A Novel Red Pigment from Marine Arthrobacter Sp. G20 with Specific Anticancer Activity

Journal of Applied Microbiology ISSN 1364-5072 ORIGINAL ARTICLE A novel red pigment from marine Arthrobacter sp. G20 with specific anticancer activity S. Afra1, A. Makhdoumi1 , M. M. Matin1,2 and J. Feizy3 1 Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran 2 Cell and Molecular Biotechnology Research Group, Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran 3 Research Institute of Food Science and Technology, Mashhad, Iran Keywords Abstract anticancer, antioxidant, Arthrobacter, carotenoids, marine, pigment. Aims: Bacterial pigments are promising compounds in the prevention and treatment of various cancers. In the current study, the antioxidant, cytotoxic Correspondence and antimicrobial effects of a red pigment obtained from a marine bacterial Ali Makhdoumi, Department of Biology, strain were investigated. Faculty of Science, Ferdowsi University of Methods and Results: Optimization of the pigment production by the marine Mashhad, Mashhad, Iran. strain was conducted using the one-factor-at-a-time approach. Chemical E-mail: [email protected] identification of the pigment was achieved by UV-visible, FTIR and HPLC 2017/1213: received 22 June 2017, revised analyses. The biological activities of the pigment were evaluated by DPPH, 15 August 2017 and accepted 25 August MTT and microbroth dilution assays. The strain was identified as Arthrobacter, 2017 and its pigment was related to carotenoids. The EC50 antioxidant activity of À the pigment was evaluated as 4Á5mgml 1. It showed moderate anticancer doi:10.1111/jam.13576 effects on an oesophageal cancer cell line, KYSE30, while no inhibition was observed on normal HDF (human dermal fibroblasts) cells. The pigment had no antibacterial effects on the four tested strains. Conclusion: The antitumour activity of a carotenoid-related pigment from Arthrobacter sp. was reported for the first time. Significance and Impact of the Study: Marine environments are interesting sources for the identification of novel bioproducts. The identification of carotenoid pigments from marine bacteria with remarkable antioxidant and anticancer activities would result in better insights into the potential pharmaceutical applications of carotenoids and marine environments. used in chemotherapy, but they usually have severe side Introduction effects as they affect both cancerous and normal cells. Cancer is a group of debilitating diseases in which cells Therefore, it is necessary to develop anticancerous com- begin to divide and spread into the surrounding tissues is pounds with selective activity against tumour cells an uncontrolled manner. The development of cancer in (Chang et al. 2011). the body is a dynamic and long-term process affected by Pigments with various chemical formulas are colourful factors like chemical pollutants, diets, hormonal and materials as a result of selective visible wavelength many other (unknown) factors (Pan and Ho 2008). The absorption. The main sources of biological pigments are increasing industrial applications of carcinogenic com- plants and flowers. They have been shown to possess var- pounds, along with changes in the lifestyle, and extended ious biological effects like antibacterial, anticancerous and life expectancy have resulted in the recognition of cancer antioxidant activity. Flavonoids and quinones obtained as a big threat to public health, and it accounts as one of from citrus and vegetables are some examples of plant the leading causes of death all over the world. Che- pigments with antitumour activity (Dai and Mumper motherapy is one of the useful methods for the clinical 2010). However, there is a growing interest in bacterial treatment of cancer patients. Some synthetic drugs are pigments in comparison to plant sources because of some 1228 Journal of Applied Microbiology 123, 1228--1236 © 2017 The Society for Applied Microbiology S. Afra et al. Arthrobacter pigment and cancer properties like rapid production regardless of seasonal- became colourless. The methanolic extract was collected geographical conditions and specific pharmaceutical char- and evaporated under reduced pressure below 40°Cina acteristics (Venil et al. 2013). Bacterial pigments have rotary evaporator (Heidolph, Germany), and then it was great applications in food, textile, cosmetic and pharma- freeze-dried (Freeze dryer, Christ Alpha 1Á2 LD Plus, Ger- ceutical industries (Rao et al. 2017). In addition to being many). Thin layer chromatography (TLC) was developed environmental friendly, these pigments can apply as natu- using silica plates (Merck, Darmstadt, Germany). The con- ral colour to foods and have antioxidant and cancer pre- centrated pigment was dissolved in a methanol solution, vention health benefit (Tuli et al. 2015). Prodiginin, a red spotted on a silica gel sheet and developed with a mobile pigment produced by Serratia spp., and violacein, a pur- phase of ethanol: Diethyl ether (1:4 v/v) at room tempera- plish black pigment produced by Chromobacterium spp., ture in the dark. For preliminary