underwent apoptosis. Apoptosis is tightly regulated by Bax (proapoptotic “gatecrashers” member) and Caspase-3 (executioner) [54]. Caspases play a critical role in apoptosis [55] in which Caspase-3 is one of the effector units found in apoptotic cells [56]. Hence, we assessed the protein levels of proapoptotic proteins to determine the efficacy of ZnONPs from RR in augmenting apoptosis in MG-63 cell line. The Bcl-2 family members mainly control the mitochondrial pathway of apoptosis. Bax and Bcl-2 are the important activators for release of cytochrome C and consequent activation of caspases [57]. Thus, in the present study, the molecular mechanism of ZnONPs in promoting apoptosis was evaluated and the protein expression of Bax, caspase-3 and caspase-9 was found to be augmented at increasing doses in cells treated with ZnONPs than the control cells. The increase in levels of Bax, caspase-3 and caspase-9 indicates that ZnONPs has induced apoptosis in MG-63 cell line through mediation of mitochondrial apoptotic pathway. Further, increased ROS production, and marked rise in apoptotic markers in cells treated with ZnONPs showed that cell death is mediated via mitochondrial apoptotic pathway [58,59].
Fig. 9. Protein expression of Bax, caspase-3 and caspase-9 in MG-63 cells treated with or without ZnONPs. MG-63 cells treated with ZnONPs at varying concentrations 30 and 50 μg/ml and Each blot is a representative of three independent observations.
the images obtained by diffraction has the similar pattern observed in XRD analysis which confirms the hexagonal structure of ZnONPs [45]. Similar XRD analysis pattern for ZnONPs was reported in earlier investigations [46,47]. Viability of cancer cells displayed that ZnONPs from RR exhibited a strong chemo preventive effect and showed diminished cellular adhesion in MG-63 cell line along with some variations in morphology. Drugs which might interfere with the apoptotic signalling cascade demands increased attention in treating the cancer cells [48]. ZnONPs synthesized from RR exhibited less or no cytotoxic effect in MG-63 cancer cell lines which is correlated with the earlier evidence reported by Seo et al. (2008) [26]. Generation of ROS plays a vital role in the pathological processes which includes proliferation of cells, oxidative defense mechanism and accountable for killing of foreign organisms [49]. Cancer cells are highly prone to increased ROS production and promotes higher rate of apoptosis than the normal cell lines [50]. Decreased or altered intracellular antioxidant defense mechanism increased the production of ROS and extensively damages cellular macromolecules such as DNA, proteins and lipids. Thus any compound which increases the production of ROS could cause increased mitochondrial damage and finally results in increased rate of apoptosis [51]. Thus, the result of the study showed that ZnONPs synthesized from RR has the ability to increase ROS production and thereby increase the rate of apoptosis and promote cancer cell death. The apoptotic effect of RR observed in the study relates with the earlier literature evidence which also reported that RR administration to cancer cells has increased ROS production and apoptosis [28]. More studies were performed to examine the apoptosis induction in MG-63 cells by ZnONPs. Treatment targeting the cancer cells at present is mainly focussed on mitochondria to promote apoptosis. Among 7
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al.
5. Conclusion
