|Year : 2018 | Volume
| Issue : 1 | Page : 31-33
Investigation of in vitro cytotoxic activity of pigment extracted from Salinicoccus sp. isolated from Nellore sea coast
V Srilekha1, G Krishna2, P Mahender3, MA Singara Charya2
1 Department of Biotechnology, Chaitanya Degree and PG College (Autonomous), Warangal, Telangana, India
2 Department of Microbiology, Kakatiya University, Warangal, Telangana, India
3 Department of Biotechnology, Kakatiya University, Warangal, Telangana, India
|Date of Web Publication||9-Jul-2018|
Department of Biotechnology, Chaitanya Degree and PG College, Warangal - 506 001, Telangana
Source of Support: None, Conflict of Interest: None
Objective: To investigate the in vitro cytotoxic activity of pigment extracted from Salinicoccus sp. isolated from Nellore sea coast. Materials and Methods: In the present study, pigment-forming bacteria were isolated from samples collected from Nellore coast, Andhra Pradesh. Among different pigmented isolates obtained on Zobell agar medium, the pinkish orange bacterium was selected for the study. The bacterium was cultured in Zobell broth medium and incubated in an orbital shaker at 120 rpm for 6 days at 25°C. After incubation, the culture broth was centrifuged at 10,000 rpm for 10 min to obtain a pellet, and the pellet was extracted using the solvent methanol and acetone (5:1). The crude pigment extract was evaluated for cytotoxic potential and was found to exhibit cytotoxic effect on A549 (human lung cancer) and MCF-7 (breast cancer) cell lines. The cell lines A549 cells, MCF-7, were cultured in Dulbecco's Modified of Eagle medium with L-glutamine and 1000 mg/L glucose supplemented with 10% fetal bovine serum, penicillin G (100 units/ml), and streptomycin sulphate (0.1 mg/ml) in a humidified atmosphere consisting of 5% CO2at 37°C. Results: The results of the present study revealed that the crude pigment extract has a strong anticancer potential, especially toward the A549 (Lung cancer cell lines) and MCF-7 (Breast cancer cell lines), respectively. Conclusion: This study clearly indicated that the pigment extract of marine Salinicoccus sp. has a strong cytotoxic activity against A549 (lung) and MCF-7 (breast) cancer cell lines which may be utilized for the drug development.
Keywords: Anticancer, cytotoxic activity, orange pigment, Salinicoccus sp.
|How to cite this article:|
Srilekha V, Krishna G, Mahender P, Singara Charya M A. Investigation of in vitro cytotoxic activity of pigment extracted from Salinicoccus sp. isolated from Nellore sea coast. J Mar Med Soc 2018;20:31-3
|How to cite this URL:|
Srilekha V, Krishna G, Mahender P, Singara Charya M A. Investigation of in vitro cytotoxic activity of pigment extracted from Salinicoccus sp. isolated from Nellore sea coast. J Mar Med Soc [serial online] 2018 [cited 2018 Dec 14];20:31-3. Available from: http://www.marinemedicalsociety.in/text.asp?2018/20/1/31/236256
| Introduction|| |
Marine microorganisms are of extensive interest as a new and promising source of biologically active compounds. For this reason, great research efforts have been made in the past decade to search for these microorganisms in the marine environment, and the results have been very productive.,,,, Marine microorganisms live in a biologically competitive environment with unique conditions of salinity, pressure, temperature, light, oxygen, pH, and nutrients. For the survival and defense mechanisms, microbes produce unique secondary metabolites. Even though these biologically active compounds are produced in response to stress, many have shown value in pharmaceutical and biotechnological applications.
Cancer is considered as one of the most severe and major health problems in both developing and developed countries worldwide, and the identification of novel targets and the development of more specific chemotherapeutic agents are the most important goals of research in cancer therapy. Therefore, there is a constant demand to develop new, effective, and affordable anticancer drugs. In this regard, natural product extracts continue to be the most promising source of new drugs for cancer.
The genus Salinicoccus in the family Staphylococcaceae is defined as moderately halophilic, aerobic, Gram-positive, nonmotile, nonsporulating, and heterotrophic cocci. A moderately halophilic Salinicoccus sp. ns1 was isolated from sea anemone Anthopleura sp and was reported to produce a carotenoid pigment. An orange color pigment was reported from Salinicoccus sp. M KJ997975 isolated from forest soil of Tungareshwar, Vasai, Thane-Maharashtra, India. The marine Salinicoccus roseus was isolated from saltpan region of Mukkani, Tuticorin District, and the extracted pigment from pellet was characterized to be lycopene and was shown to exhibit antioxidant, anticancer, and antidiabetic activity. Keeping this in view, the present study was an attempt to extract pigment from marine bacteria collected from the coastal areas of Nellore, Andhra Pradesh, India and evaluate its cytotoxic potential against two different human cell lines.
| Materials and Methods|| |
Sample collection and isolation of bacteria
The marine sea water samples were collected from the coastal areas of Nellore, Andhra Pradesh, India. Samples were collected in sterile containers and transferred to the laboratory immediately. The collected samples were spread over surface of plates containing Zobell marine agar medium and incubated at 25°C for 2 days. The colonies showing pigment production were selected and purified. Stock cultures were maintained on Zobell marine agar slants and stored at 4°C in refrigerator. The pinkish orange-pigmented marine bacterium was selected for further study.