chemical characterization, are two well-known bacterial pigments with antibacterial the UV-visible spectrum of the pigment was recorded (Lapenda et al. 2015), antiviral (Cazoto et al. 2011) and between 200 and 800 nm, using methanol as a blank. anticancer (Montaner et al. 2000) properties. High-performance liquid chromatography (HPLC) system Marine environments cover two-thirds of the planet’s (Waters, Milford, MA, USA) consisting of a 1525 binary surface; they are interesting sources for the identification pump and a 2489 UV/vis detector set at 440 nm, was used. of novel bioactive compounds. Mudit and El Sayed Separation was conducted on an InertSustainSwift C18 recently reviewed marine- derived compounds with column (150 mm length, 4Á6-mm internal diameter and 5- potential anticancer activities (Mudit and El Sayed 2016). lm particle size) and the data were analysed with the Several of these entities have bacterial sources (Chang Breeze 2 software (Waters). Linear gradient elution chro- et al. 2011). Aqueous environments are also interesting matography was done using solvent systems A (methanol) habitats for isolating cold adaptive micro-organisms in and B (water) at ambient temperature. The gradient pro- which pigmentation is a common trait for various pro- gram used was as follows: initial, A–B (20:80, v/v), and 0– tection purposes (De Maayer et al. 2014). The aim of the 60 min, linear change to A–B (80:20, v/v) at 1% methanol/ current study was to explore the biological activities of a min gradient speed. The flow rate was kept constant at À red pigment extracted from a psychrophilic bacterial 1mlmin 1, and 20 ll of the methanol-water (50%, v/v) strain isolated from the Caspian Sea. extract of pure pigment was applied. Crocin—a well- known plant carotenoid—was used as the reference pigment. Fourier transform infrared spectroscopy (FTIR) was Materials and methods measured in cuvettes with windows of calcium florid. The transformed spectres were background-corrected for the Isolation and identification of pigmented bacterium solvent (Aust et al. 2003). Samples were obtained from 15 m depth of the Caspian Sea (36Á46°N51Á02°E). They were aseptically pooled and À Enhancing pigment production diluted up to 10 5, and inoculated on the artificial sea À water (ASW) agar medium containing (g l 1): NaCl 10, To increase the pigment production, amounts of different MgSO4 0Á52, FeSO4 0Á01, MgCl2Á6H2O0Á19, (NH4)2SO4, factors including the carbon source (1% w/v of glucose, – ° 0Á98, CaCl2Á2H2O0Á19, KCl, 0Á15, peptone 10, yeast fructose, maltose and starch), temperature (10 40 C), pH extract 5, glucose 2 and agar 15 (Stafsnes et al. 2010). (6–9) and incubation time (0–120 h) were varied one at a The pH was adjusted to 8, and the plates were kept at time in an ASW basal broth medium (20°C, pH 8). Each 20°C for at least 1 month. A red-pigmented bacterium subsequent factor was examined after taking into account designated as strain G20 was obtained in the isolation the previously optimized factor(s). In each experiment, the procedure. The extraction of genomic DNA, PCR ampli- absorbance of methanolic extract was measured at 490 nm, fication of 16S rRNA and phylogenetic analysis were car- and the bacterial growth was determined by the measure- ried out as described later (Makhdoumi et al. 2015). ment of the absorbance at 600 nm (Shimadzu, Japan). Preparation of the pigment extract Antioxidant activity The bacterial cells were grown in the ASW broth medium The antioxidant capacity of the pigment was studied fol- in a 1000-ml Erlenmeyer flask and kept in a shaker incu- lowing a method described by Blois (1958). Briefly, two- bator (150 rpm and 20°C) until the culture medium fold serial dilutions of the pigment prepared in dimethyl turned red. The bacterial cells were harvested following sulfoxide were added to a 0Á1 mM DPPH (1,1-diphenyl- centrifugation at 4000 rpm for 20 min. The cell pellets 2-picrylhydrazyl) (Sigma, USA) methanolic solution, and were extracted with 95% (v/v) methanol until the pellets then left in darkness at 30°C for 30 min. The absorbance Journal of Applied Microbiology 123, 1228--1236 © 2017 The Society for Applied Microbiology 1229 Arthrobacter pigment and cancer S. Afra et al. of the resulting solutions was measured colorimetrically aeruginosa (PTCC1074) and Escherichia coli (PTCC 1330) at 517 nm. A sample containing only the solvent and the were selected as reference strains. Microbial growth was DPPH without the pigment was used as a control. The determined by measuring the turbidity at 600 nm. scavenging effect on the DPPH was determined as follows: Data analysis DPPH scavenging effect ð%Þ¼½ðA0 À A1Þ=A0Â100 All quantitative experiments were repeated at least three where A0 was the absorbance of the control reaction and times. The data analysis was conducted using GraphPad A1 was the absorbance in the presence of the pigment Prism version 6Á0 and the IC50 and EC50 values were extract. obtained from nonlinear regression (Mosaddik et al. 2004; Deng et al. 2013).

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