[21] P.K. Mishra, H. Mishra, A. Ekielski, S. Talegaonkar, B. Vaidya, Zinc oxide nanoparticles: a promising nanomaterial for biomedical applications, Drug Discov. Today 22 (2017) 1825–1834. [22] F. Ghelfi, M. Tieri, S. Gori, F. Nicolis, M.C. Petrella, A. Filiberti, G. Apolone, L. Titta, Do cancer patients change their diet in the e-health information era? A review of the literature and a survey as a proposal for the Italian population, Food Res. Int. 104 (2018 Feb 1) 59–68. [23] C.S. Yook, Herbal medicine II, Gwangmyunguihagsa Publishers, Seoul, 1992, p. 141 Korean. [24] M.K. Shin, Clinical herb, Namsandang Publishers, Seoul, 1986, p. 218 Korean. [25] S.I. Lee, Clinical Application of Korean Medicine, Sungbosa Publishers, Seoul, 1986, pp. 199–201 Korean. [26] H.S. Seo, The experimental study on anti-inflammation and anti-oxidation of indigo naturalis and Rehmanniae Radix, J. Korean Orient. Med. Ophthalmol. Otolaryngol. Dermato. 21 (3) (2008) 104–110. [27] Y.H. Lee, J.W. Kim, S.K. Chae, J.H. Ahn, G.Y. Do, D.B. Koo, Anti-apoptotic effects of catalpol on preimplantaion porcine embryos, J. Emb. Trans. 30 (1) (2015) 23–31. [28] S.H. Kim, T.H. Yook, J.U. Kim, R. Radix, An effective treatment for patients with various inflammatory and metabolic diseases: results from a review of Korean publications, Aust. J. Pharm. 20 (2) (2017 Jun) 81–88. [29] J.D. Lim, H.Y. Kim, M.J. Kim, D.C. Yang, S.J. Yun, I.M. Chung, et al., Biological activities of resveratrol-3-O-ß-D-giucoside in transgenic Rehmanniae Radix, J. Plant. Biotechnol. 5 (1) (2005) 129. [30] C.H. Jin, Y.M. Lee, M.A. Kang, Y.D. Park, D.S. Choi, M.W. Byun, et al., Anti-inflammatory activities of ethylacetate extract of rehmannia glutinosa in LPS-induced RAW 264.7 cells, Food Sci. Biotechnol. 18 (4) (2009) 923–927. [31] Y.S. Ki, Y.J. Son, Y.J. Lee, Effects of Rehmanniae Radix on the rat liver damaged with CCl4, Kor. J. Herbol 15 (1) (2000) 45–51 Korean. [32] J.Y. Kim, O.H. Jo, C.M. Choi, H.B. Jo, Rhizoma Rehmanniae induced apoptosis in human cervical carcinoma HeLa cells, J. Orient. Obstet. Gynecol 19 (1) (2006) 69–80 Korean. [33] W. Gu, E. Yeo, N. McMillan, C. Yu, Silencing oncogene expression in cervical cancer stem-like cells inhibits their cell growth and self-renewal ability, Cancer Gene Ther. 18 (12) (2011 Dec) 897–905. [34] H. Huang, J. Chen, H. Lu, M. Zhou, Z. Chai, Y. Hu, Two mTOR inhibitors, rapamycin and Torin 1, differentially regulate iron-induced generation of mitochondrial ROS, Biometals. 30 (6) (2017 Dec 1) 975–980. [35] D.S. Yoon, M.H. Lee, D.S. Cha, Measurement of intracellular ROS in Caenorhabditis elegans using 2′, 7′-dichlorodihydrofluorescein diacetate, Bio. Protoc. 8 (6) (2018 Mar 20) e2774. [36] Jinhua Zhu, Meihui Liu, Yuanfen Liu, Yiting Zhang, Bing Yang, Wei Zhang, Zoledronic Acid Regulates Autophagy and Induces Apoptosis in Colon Cancer Cell Line CT26, Biomed. Res. Int. 2017 (2017) 6. Article ID 7203584. [37] M. Kurokawa, S. Kornbluth, Caspases and kinases in a death grip, Cell. 138 (5) (2009 Sep 4) 838–854. [38] C. Gorrini, I.S. Harris, T.W. Mak, Modulation of oxidative stress as an anticancer strategy, Nat. Rev. Drug Discov. 12 (12) (2013 Dec) 931–947. [39] D. Trachootham, J. Alexandre, P. Huang, Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat. Rev. Drug Discov. 8 (7) (2009 Jul) 579–591. [40] Y.D. Jin, J.P. Yang, P.L. Heremans, et al., Single-layer organic light-emitting diode with 2.0% external quantum efficiency prepared by spin-coating, Chem. Phys. Lett. 320 (5–6) (2000) 387–392. [41] D.H. Zhang, Z.Y. Xue, Q.P. Wang, Formation of ZnO nanoparticles by the reaction of zinc metal with aliphatic alcohols, J. Phys. D 35 (21) (2002) 2837–2840. [42] C.N.R. Rao, Chemical Applications of Infrared Spectroscopy, Academic Press, New York and London, 1963. [43] Markova-Deneva, J. Uni. Chem. Technol. Metal. 45 (2010) 351–378. [44] V. Parthasarathi, G. Thilagavathi, International journal of pharmaceutical, Science 3 (2012) 1–7. [45] G. Voicu, O. Oprea, B.S. Vasile, E. Andronescu, Digest J. Nanomater. Biostruct. 