INTRODUCTION:

Biopigments are substances produced by living organisms that absorb light in the wavelength range of the visible region. Biological pigments include those produced by plants, animals, fungi and microorganisms (Delgado-Vargas et.al., 2000). Biopigments produced from microbes are preferred over those from plants because of their availability; stability; cost efficiency; labor; yield and easy downstream processing (Joshi et.al., 2003).

Among microbes, bacteria have immense potential to produce diverse bioproducts. Bacterial pigment production is now one of the emerging fields of research to demonstrate its potential for various industrial applications. Varieties of bio pigments have been produced such as carotenoids, melanins, flavins, quinines, monascins, violancein using microorganisms (Duffose et.al., 2006).

The various microorganisms such as Micrococcus, Bacillus, Rhodotorula, Monascus, Phaffia, Sarcina and Achromobacter have the capability to produce different pigments. These colors have numerous beneficial properties like anti-cancerous, immunosuppressive, antibiotic, anti-proliferative, bio-degradability etc. Further, they have broad area of application mainly in food, dairy, printing, textile and pharmaceutical industries etc (Heer et.al.,2017).

Biopigments produced from microorganisms can be an alternative for synthetic compounds in food and pharmaceutical technology so as to develop new drugs in order to treat various pathological disorders including cancer (Cragg et.al., 1999). Cancer is a deadly disease known as the second leading cause of death after cardiovascular diseases, in which cervical cancer is one of the leading cancers affecting women population. (Rebecca et.al., 2018, Kumar Naina, 2016)

Treatment of cervical cancer depends on many factors including the stage of cancer diagnose. Research is ongoing not only to improve treatment but also to improve methods of treating pre-cancer and detecting it in early treatable stages.

With this regard the present study focus on the isolation of a wild strain yellow pigment producing bacterium, extraction of pigment from it and evaluation of its applicability as an anticancer and apoptotic drug.

MATERIALS AND METHODS:

Materials:

HeLa, Cervical Cancer Cell lines were procured from NCCS, Pune and were maintained in Dulbecco’s Modified Eagles Medium (DMEM, HiMedia) supplemented with 10% Foetal Bovine Serum (FBS, Invitrogen, USA) and grown under humidified conditions in a CO₂ incubator (NBS, Eppendorf, Germany) with a supplementation of 5% CO₂ at 37˚C.

METHODS:

Dairy products collected from local areas of Trivandrum were used for the isolation of pigment producing bacteria. Serially diluted milk (10^-5) samples were swabbed on Nutrient agar medium and were incubated at 37˚C for 24-48 hrs of incubation. The plates were observed for characteristic colony formation and were then sub-cultured to obtain pure colonies.

The isolated colonies were further characterized using Gram staining and other biochemical characterization including IMViC Test, Urease Test, Starch hydrolysis Test and Gelatin Hydrolysis Test (Bregey’s Manual of Determinative Bacteriology, 1994).

The isolated bacteria were then cultured in Nutrient Broth for 72 hrs to enhance the production of pigment. After incubation the culture was centrifuged and pellet was collected, 4 ml chloroform was mixed with pellet. Upper layer was pipetted out into a petriplate and dried.

The pigment was then checked for determining its anticancer activity against cervical cancer using HeLa cell lines as in vitro model. Cell lines were grown in Dulbecco’s modified eagle medium (DMEM) supplemented with FBS, Penicillin and Streptomycin. The cells were sub cultured after trypsination with 0.25% in 0.5 mm EDTA and were cultured in an atmosphere containing 5% CO2 and 90% humidity at 37˚C.

The cells were allowed to grow to a confluency of 80% and samples were added in different concentration (6.25µg, 12.5µg, 50µg and 100µg). Untreated cells were kept as control and all the cells were allowed to incubate in a CO₂ incubator with a supplementation of 5% CO₂ at 37˚C for 24hrs.

MTT cell viability assay was further done in the cells after incubation. Cells were washed with PBS and were added with MTT (0.5mg/ml). Incubated for 3hrs at 37°C, until intracellular purple formazan crystals were visible under microscope. MTT was then removed and DMSO was added. Again incubated at room temperature for 30 minutes, until cells have lysed and purple crystals have dissolved and absorbance at 540 nm was measured and was expressed in terms of percentage viability (Mosmann, 1983).

