8. CHAPTER IV. ANTICANCER ACTIVITY OF BIOSYNTHESIZED SILVER NANOPARTICLES

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1 8. CHAPTER IV. ANTICANCER ACTIVITY OF BIOSYNTHESIZED SILVER NANOPARTICLES 8.1. Introduction Nanobiotechnology, an emerging field of nanoscience, utilizes nanobased-systems for various biomedical applications. Nanoscale particles and molecules are a potential alternative for treatment of disease because they have unique biologic effects based on their structure and size, which differ from traditional small-molecule drugs (Wagner et al., 2006). This rapidly developing field of nanoscience has raised the possibility of using therapeutic nanoparticles in the diagnosis and treatment of human cancers (Yezhelyev et al., 2006). Cancer is an abnormal type of tissue growth in which the cells exhibit an uncontrolled division, relatively in an autonomous fashion, leading to a progressive increase in the number of dividing cell (Kanchana and Balakrishna, 2011). Discovery of anticancer drugs that must kill or disable only tumor cells without undue toxicity is an extraordinary challenge (Hameed et al., 2009). Microorganisms are good sources for anticancer compounds. Toxicity of microbial material is considered as the presence of antitumor compounds (Bhimba et al., 2012). In recent years, an increasing number of marine natural products have been reported to display antimicrobial activities (Joel and Bhimba, 2010) and anti-tumor compounds from sponges, tunicates, algae and other organisms (Chapman and Gellenbeck, 1983). In most cases, the evaluation of the anticancer potential of crude extracts from different sea organisms has been carried out by in vitro cytotoxicity tests in malignant cell cultures (Russell, 1963). Hundreds of potential anti-tumor agents have been isolated from marine origin (Adams, 1994; Fadli et al., 1991). Isolation of

2 cytotoxic anti-tumor substances from marine organisms has been reported in several references during the last 40 years (Gonzalez et al., 1982). There is increasing demands for anticancer therapy (Unno et al., 2005). In vitro cytotoxicity testing procedures reduces the use of laboratory animals (Abraham et al., 2004) and hence use of cultured tissues and cells have increased (Byrd et al., 2003). The discovery and identification of new antitumor drug with low side effects on immune system has become an essential goal in many studies of immuno-therapies (Xu et al., 2009). Despite many efforts, multi drug resistance is still considered as a major drawback in chemotherapy of cancer which has been the subject of exhaustive experiments recently (Gottesman et al., 2002). Consequently, many attentions have been paid to natural compounds in marine organism and microorganisms. Many medically relevant nanoparticles such as AgNPs were investigated for their cytotoxicity aspect. AgNPs showed different degrees of in vitro cytotoxicity (Hsin et al., 2008). Many attempts have been made to use silver nanoparticles as an anti-cancer agent and they have all turned up positive (Vaidyanathan et al., 2009). The role of silver nanoparticles as an anti-cancer agent should open new doors in the field of medicine. Recently, AgNPs were also found to play an effective role in tumour control via their cytotoxic effects (Lara et al., 2010; Sukirtha et al., 2012). Therefore the present study aimed to evaluate anti-cancer effect of biologically synthesized AgNPs through its cytotoxic activity Materials and methods Reagents 3-(4,5-dimethylthiaozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS), cell culture chemicals such as Dulbecco's modified