Preparation of bacterial culture and crude extracts
The selected marine bacterium was cultured in 300 ml of Zobell Marine Broth in 500 ml Erlenmeyer flasks for the production of pigment. Flasks were incubated on a rotary shaker at 25°C at 120 rpm for 6 days. After 6 days, the broth was centrifuged at 10,000 rpm at 4°C for 10 min to remove the cells, and the cell pellet was extracted with suitable solvent. Different solvents were tested to standardize the best solvent for extraction of the pigment. Among various solvents tested, the methanol and acetone in the ratio of 5:1 gave best extraction of the pigment. The pigment was extracted from cell pellet using methanol and acetone in 5:1 ratio, and the extracted pinkish orange pigment was filtered by Whatman no. 1 filter paper. The filtered crude pigment extract was concentrated by evaporation and was used for anticancer assay.
Cell lines and culture
The present work was carried out on A549 and MC-7 (human lung and breast cancer cell lines, respectively). The cell lines were obtained from National Centre for Cell Science, Pune, Maharashtra, India. The cell lines were cultured in Dulbecco's Modified Eagle's medium with L glutamine and 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% CO2 at 37°C.
Cytotoxicity determination by 3-(4, 5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay
The effect of crude compound on cell viability was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay. A549 and MC-7 cells were seeded at a concentration of 5 × 105 cells/well in 96-well plates and incubated at 37°C in 5% CO2 for 24 h. Once the cells reached 70% confluency, media with different concentrations of compound (1, 10, 25, 50, 75, 100, 500,1000 μM) were added and incubated at 37°C in 5% CO2 for 24 and 48 h. The samples also included a “blank” (medium alone) and “control” (DMSO alone). After 24 and 48 h, the absorbance recorded at 570 nm using microtiter plate reader. The percentage viability was calculated as follows:
Cell Viability = Optical density of samples/Optical density of control × 100
| Results and Discussion|| |
Marine environment covers two-thirds of the planet's surface and has recently become an attractive source for the identification of novel biologically active metabolites due to its rich biodiversity. Marine bacteria are considered highly valuable as they produce various antibiotics and other therapeutically useful compounds with diverse biological activities. Microorganisms are known to produce a variety of pigments. Recent studies have shown that microorganisms are a promising source for natural colors. Marine bacteria are one of the prolific biological sources for the natural pigments. Biopigments are also known to possess antimicrobial and antitumor activity. In the present study, the pinkish orange bacterium was selected for the study from the various distinct colored bacteria isolated from sea water samples collected from different areas along the sea coast of Nellore (Mypadu and Kotha koduru beach area). Identification of selected bacterial strain was done by studying morphological, microscopic as well as biochemical characters of the organism.
Cancer still represents one of the most serious human health problems despite the great progress in understanding its biology and pharmacology. Although numerous advances have been made in prevention, diagnosis, and treatment of the disease, it still continues to torment mankind. In the present study, the cytotoxic potential of pinkish orange pigment extract from Salinicoccus sp. isolated from sea water was investigated. The inhibitory effect of pigment treated cells was observed after 24 h of incubation. The maximum cell growth inhibition (80.45%) was observed at a concentration of 125 μg/ml for A549 cells, and the pigment extract caused 85.36% inhibition of (MCF-7) cell line at a concentration of 125 μg/ml. No cytotoxicity was detected against standard untreated cell. Many of the microbial pigments not only act as coloring agents in various food processing and cosmetics industry but also possess anticancer, antioxidant, anti-inflammatory, and antimicrobial activities. The cytotoxic activity of crude pigment extract was tested against human lung cancer cell line (A549) and breast cancer cell line (MCF-7) using MTT assay. Decrease in proliferation of pigment extract treated cells was observed when compared to the untreated control. The role of microbial pigments to induce apoptosis and cell cycle inhibition has been reported by many studies., It is inferred from the present study that the crude pigment extract of marine Salinicoccus sp. showed a significant and potential anticancer activity against A549 and MCF-7 cell lines.
| Conclusion|| |
In the current scenario, there is a lot of interest in the scientific community around the world in exploiting novel and effective anticancer drugs to treat cancer. The increasing incidence of cancer prompted researchers to investigate the efficacy of natural products in the treatment of cancer and search for cheaper, safer, and potent medicines to challenge the dreadful human disease. Owing to a diverse chemical ecology, marine-derived bacteria appear to have immense potential as a source of anticancer compounds and are worthy of further exploration as novel anticancer drugs and deserve an extensive investigation. In this context, the present study was an initiative approach toward the use of the pigment produced from Salinicoccus sp. for the development of anticancer drugs. The crude pigment was found to exhibit inhibitory action against the growth of human cancer cell lines. Further, the pigment obtained in this study has to be characterized and would be tested for its applications in diverse fields as an alternative for synthetic chemicals.