8 (2013) 667–675. [46] B.Y. Chena, P. Liu, J.F. Liu, L. Wang, J. Ceram. Process. Res. 12 (2011) 420–425. [47] Swee-Yong Pung, Wen-Pie Lee, Azizan Aziz, Int. J. Inorg.Chem. 2012 (2012) 1–9. [48] P.N. Kelly, A. Strasser, The role of Bcl-2 and its pro-survival relatives in tumourigenesis and cancer therapy, Cell Death Differ. 18 (9) (2011 Sep) 1414–1424. [49] L. He, T. He, S. Farrar, L. Ji, T. Liu, X. Ma, Antioxidants maintain cellular redox homeostasis by elimination of reactive oxygen species, Cell. Physiol. Biochem. 44 (2) (2017) 532–553. [50] M.A. Gallego, C. Ballot, J. Kluza, N. Hajji, A. Martoriati, L. Castéra, C. Cuevas, P. Formstecher, B. Joseph, G. Kroemer, C. Bailly, P. Marchetti, Overcoming chemoresistance of non-small cell lung carcinoma through restoration of an AIF-dependent apoptotic pathway, Oncogene. 27 (14) (2008 Mar 27) 1981–1992. [51] K.C. Pramanik, S.R. Boreddy, S.K. Srivastava, Role of mitochondrial electron transport chain complexes in capsaicin mediated oxidative stress leading to apoptosis in pancreatic cancer cells, PLoS One 6 (5) (2011) e20151. [52] T. Sithara, K.B. Arun, H.P. Syama, T.R. Reshmitha, P. Nisha, Morin inhibits proliferation of SW480 colorectal cancer cells by inducing apoptosis mediated by reactive oxygen species formation and uncoupling of Warburg effect, Front. Pharmacol. 8 (2017 Sep 12) 640. [53] K. Liu, P.C. Liu, R. Liu, X. Wu, Dual AO/EB staining to detect apoptosis in osteosarcoma cells compared with flow cytometry, Med. Sci. Monit. Basic Res. 21 (2015 Feb 9) 15–20. [54] Z.N. Oltvai, C.L. Milliman, S.J. Korsmeyer, Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death, Cell. 74 (4) (1993 Aug 27) 609–619.
Our study has enlightened that MG-63 cells that are supplemented with ZnONPs at varying concentrations have brought increased synthesis of ROS. Increased ROS generation has led to altered mitochondrial membrane potential and thus resulted in apoptosis. Thus, ZnONPs have the efficacy to attenuate the progress and survival of cancer cells via mediating mitochondrial pathway of apoptosis. Induction of apoptosis as well as prevention of metastasis collectively might have resulted in the potent anticancer activity of ZnONPs from RR in MG-63 cells. Considering all these evidences, it suggests that ZnONPs synthesized from RR can be used as an potent therapeutic regime in cancer treatment. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgment The authors extend their appreciation to The Researchers Supporting Project number (RSP-2019/56) King Saud University, Riyadh, Saudi Arabia. References [1] T.S. Park, V.S. Donnenberg, A.D. Donnenberg, E.T. Zambidis, L. Zimmerlin, Dynamic interactions between cancer stem cells and their stromal partners, Curr. Pathobiol. Rep. 2 (2014) 41–52. [2] G.D. Roodman, Mechanisms of bone metastasis, N. Engl. J. Med. 350 (2004) 1655–1664 (CrossRefMedlineGoogle Scholar). [3] R.E. Coleman, Metastatic bone disease: clinical features, pathophysiology and treatment strategies, Cancer Treat. Rev. 27 (2001) 165–176. [4] V.O. Lewis, What’s new in musculoskeletal oncology, J. Bone Joint Surg. Am. 91 (2009) 1546–1556. [5] J. Fornetti, A.L. Welm, S.A. Stewart, Understanding the bone in cancer metastasis, J. Bone Miner. Res. 33 (2018) 2099–2113. [6] R. Gowda, N.R. Jones, S. Banerjee, G.P. Robertson, Use of nanotechnology to develop multi-drug inhibitors for cancer therapy, J Nanomed Nanotechnol. 4 (6) (2013). [7] R. Wang, P.S. Billone, W.M. Mullett, Nanomedicine in action: an overview of cancer nanomedicine on the market and in clinical trials, J. Nanomater. 