3 of Eagle medium with L-glutamine (DMEM), Fetal bovine serum (FBS), Penicillin, streptomycin, were obtained from Sigma Aldrich Chemicals, USA Cell lines and culturing The present work was carried out in A549 (human lung cancer) cell line, Hela (cervical cancer) cell line and Vero (kidney epithelial cells of the African Green Monkey) cell line were obtained from National Centre for Cell Science (NCCS), Pune, India. A549 cells, Hela cell line, and Vero cell line were cultured in DMEM (Dulbecco's modified of Eagle medium with L-glutamine & 1000 mg/l glucose) supplemented with 10% fetal bovine serum, penicillin G (100 units/ml) and streptomycin sulfate (0.1 mg/ml) in a humidified atmosphere consisting of 5% CO 2 at 37 C Cytotoxic Assessment Using MTT Assay The cytotoxic activity of AgNPs on A549 cells, Hela cell line, and Vero cell line was determined by MTT assay based on the detection of mitochondrial dehydrogenase activity in living cells (Mosmann, 1983) Principle MTT (3-(4, 5-Dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide), is cleaved by NAD-dependent mitochondrial dehydrogenase of viable cells, yielding a measurable purple formazan product. This formazan production is proportionate to the viable cell number and inversely proportional to the degree of cytotoxicity Reagents MTT stock solution (5mg/ml) was prepared using the following procedure. 50 mg of MTT was added to 10 ml PBS. Then it was vortexed for 20 minutes and it was filtered through 0.45 micron filter. The bottle was wrapped with aluminium foil for preventing light because MTT is light-sensitive. Finally, the preparation was stored at 4 0 C.

4 Procedure Yellow coloured MTT is converted to the blue formazan product only by metabolically active mitochondria and the absorbance is directly proportional to the number of viable cells. A549, Hela, and Vero cells were plated in 96 well flat bottom tissue culture plates at a density of approximately 1.2 X 10 4 cells/well and allowed to attach overnight at 37 o C. The medium was then discarded and cells were incubated with different concentrations of the AgNPs for 24 hours. After the incubation, medium was discarded and 100µl fresh medium was added with 10µl of MTT (5mg/ml). After 4 hrs, the medium was discarded and 100µl of DMSO was added to dissolve the formazan crystals. Then, the absorbance was read at 570nm in a microtitre plate reader. Cell survival was calculated by the following formula: Viability % = (Test OD/ Control OD) X 100 Cytotoxicity % = 100 Viability% 8.3. Results Cytotoxicity of silver nanoparticles (MTT assay) The In vitro cytotoxic activity of silver nanoparticles was evaluated against human lung cancer (A549) and human cervical (HeLa) cancer cell line at different concentrations. The AgNPs exhibited potent cytotoxicity/anticancer activity in the tested cell lines. The effect was compared with Vero cell lines (normal cell lines). Results showed that at higher concentrations there is significant cell mortality. The inhibitory effect was observed after 24 of incubation. Fig 17 shows the changes in the percentage of inhibition in nanoparticles treated A549 cells. The treatment of µg/ml of sample with AgNPs significantly inhibited A549 cells. The highest cell growth inhibition (80.54%) was observed in the concentration 125µg/ml. The silver nanoparticles were able to reduce viability of the A549 cells in a dose-dependent manner as

5 shown in Table 5. Similarly AgNPs induced 85.36% death of Hela cells at a concentration of 125µg/ml (Table. 6, Fig. 19). Less cytotoxicity of synthesized AgNPs was observed against normal Vero cell line with 54.34% at 125µg/ml (Table. 7, Fig. 21). The results also showed that A549 cells were inhibited by AgNPs with an IC 50 value of 40 g/ml of the concentration, Hela cells with an IC 50 value of 30 g/ml of the concentration and Vero cells with an IC 50 value of 115 g/ml of the concentration. Anticancer activity of A549 cell line, Hela cell line and Vero cell line at different concentrations was shown (Fig.18, 20 and 22 respectively). Thus the synthesized nanoparticles were found to be potently cytotoxic agent against Hela (cervical cancer) cell line than the A549 (human lung cancer) cell line. Therefore these results indicate that the sensitivity of human cancer cell line for cytotoxic drugs is higher than that of Vero cell line for the same cytotoxic agents. Table.5. MTT assay test on A549 cell line after treatment with different concentrations of synthesized AgNPs Concentration (µg/ml) A549 cell line % Cell viability % Cell inhibition ± ± ± ± ± Control 100 0

6 Fig.17. Cytotoxic effect of AgNPs on A549 cell line The values are given as mean ± SD of three experiments in each group

7 Fig.18. Cytotoxic activity of AgNPs on A549 cell line at different concentrations Table.6. MTT assay test on Hela cell line after treatment with different concentrations of synthesized AgNPs Concentration (µg/ml) Hela cell line % Cell viability % Cell inhibition ± ± ± ± ± Control 100 0

