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In Vitro Anticancer Property of Yellow Pigment from Streptomyces Griseoaurantiacus JUACT 01

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In Vitro Anticancer Property of Yellow Pigment from Streptomyces Griseoaurantiacus JUACT 01 869 Vol.58, n.6: pp. 869-876, November-December 2015 BRAZILIAN ARCHIVES OF http://dx.doi.org/10.1590/S1516-89132015060271 ISSN 1516-8913 Printed in Brazil BIOLOGY AND TECHNOLOGY AN INTERNATIONAL JOURNAL In vitro Anticancer Property of Yellow Pigment from Streptomyces griseoaurantiacus JUACT 01 Kuruvalli Prashanthi1*, Sandeep Suryan2 and Kilinger Nadumane Varalakshmi1 1Department of Biotechnology; Centre for Post Graduate Studies; Jain University, Jayanagar, Karnataka - India. 2Centre for Emerging Technologies; Jain University; Jain Global Campus; Kanakapura Taluk, Karnataka - India ABSTRACT Despite the complications in isolation of pigments, microbial pigments are increasingly gaining the attention of researchers because of their broad range therapeutic potentials, especially against cancer. In this study the cytotoxic and anti proliferative potentials of yellow pigment from Streptomyces griseoaurantiacus JUACT 01 isolated from soil are investigated. The effect of pigment treatment on the growth and proliferation of in vitro cervical cancer cells (HeLa) and liver cancer cells (Hep G2) was tested by various methods. Significant cytotoxicity was observed with IC 50 values as low as 1.5 and 1.8 µg /mL with HeLa and Hep G2 cells respectively. The pigment exhibited non toxic effects on human lymphocytes. Decrease in the number of viable cells, presence of apoptotic bodies, nuclear condensation and sheared DNA were distinctly observed in pigment treated cancer cells. The biochemical test and the infrared (IR) spectra indicated the probable carotenoid presence in the TLC purified pigment fraction. High Performance Liquid Chromatography (HPLC) analysis of the TLC purified yellow pigment showed a single large peak with a retention time of 9.90 min and m/z value corresponding to the peak was found to be 413.22 showing 100% relative abundance. Key words: Anticancer activity, ESI-MS, Fluorescence microscopy, Streptomyces griseoaurantiacus JUACT 01, yellow pigment INTRODUCTION producers of large number of bioactive molecules (Berdy 2005). Actinomycetes and, in particular the “Chemotherapy “defined as use of chemicals to genus Streptomyces, have been well known during treat disease is one of the most popular modes of the last seventy years as prolific producers of new treating cancer (Ferlay et al. 2013). The age-old bioactive compounds. These bioactive molecules practice of surgery and radiotherapy became are extensively being used as antibiotics, antitumor unsatisfactory with the increase in major agents, immunosuppressive agents and enzymes ramifications with cancer. Search for novel drugs (Solanki et al. 2003). Specifically, bacteria of this has become a priority goal for cancer therapy, due group produce antitumor compounds such as to the fast development of resistance to multiple anthracyclines (aclarubicin, daunomycin and chemotherapeutic drugs. A large number of doxorubicin), glycopeptides (bleomycin and anticancer compounds are natural products or their actinomycin D), aureolic acids (mithramycin), derivatives, mainly produced by microorganisms. enediynes (neocarzinostatin), antimetabolites Actinomycetes which are filamentous gram (pentostatin), carzinophilin, mitomycins and others positive bacteria are popularly known as the (Olano et al. 2009). Even the pigments of *Author for correspondence: [email protected] Braz. Arch. Biol. Technol. v.58 n.6: pp. 869-876, Nov/Dec 2015 870 Prashanthi, K. et al. actinomycetes have bioactive potentials (Soliev et dried under vacuum conditions. The dry pigment al. 2011). In the current study, we attempted to was re-dissolved in a suitable solvent for isolate a yellow pigment from a soil actinomycete spectrophotometric quantification. Pre coated and evaluate its anticancer potentials. silica TLC sheets (60 F 254, Merck) were used to fractionate the pigment. A number of solvents like n-hexane, chloroform, ethyl acetate, acetone, MATERIAL AND METHODS methanol, water and acetonitrile in different proportions were tested as solvent system. After Isolation and identification activating the TLC sheet at 100˚C the extracts Soil samples for isolation of pigmented were spotted half centimetre above the bottom actinomycetes were collected from a variety of edge and allowed to run till the solvent system sources like rhizosphere of orchids, leaf litter soil, reaches half centimetre below the top edge. The garden soil etc. Serial dilution and spread plate separated fractions were detected by observing the technique using Starch Casein Nitrate Agar TLC sheet under UV – light and then exposing the (SCNA) media containing fluconazole (25 mg/L) sheet to iodine vapour. As we were