Antiproliferative effect of silver nanoparticles synthesized using amla on Hep2 cell line BY stanleY00086 Asian Pacific Journal of Tropical Medicine (2012)1-10 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine journal homepage:www. elsevier. com/locate/ap]tm Document heading dot: Antiproliferative effect of silver nanoparticles synthesized using amla on Hep2 cell line Fathima Stanley Rosarinl , Vadivel Arulmozhil , Samuthira NagaraJan2, Sankaran Mirunalinil* Department of Biochemistry and Biotechnology, Annamalai University, Annamalai nagar-608 002, Tamilnadu, India Department of Chemistry, Annamalai
Results: PE-AgNPs was synthesized and confirmed through kinetic behavior of NPs. The shape of PE-AgNPs was spherical and ubic since it was agglomerated, and the nanoparticle surface was complicated. Average particle size distribution of PE-AgNPs was found to be 188 nm. Potent biomolecules of P. emblica such as polyphenols were capped with AgNPs and reduced its toxicity. In cytotoxicity assay the concentration in which the maximum number of cell death was 60 fig/mL and 50 fig/mL for P. emblica (alone) and AgNPs, respectively and IC50 values were fixed as 30 fig/mL and 20 fig/mL.
ROS generation, apoptotic morphological changes, mitochondrial depolarization, DNA damage and oxidative stress was observed as more in AgNPs treated cells than in P. mblica (30 g/mL) (alone) treated cells and 5-FU treated cells gave similar result. Conclusions: The results suggest that the AgNPs are capped with biomolecules of amla enhanced cytotoxicity in laryngeal cancer cells through oxidative stress and apoptotic function on Hep2 cancer cells. Keywords: Amla AgNPS Oxidative stress Cytotoxicity Antiproliferation 1.
Introduction Cancer is considered as one of the most deadly disease in the world with high mortality. Since there are many cancer therapies available, chemotherapy has become an integral component of cancer treatment for most cancers. In the area f oncology drug discovery, conventional chemotherapeutic agents still exhibit poor specificity in reaching tumor tissue and are often restricted by dose-limiting toxicity. The combination of developing controlled-release technology and targeted drug delivery may provide a more efficient and less harmful solution to overcome the limitations in *Corresponding author: Dr.
S. Mirunalini, assistant professor, Depatment of Biochemistry, Annamalai University, Annamalai nagar – 608 002, Tami nadu, India. Tel: Fax: conventional chemotherapy. R ecent interest has been are capable of controlling the release of chemotherapeutic gents directly inside cancer cells[l]. Nanomaterials are expected hopefully to revolutionize the cancer diagnosis and therapy. Nanoscale particles decorated with multiple functionalities are able to target and visualize tumor site via an imaging technology, thereby allowing for the early detection of cancer.
Furthermore, intelligent nanosystems can be constructed as controlled delivery vehicles which are capable of delivering anticancer drugs to a predetermined site and then releasing them with a programmed rate, which can improve therapeutic efficacy. In inorganic nanoparticles, metal nanoparticles have eceived considerable attention in recent years because of their unique properties and potential applications in Fathima Stanley Rosarin et al. /Asian Pacific Journal of Tropical Medicine (2012)1-10 catalysis, photonics, optoelectronics, biological tagging and pharmaceutical applications.
A number of approaches are available for the synthesis of silver nanoparticles (AgNPs). For example, silver ions are reduced by chemical, electrochemical, radiation, photochemical methods, Langmuir-Blodgett and biological techniques[3-9]. Among these methods, biological synthesis is a good way to fabricate benign nanostructure aterials. Biological technique is less toxic and eco friendly, in the synthesis of nanoparticles capping agents are used. Capping agent is absorbed by nanoparticles. They are usually organic molecules, and used to aid stabilization of nanoparticles.
Silver nanoparticles were synthesized by various plant materials as capping agents such as papaya and neem. Polyphenols of Phyllanthus emblica (P. emblica) includes hydrolysable tannins, flavonoids, alkaloids, gallic acid, ellagic acid and quercetin [1 2], and this plant exhibits antioxidant, adaptogenic, and hepato-protective action[1 5]. Due to the existence of the conjugated ring structures and hydroxyl groups, many phenolic compounds have the potential to function as antioxidants by scavenging superoxide anion and singlet oxygen.
Metal nanoparticles can be synthesized in the average size of more than 50 nm, therefore their large surface area has the ability to carry a relatively high drug dose. Functionalizing the surface of conventional metallic nanoparticles like gold and silver to carry drugs is under investigation. investigate the anticancer effect of PE-AgNPs and drug delivery efficacy of Ag nanoparticles on Hep2 (laryngeal pidermoid carcinoma, a common malignant tumor of head and neck) cell line which has not been previously studied.