8 Fig.19. Cytotoxic effect of AgNPs on Hela cell line The values are given as mean ± SD of three experiments in each group Fig.20. Cytotoxic activity of AgNPs on Hela cell line at different concentrations Control 100µg 75µg 125µg

9 Table.7. MTT assay test on Vero cell line after treatment with different concentrations of synthesized AgNPs Concentration (µg/ml) Vero cell line % Cell viability % Cell inhibition ± ± ± ± ± Control Fig.21. Cytotoxic effect of AgNPs on Vero cell line The values are given as mean ± SD of three experiments in each group

10 Fig.22. Cytotoxic activity of AgNPs on Vero cell line at different concentrations 100µg 8.4. Discussion The biomedical applications of silver nanoparticles are promising with their tremendous effects in the fields of medicine, drug delivery and anti angiogenic property of cancer (Sahoo et al., 2004; Gurunathan et al., 2009). In the present study the cytotoxic effect of biosynthesized AgNPs was tested against human lung cancer cell line (A549), human cervical (HeLa) cancer cell line and Vero cell line using MTT assay. The viability of the cell lines was observed after 24

11 h of treatment with AgNPs. Results showed that the reduced cell viability of cancer cell lines with increasing concentrations ( g/ml) of AgNPs at 24 h. The reduction of cell viability observed in this study is suggestive of anticancer effects of AgNPs. In this study, dose dependent approach was used to evaluate the toxicity of the nanoparticles on A549, HeLa and Vero cell lines. The viability of cells considerably decreased with increasing concentration of nanoparticles. The mortality data obtained in this study concluding that AgNPs synthesized by marine E. coli having potential anticancer activity. The present findings are similar to the observation of Nagajyothi et al., (2014) in which they reported antiproliferative activity of AgNPs against A549 human lung cancer cell line and MCF-7 human breast cancer (HTB-22) cell line. AgNPs significantly inhibited the cell proliferation in A549 (51.61%) and MCF-7 (59.11) cells. In another study, silver nanoparticles synthesized from Iresine herbstii showed potent cytotoxicity against HeLa cervical cell lines. IhAgNPs induced over 88% death of HeLa cells at a treatment concentration of 300µg/ml (Dipankar and Murugan, 2012). Similarly, Verma et al., (2013) reported anticancer activity of silver nanoparticles biosynthesized by Penicillium spp against human colon adenocarcinoma (HT 29) cell lines. They also observed the effect of cytotoxicity on normal Vero cell line. Shanmugasundaram et al., (2013) studied cytotoxic effects of actinobacterially synthesised silver nanoparticles on HeLa cell line. The biosynthesized silver nanoparticles are found to be effective in cytotoxic activity against tumor cells. Despite the widespread use of synthetic AgNPs, very few studies are conducted to determine the cytotoxic effects of biosynthesized AgNPs. The MTT assay was used to assess the effect of AgNPs on proliferation of HeLa cells and normal HBL 100 cells. The AgNPs cytotoxicity was tested against cancer cell lines. As the concentration increased the cell

12 proliferation percentage decreased. It was hypothesized that cell apoptosis was the mechanism induced by the biosynthesized AgNPs (Sukirtha et. al., 2012). In another study, in vitro cytotoxic activity on human colon adenocarcinoma (HT29) was assessed. The AgNPs were used to check the cytotoxic activity on HT-29 cell lines. The AgNPs showed potential cytotoxic activity against the colon cancer cell lines (Bhimba et al., 2012). At present cancer claims the lives of approximately seven million people worldwide on annual basis. Hence, in recent years, the search for the cancer therapeutics from natural products has been increasing day by day. Bioactive compounds in marine organisms have been reported against various cancer cell lines. In conclusion, anticancer activity of silver nanoparticles synthesized by marine E. coli VM1 showed considerable cytotoxic effect against human cancer cell lines. Therefore, the synthesized silver nanoparticles could be considered as an effective anticancer agent. However, detailed study regarding interaction of microbial synthesized AgNPs with the cancer cell lines need to be ascertained before the extensive using medical applications.