interested in and tetracycline (50 mg/L) were used for selective the pigment fraction, the coloured fraction was isolation of the actinomycetes. Preliminary visible to the naked eye. The distance moved by identification was based on the microscopic and each fraction from the origin was measured and cultural observations as per the International corresponding Rf values were calculated (Kirchner Streptomyces Project (ISP) (Shirling and Gottlieb et al. 1951).The separated fractions were 1966). Molecular identification was performed by recovered by scraping the spot on the silica sheet sequencing the conserved 16S ribosomal RNA and suspending in suitable organic solvent. The gene encoding DNA fragment of 1500 bp, silica was removed by centrifugation and the obtained after specific PCR amplification using supernatant organic solvent was evaporated to the universal primers (Schwieger and Tebbe obtain the purified fraction (Kirchner 1973). 1998). The sequence obtained was compared with Further, to detect the presence of carotenoids similar sequences retrieved from the GenBank biochemical test was conducted as illustrated by nucleotide sequences database (Altschul et al. Ajayi et al. (2011). The purified pigment was 1990). The sequence was deposited in GenBank analysed using Attenuated Total Reflectance Infra with the accession number KJ774106. A distance Red (ATR-IR) (Bruker Alpha ECO-ATR matrix tree was constructed using the neighbor- spectrometer) to obtain the functional group joining method (Saitou and Nei 1987), and the absorption frequencies (Coates 2000). topology of the phylogenetic tree was built by bootstrap analysis (Felsenstein 1985) using Screening for anticancer activities MEGA6.2 (Tamura et al. 2011). The effect caused by the TLC purified yellow pigment on the growth and proliferation of Human Media optimization and pigment extraction cervical cancer cell line (HeLa) and Human liver Yellow pigment producing Streptomyces cancer cell line (HepG2) was tested by: 3-(4, 5- griseoaurantiacus JUACT 01 was selected for the dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium study. The best ISP media suited for maximum bromide) (MTT) cell viability assay, caspase pigment yield was identified by growing the activity assay, lactate dehydrogenase (LDH) organism in all the seven ISP media (Shirling and activity assay, DNA fragmentation assay, flow Gottlieb 1966). The pigment was extracted by cytometry and trypan blue cell counting assay. solvent extraction as previously reported MTT assay was performed with HeLa, Hep G2 (Selvameenal et al. 2006) with minor and human lymphocytes. The cells were seeded in modifications. Instead of wheat flakes, 10 gm of 96-well flat-bottom microtiter plates and incubated oat flakes were used as substrate in the solid state in a CO incubator at 37°C with 5% CO and 95% fermentation process. Ethyl acetate and methanol 2 2 air overnight for cell adhesion. Different were used for pigment extraction from concentrations of the pigment (2.5, 5, 10 and 20 Streptomyces griseoaurantiacus JUACT 01 µg /mL) were added in quadruplicates and cultured under submerged fermentation. The incubated for 24, 48 and 72 h. Following biomass was homogenised using the solvents and incubation, 100 μL of MTT solution was added. filtered. The filtrate containing the pigment was The culture was then incubated for 3 h in a dark. Braz. Arch. Biol. Technol. v.58 n.6: pp. 869-876, Nov/Dec 2015 Cytotoxic yellow pigment from Streptomyces griseoaurantiacus JUACT 01 871 After the incubation period the supernatant was aspirated and 100 μL of DMSO was added to Fluorescence microscopic analysis dissolve the formazan. The absorbance was Cells morphology is one of the indicative factors recorded at 540 nm with the help of an ELISA to assign the healthy status of the cell. The cells plate reader (Mosmann 1983). The percentage treated with pigment for 24 h were harvested by viability was calculated using the following centrifugation and stained with AO/EB dye (each formula: 100 mg/L in PBS). The cells were examined and photographed using a fluorescence microscope. Percentage viability (%) = (A540 of the test sample) / (A540 of the control) × 100 The effect of pigment on normal cells was tested Flow cytometry by treating the yellow pigment on healthy The effect of pigment fraction on cell cycle was lymphocytes. Lymphocyte isolation was determined by Flow cytometry with PI Staining performed based on the method described by (Pozarowski et al. 1995).The treated HeLa cells Nadumane et al. (2013) and cytotoxicity was were harvested by trypsinisation and tested by MTT assay as mentioned above. centrifugation. Required quantity of PBS was added to the cell pellet to obtain a final DNA fragmentation analysis concentration of 1 - 2×106 cells /ml. The cells were For analysing the DNA fragmentation in the fixed in chilled 70% ethanol at 4°C overnight. treated
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