The biological applications of silver nanoparticles (AgNPs), antimicrobial properties in particular have been widely studied. AgNPs are known to be cytotoxic to both normal and cancer cells in mammals  and the modes of interactions of AgNPs have been investigated in different prokaryotic and eukaryotic systems [21-23]. Since nanoparticles ( NPs) are more biocompatible than the conventional therapeutics, they are exploited for drug encapsulation and delivery.
It has been stressed over he years that size reduction of NPs play an important role in improving their bioavailability as well as compatibility for therapeutical applications in diseases like cancer. Silver nanoparticles (AgNPs) have a great potential in cancer management because it selectively involved in disruption of the mitochondrial respiratory chain by AgNPs leading to production of ROS and interruption of ATP synthesis, which in turn cause DNA damage[26,21].
Based on the conflicting results, here is an urgency to evaluate cytotoxicity and apoptotic properties of PE-AgNPs on hep2 cells. In order to study the properties of PE-AgNPs to induce apoptosis n cancer cells, it was compared with a standard reference drug 5-flourouracil (5-FU) and we also compared P. emblica fruit extract (alone) with its encapsulated AgNPs. For this study, we employed a well characterized AgNPs to access its apoptotic function via its cytotoxicty and oxidative stress. Dose was fixed and applied in the Hep2 cells by the cytotoxicity test.
Based on this study and our ability to access the interaction and interference with a wide range of biological functions, PE-AgNPs employed in the present study provide a unique opportunity to investigate oxidative stress, intracellular ROS generation, apoptotic bodies and poptotic DNA fragmentation, and mitochondrial membrane potential and toxicity in human laryngeal carcinoma cells (Hep2 cell line). Though the AgNPs induces mitochondrial mediated apoptosis, we have made an attempt to cap the AgNPs with potent biomolecules of P. mblica and we assessed the toxicity and examined the apoptotic function of PE-Ag NPs which comprised of biomolecules of P. emblica and silver precursors. 2. 1 . Silver nanoparticles The silver nanoparticles were synthesized using previously published procedure in which AgNPs were produced through ion reduction and subsequent stabilization using aqueous xtract of P. emblica fruits. In this method P. emblica pericorps were initially rinsed thrice in distilled water and dried on paper toweling.
About 25 g of fruit were cut into fine pieces and boiled with 100 mL sterile distilled water for 5 minutes and filtered through Whatman No. l filter paper twice. The filtrate was stored at 4 C and used for the present study. About 10 mL of aqueous fruit extract was added into the 100 mL aqueous solution of 1 mM AgN03 (AR). The 100 mL of 1 mM silver nitrate solution was reduced using 10 mL of P. emblica extract at room temperature within 10 min. Ag ion reduction was monitored by measuring the UV-vis spectrum of the reaction medium at various time intervals (5 min to 78 h) in room temperature.
The pellet of AgNPs obtained after centrifugation was air dried and mixed with KBr and the KBr-AgNPs pellet was subjected to FTIR to ensure the formation of silver nanoparticles with encapsulation of biomolecules of P. emblica. A scanning electron microscope was used to record the micrograph images of synthesized AgNPs, the particle size distribution of AgNPs was evaluated using dynamic light scattering measurements. These well characterized silver nanoparticles ere further used for cytotoxicity and oxidative stress on cancer cells. 2. 2.
Cell culture The human laryngeal carcinoma cell line ( Hep2) was purchased from National Centre for Cell Science, Pune, I ndia. T he cells were cultured as monolayer in MEM supplemented with 10% FBS, 1% glutamine and 100 IJ/mL penicillin-streptomycin at 37 C in 5% C02 atmosphere, stocks were maintained in 25 cm2 tissue culture flasks. A stock solution of PE (1 mg/mL) and PE-AgNPs (1 mg/mL) was prepared in 0. 5% dimethyl sulphoxide (DMSO) (w/v) and stored at 4 ‘C further dilution was made in culture media to obtain the desired oncentrations.
The final concentrations of DMSO in the culture medium were not more than 0. 01% (wv). 0. 01% DMSO was used as a sham control. C ells were treated with different concentration of PE and PE-AgNPS (1, 5, 10, 20, 30, 40, 50 fig,’t-nL) and the cytotoxicity was observed by (3-4, 5-dimethyl thiaz012yl)-2, 5-di phenyl tetrazolium bromide (MTT) assay. MTT assay was first proposed by mossmon. IC50 value was calculated and optimum dose of PE and PE-AgNPs was fixed in this assay for further study. It is a colorimetric assay for measuring the activity of enzymes that reduce MTT to purple color.
Formazan product is directly proportional to viable cells. 10 GL of MTT solution (5 mg/mL in PBS) was added to each culture well after 24 hours of incubation with PE and PE-AgNPs treatment. The color was allowed to develop for additional 4 hours incubation. An equal volume of DMSO was added to stop the reaction and to solubilize the blue crystals. The absorbance was taken using UV-visible spectrophotometer (Elico SLl 59, India) at a wavelength of 570 nm. OD value was subjected to sort-out percentage of viability by using the following formula, OD value of experimental samples.