The apoptotic ability of isolated pigment was then checked in the treated cells using Acridine Orange and Ethidium Bromide double staining method. After treatment with 100 µg/ml of sample per 24 hrs, the cells were washed by cold PBS and then stained with mixture of AO and EtBr at room temperature for 10 mins. The stained cells were washed twice with 1X PBS and observed by a fluorescent microscope (Olympus CK X 41 with Optica Pro5 CCD Camera) in blue filter. The cells can be divided into four categories accordingly as; Living cells, Early apoptotic, late apoptotic and necrotic cells (Wu et.al., 2015).

RESULT:

The wild strains of yellow pigment producing bacteria were isolated from milk samples and were sub-cultured on Nutrient agar medium. The organism was characterized using Gram staining. The microscopic examination revealed the organism as Gram Negative Cocci, under 40X.

The organism was further characterized using several biochemical analysis including IMViC Test, Urease Test, Starch hydrolysis Test and Gelatin Hydrolysis Test. The results are tabulated as follows;

Table 1: Biochemical Characterization of Isolated Organism

SL NO	TEST	OBSERVATION	INFERENCE
1	Indole test	No formation of red color ring	Negative
2	Methylred test Voges-proskauer test	Formation of red color ring No formation of red color ring	Positive Negative
3	Citrate test	Appearance of royal blue	Positive
4	Urease test	Appearance of pink color formation	Positive
5	Starch test	No appearance of clear zone	Negative
6	Gelatin hydrolysis	Appearance of white zone	Positive

The isolated bacteria was sub-cultured for pigment production and the pigment was extracted using Chloroform

The pigment was then checked for its anticancer potential. The normal dosages of yellow pigment on the cells were demonstrated by incubating cells with different concentration of white pigment.

Fig 4 Phase contrast images of HeLa cell lines treated with (A) 6.25 µg, (B) 12.5 µg, (C) 25 µg, (D) 50 µg, (E) 100 µg, (F) Control cells.

In the study, it was observed that as the concentration increased percentage of viability decreased from 86% (6.25µg) to 44% (100µg), with a LD 50 value of 81.838µg/ml (ED50 Plus V 1.0 software).

Table 2: MTT Cell Viability Assay

CONCENTRATION(µg)	OD at 540nm	% VIABILITY
6.25	0.3707	86.04
12.5	0.3640	84.49
25	0.3409	79.13
50	0.2431	56.42
100	0.1931	44.82
CONTROL	0.4308	100

Fig 5 Percentage Viability of Hela cells treated with different concentrations of pigment.

The apoptotic effect of pigment was determined by using EtBr/AO staining in which apoptotic and live cells were differentially stained. Live cells were viewed with green fluorescent nuclei due to uptake of acridine orange whereas apoptotic cells with compromised membrane integrity showed red fluorescence, under fluorescence microscopy (Olympus CK X 41 with Optica Pro5 CCD Camera). The control cells were observed to have green fluorescence and the treated group had cells emitting red fluorescence indicating the apoptotic effect of isolated pigment.

Fig 6 Fluorescent microscopic images of HeLa cell lines stained with EtBr/AO of, (A) Control cells, (B) treated with 40µg, (C) treated with 80 µg.

SUMMARY AND CONCLUSION:

Bioprofiling of bacterial pigments for different therapeutics is an area of recent research interest .The present study attempts to validate the anti cancer potential of Microbial pigment using HeLa cells. The organisms were already isolated from dairy products and yellow colour pigment producing bacteria was used for pigment extraction. Biochemical identification studies has confirmed the organism to be gram negative Cocci with distinct yellow colour. The presence of yellow pigment producing bacteria is previously reported by workers such as Indra et al, 2014. Further studies are warranted to identify the organism which needs to be studied in detail.

We used in vitro cultured cells HeLa cells to determine the anti cancer activity of pigments. MTT assay is one of the most widely used methods for measuring the cell viability and our results depict dose dependent decrease in cell viability with increase in concentration. Induction of apoptosis is critical in anti cancer activity of compounds and the Live dead assay ascertained the apoptotic activity of pigments. The presence study envisages the anticancer potential of bacterial pigments isolated from dairy products which can find applications in therapeutic regimens.

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Received on 08.08.2018 Accepted on 28.09.2018

Asian J. Pharm. Tech. 2018; 8 (4):244-247.

DOI: 10.5958/2231-5713.2018.00037.5