2013 (2013) 12. [8] S.E. McNeil, Nanoparticle therapeutics: a personal perspective, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 1 (3) (2009) 264–271. [9] T. Ates, C. Tatar, F. Yakuphanoglu, Preparation of semiconductor ZnO powders by solegel method: humidity sensors, Sens. Actuator A 190 (2013) 150e160. [10] M.J. Akhtar, M. Ahamed, M.A. Majeed Khan Sudhir Kumar, Javed Ahmad, S.A. Alrokayan, Zinc oxide nanoparticles selectively induce apoptosis in human cancer cells through reactive oxygen species, Int. J. Nano Med. 7 (2012) 845e857. [11] R. Wahab, M.A. Siddiqui, Q. Saquib, S. Dwivedi, J. Ahmad, J. Musarrat, A.A. AlKhedhairy, H.S. Shin, ZnO nanoparticles induced oxidative stress and apoptosis in HepG2 and MCF-7 cancer cells and their antibacterial activity, Colloids Surf. B: Biointerfaces 117 (2014) 267e276. [12] I. Pujalte, B. Passagne, M. Brouillaud, E. Treguer, C. Durand, Ohayon-Courtes, Beatrice L’Azou, cytotoxicity and oxidative stress induced by different metallic nanoparticles on human kidney cells, Part. Fibre Toxicol. 3 (2011) 8e10. [13] R. Guan, T. Kang, F. Lu, Z. Zhang, H. Shen, M. Liu, Cytotoxicity, oxidative stress, and genotoxicity in human hepatocyte and embryonic kidney cells exposed to ZnO nanoparticles, Nanoscale Res. Lett. 7 (2012) 602e610. [14] T. Kang, R. Guan, X. Chen, Y. Song, H. Jiang, J. Zhao, In vitro toxicity of differentsized ZnO nanoparticles in Caco-2 cells, Nanoscale Res. Lett. 8 (2013) 496e501. [15] M. Liong, J. Lu, M. Kovochich, T. Xia, S.G. Ruehm, A.E. Nel, et al., Multifunctional inorganic nanoparticles for imaging, targeting, and drug delivery, ACS Nano 2 (2008) 889–896. [16] D. Selvakumari, R. Deepa, V. Mahalakshmi, P. Subhashini, N. Lakshminarayan, Anticancer activity of ZnO nanoparticles on MCF7 (breast cancer cell) and A549 (cancer cell). ARPN, J. Eng. Appl. Sci. 10 (2015) 5418–5421. [17] P. Amornpitoksuk, S. Suwanboon, S. Sangkanu, A. Sukhoom, J. Wudtipan, K. Srijan, S. Kaewtaro, Synthesis, photocatalytic and antibacterial activities of ZnO particles modified by diblock copolymer, Powder Technol. 212 (2011) 432–438. [18] P. Yousefpour, F. Atyabi, E.V. Farahani, R. Sakhtianchi, R. Dinarvand, Polyanionic carbohydrate doxorubicin-dextran nanocomplex as a delivery system for anticancer drugs: in vitro analysis and evaluations, Int. J. Nanomedicine 6 (2011) 1487–1496. [19] S. Ibsen, E. Zahavy, W. Wrasdilo, M. Berns, M. Chan, S. Esener, et al., A novel doxorubicin prodrug with controllable photolysis activation for cancer chemotherapy, Pharm. Res. 27 (2010) 1848–1860. [20] A. Chahal, A.K. Saini, A. Kumar, R.V. Saini, Natural antioxidants as defense against cancer, Asian J. Pharm. Res. 11 (2018) 38–44.
8
Journal of Photochemistry & Photobiology, B: Biology 202 (2020) 111644
J. Cheng, et al. [55] D.S. Chelur, M. Chalfie, Targeted cell killing by reconstituted caspases, Proc. Natl. Acad. Sci. U. S. A. 104 (7) (2007 Feb 13) 2283–2288 Epub 2007 Feb 5. Erratum in: Proc Natl Acad Sci U S A. 2008 Jan 15;105(2):824. [56] M. Masumura, R. Hata, I. Nishimura, T. Uetsuki, T. Sawada, K. Yoshikawa, Caspase3 activation and inflammatory responses in rat hippocampus inoculated with a recombinant adenovirus expressing the Alzheimer amyloid precursor protein, Brain Res. Mol. Brain Res. 80 (2) (2000 Sep 15) 219–227. [57] C.C. Su, J.Y.F. Chen, Z.H. Din, J.H. Su, Z.Y. Yang, Y.J. Chen, R.Y. Wang, Y.J. Wu, 13-acetoxysarcocrassolide induces apoptosis on human gastric carcinoma cells
through mitochondria-related apoptotic pathways: P38/JNK activation and PI3K/ AKT suppression, Mar. Drugs 12 (2014) 5295–5315. [58] K. Schulze-Osthoff, A.C. Bakker, B. Vanhaesebroek, R. Beyaert, W.A. Jacob, W. Fiers, Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions: evidence for the involvement of mitochondrial radical generation, J. Biol. Chem. 267 (1992) 5317–5323. [59] T.M. Buttke, P.A. Sandstrom, Oxidative stress as a mediator of apoptosis, Immunol. Today 15 (1994) 7–10.
9