Percentage of cell viability= OD value of experimental sample (AgNPs) OD value of experimental control (untreated) 2. 4. Cell treatment The Hep2 cells were treated with PE and PE-AgNPs in the following concentrations as revealed by MTT assay. They are Group l- Control (untreated cancer cells), Group ll- Hep2 cells (30 Gg/mL PE), Group Ill – Hep2 cells + 20 fig/mL PE-AgNPs and Group IV – Hep2 cells + 30 Gg/mL 5-FU. After treatment they were incubated at 37 ‘C in 5% C02 incubator after 24 h incubation. The cells were harvested by trypsinization for further experiments. . 5. Reactive oxygen species generation R eactive oxygen species was assessed following the rocedure described by J esudason et al . C ells were seeded in 96 well plate and incubated with PE extract (30 figmL), PE-AgNPS (20 fig,’t-nL) and 5-FU (30 for 24 h. A fter incubation, fluorescent dye 2 ‘, 7 ‘dichlorfluorescein-diacetate (DCFH- DA) a non-fluorescent probe that can penetrate into the intracellular matrix of cells, which were then kept in incubator for 30 min. Then the cells were washed with PBS to remove the excess dye. . 6. Mitochondrial membrane potential Mitochondrial membrane potential was evaluated following the procedure described by B hosle et al  using the Rhodamine-123 (Rh-123) which is a lipophilic cationic dye, 3 highly specific for mitochondria. The cells were seeded in 96 well plate and treated with PE (30 fig/mL), PE-AgNPs (20 fig/mL) and 5-FIJ (30 fig/mL) and incubated for 24 h. A fter incubation of the cells, fluorescent dye R h- 123 (5 mmol/L) was added to the cells and kept in incubator for 30 min.
Then the cells were washed with PBS and viewed under fluorescent microscope using blue filter. 2. 7. Apoptotic morphological changes The apoptotic bodies which were the result of treatment with PE-Ag NPs was assessed by the method of Lakshmi et al. Apoptotic nuclei exhibits typical changes such as nuclear condensation and fragmentation were stained by AO/EtBr to know the dead apoptotic cells. Cells were treated with PE (30 Gg/mL), PE- AgNPS (20 fig/’TlL) and 5-FIJ (30 fig/mL), and incubated in C02 incubator for 24 h.
The cells were fixed in methanol: glacial acetic acid (3:1) for 30 min at 37 C. The cells were washed with PBS and stained in 1:1 ratio of AO/EtBr, stained cells were immediately washed and viewed under a fluorescent microscope with a magnification of 40x. 2. 8. Apoptotic DNA fragmentation A poptotic DNA fragmentation is a key feature of rogrammed cell death and also occurs in certain stages of necrosis. DNA damage was estimated by agarose gel electrophoresis of DNA fragmentation. Hep2 cells were treated with PE ( 30 g/m L), PE – A g NP s (20 g/m L) and 5-FU (30 Gg/mL).
Treated and untreated cells were collected by centrifugation at 3 000 rpm for 15 min at 4 C. The cell pellet was suspended in cell lysis buffer (Tris Hcl 10 mmol/L pH 7. 4, Triton-xlOO, 0. 5%) and kept at 4 C for 20 minutes. The supernatant was incubated with RNAase of 40 fig/L at 37 for 1 h then incubated with proteinase K 40 Gg/L at 37 for 1 h. To he final aqueous phase 40 GL of 3. 5 M ammonium acetate was added, to this ice cold isopropanol was added and centrifuged at 25 000 rpm for 15 min and dried.
After drying, DNA was dissolved in TE buffer and separated by 2% agarose gel electrophoresis at 100 V for 50 min and the DNA Damage was analyzed by gel documentation (alpha innotech image analyzer). 2. 9. Lipid peroxidation Accumulation of lipid peroxides in the cell is associated cellular stress which leads to cancer cell death. The cells were harvested by trypsinization, the cell pellet obtained was suspended in PBS . T he suspension was taken for iochemical estimations. The level of lipid peroxidation was determined by analyzing TBA-reactive substances (TBARS).
The pink chromogen formed by the reaction of 2-TBA with breakdown products of lipid peroxidation was Figure 1 . Solution of fruit extract, AgNO 3 solution and Ag nanoparticles (From left to right). 0. 200 The statistical analysis was done among the experimental groups with control and normal groups using SPSS software Version 16 (SPSS Inc. , Chicago, IL, USA). The One-way ANOVA was done for expressing experimental significance of the present study. Statistical significance was accepted t a level of P ; 0. 05. Abs. 3. 1 . Nanoparticle synthesis and characterization 0. 050 When aqueous extract of P. mblica was added to silver nitrate solution and stirred for 1 h, the resultant solution was brownish orange (F igure 1 ).