Financial support and sponsorship
The financial support by way of the grant provided by University Grants Commission-South Eastern Regional Office, Hyderabad (MRP-6073/15) was greatly acknowledged by the authors.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Faulkner DJ. Marine natural products. Nat Prod Rep 2002;19:1-48.
Mincer TJ, Jensen PR, Kauffman CA, Fenical W. Widespread and persistent populations of a major new marine actinomycete taxon in ocean sediments. Appl Environ Microbiol 2002;68:5005-11.
Feling RH, Buchanan GO, Mincer TJ, Kauffman CA, Jensen PR, Fenical W, et al.
Salinosporamide A: A highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus Salinospora
. Angew Chem Int Ed Engl 2003;42:355-7.
Maldonado LA, Stach JE, Pathom-aree W, Ward AC, Bull AT, Goodfellow M, et al.
Diversity of cultivable actinobacteria in geographically widespread marine sediments. Antonie Van Leeuwenhoek 2005;87:11-8.
Jensen PR, Mafnas C. Biogeography of the marine actinomycete Salinispora
. Environ Microbiol 2006;8:1881-8.
Jensen PR, Williams PG, Oh DC, Zeigler L, Fenical W. Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora
. Appl Environ Microbiol 2007;73:1146-52.
Wenzel SC, Müller R. Recent developments towards the heterologous expression of complex bacterial natural product biosynthetic pathways. Curr Opin Biotechnol 2005;16:594-606.
Pandey R, Chander R, Sainis KB. Prodigiosins as anti cancer agents: Living upto their name. Curr Pharm Des 2009;15:732-41.
Coseri S. Natural products and their analogues as efficient anticancer drugs. Mini Rev Med Chem 2009;9:560-71.
El-Gendy MM, Shaaban M, El-Bondkly AM, Shaaban KA. Bioactive benzopyrone derivatives from new recombinant fusant of marine Streptomyces
. Appl Biochem Biotechnol 2008;150:85-96.
Ventosa AM, Ruizberraquero MC, Kocur FM. Salinicoccus roseus
gen. nov, sp. nov, a new moderately halophilic Gram-positive coccus. Syst Appl Microbiol 1990;13:29-33.
Narmatha K, Thangaviji V, Velmurugan S, Donio MT, Jenifer JS, Babu MM, et al
. Carotenoid producing Salinicoccus
sp. ns1: A moderately halophilic bacterium isolated from sea anemone Anthopleura
sp. Blue Biotechnol J 2012;4:481.
Bhat MR, Marar T. Media optimization, extraction and partial characterization of an orange pigment from Salinococcus
sp. MKJ 997975. Int J Life Sci Biotechnol Pharm Res 2015;4:85-9.
Ramanathan G, Ramalakshmi P. Studies on efficacy of marine bacterium Salinicoccus roseus
pigment for their bioactive potential. Eur J Biomed Pharm Sci 2017;4:330-4.
Malik K, Tokas J, Anand RC. Characterization and cytotoxicity assay of pigment producing microbes. Int J Curr Microbiol Appl Sci 2016;5:370-6.
Sasidharan P, Raja R, Karthik C, Sharma R, Indra Arulselvi P. Isolation and characterization of yellow pigment producing Exiguobacterium
sps. J Biochem Technol 2013;4:632-5.
Ramesh S, Mathivanan N. Screening of marine actinomycetes isolated from the Bay of Bengal, India for antimicrobial activity and industrial enzymes. World J Microbiol Biotechnol 2009;25:2103-11.
Vishnu TS, Palaniswamy M. Isolation and identification of Chromobacterium
sp. from different ecosystems. Asian J Pharm Clin Res 2016;9:253-7.
Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70.
Venil CK, Lakshmanaperumalsamy P. An insightful overview on microbial pigment, prodigiosin. Elect J Biol 2009;5:49-61.
Montaner B, Navarro S, Piqué M, Vilaseca M, Martinell M, Giralt E, et al.
Prodigiosin from the supernatant of serratia marcescens induces apoptosis in haematopoietic cancer cell lines. Br J Pharmacol 2000;131:585-93.
Pandey R, Chander R, Sainis KB. Prodigiosins: A novel family of immunosuppressants with anti-cancer activity. Indian J Biochem Biophys 2007;44:295-302.