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Int J Med Res. 2011; 1(4):198-203 ISSN: 0976-8971 IJMBR

International Journal of Medicobiological Research

(An International peer review journal)

Journal homepage: ijmedres.com

Research article

Cytotoxic activity of partially purified 7-demethoxy rapamycin derived from Streptomyces hygroscopicus BDUS 49

S. Parthasarathi1, S. Sathya2, R. Durai Samy3, Ram Mohan1, G. Selva Kumar 1, M. Manikandan4, T. Manikandan1 M. Ramesh5 and K. Balakrishnan1

1Department of Biotechnology, Bharathidasan University, Tiruchirappalli-620 024., Tamil Nadu, India. 2Department of Bionanotechnology, Kyungwon University, Gyeonggi-Do, South Korea - 461 701. 3Department of Biotechnology, Vysya College of Arts and Science, Salem-636 103. Tamil Nadu, India. 4Sri Krishna Arts and Science college, Kuniamuthur, Coimbatore - 641 008. Tamil Nadu, India. 5 SRM University, College of nursing, Chennai – 603 203, Tamil nadu, India.

Corresponding author: [email protected], [email protected]

Article information ABSTRACT Keywords: FTC-133, 7-Demethoxy rapamycin, Immunofluorescence staining, MTT assay, Western blotting

Received on: 23.09.2011 Revised on: 30.09.2011 Accepted on: 05.10.2011

Marine actinomycetes are often taxonomically unique, which makes them interesting as potential sources of new drug leads. One of the major areas of research on marine natural products is devoted to the discovery of new anticancer drugs. Ours earlier study reported the extraction of partially purified bioactive substance 7-demethoxy rapamycin from the culture supernatant of Streptomyces hygroscopicus BDUS 49 isolated from Bigeum Island, South West Coast of South Korea. The present study investigated the cytotoxic activity of 7-demethoxy rapamycin compound on FTC- 133 cancer cell line. The cytotoxic activity results were confirmed by immunoflourescence staining, MTT assay and Western blotting studies related with tumour suppressor gene (p53) expression. To exploit these findings for human welfare, it is necessary to carry out clinical trials for further confirmation.

1. INTRODUCTION

Cancer still represents one of the most serious human health problems despite the great progress in understanding its biology and pharmacology. The usual therapeutic methods for cancer treatment are surgery, radiotherapy, immunotherapy and chemotherapy.[1] These techniques are individually useful in particular situations and when combined, they offer a more efficient treatment for tumors. An analysis of the number of chemotherapeutic drugs and their sources indicates that over 60% of approved drugs are derived from natural compounds,[2]

[3] and many have been extracted from actinomycetes.[4] Actinomycetes are an important source of new bioactive compounds such as antibiotics and enzymes,[5,6,7,8] which have diverse clinical effects and are active against many kinds of organisms (bacteria, fungi, parasites etc.,). In fact more than 50% of the known natural antibiotics produced are from actinomycetes.[9] Antitumor antibiotics produced by actinomycetes are among the most important cancer chemotherapeutic agents including members of the anthracycline, bleomycin, actinomycin, mitomycin and aureolic

acid families.[10] In screening for actinomycetes able to produce bioactive compounds, the exploration of new soils and habitats has been recommended.[11,12] Among the actinomycetes, Streptomycetes, Gram (+) filamentous bacteria, are widely distributed in a variety of natural and man-made environments and constitute a significant component of the microbial population in most soil and marine habitats.[13]

In our studies, Follicular Thyroid Carcinoma (FTC-133) has been studied because of their malignant (aggressive) activity than papillary carcinoma. It occurs in a slightly older age group than papillary and is less common in children. In contrast to papillary cancer, it occurs only rarely after radiation therapy. Mortality is related to the degree of vascular invasion. Age is a very important factor in terms of prognosis. Patients over 40 have a more aggressive disease and typically, the tumor does not concentrate iodine as well as in younger patients. FTC originates in follicular cells and is the second most common cancer of the thyroid, after papillary carcinoma. Follicular and papillary thyroid cancers are considered to be differentiated thyroid cancers; together they make up 95% of thyroid cancer

K. Balakrishnan, et al.,/Int J Med Res. 2011; 1(4):198-203 199


cases.[14] Vascular invasion is characteristic for follicular carcinoma and therefore distant metastasis is more common. Managing follicular thyroid cancer is challenging, as no prospective randomized trials exist. The successful studies on novel compounds derived from marine microbial resources, their activities and the unique structures that were previously described raised our interest in exploring and isolating bioactive chemicals from such habitats.

There are very few reports on antimicrobial activity and

cytotoxic activity from marine microorganisms from unpolluted Bigeum Island, South West Coast of South Korea. In Bio prospecting point of view, Bigeum Island marine soil samples possess neutral to alkaline with pH ranging from 6.33 to 7.94 and CaCO3 concentration in soil samples ranged from 1,200 to 9,600 ppm. Syed et al.,[15,16,17] isolated few novel microorganisms namely, Nocardioides islandiensis sp. nov., Frigoribacterium mesophilum sp. nov., and Nocardioides halotolerans sp. nov., have been isolated from the Bigeum Island sample. Thus, it is crucial that new groups of marine microorganisms from hitherto unexplored habitats be considered as sources of novel bioactive secondary metabolites. Hence, the present investigation deals with the screening and characterization of producers of antimicrobial substances by marine microorganisms from the seawaters of Bigeum Island, South West Coast of South Korea.

Keeping the above point, the present work investigating on

the cytotoxic activity of partially purified compound 7-demethoxy rapamycin derived from Streptomyces hygroscopicus BDUS 49 isolated from Bigeum Island, South West Coast of South Korea. The cytotoxic activity were further confirmed by immunofluorescence staining, MTT assay and Western blot analysis on p53 tumour suppressor gene for the Follicular Thyroid carcinoma (FTC-133) tumor cell line.

2. MATERIALS AND METHODS 2.1. Screening, characterization and extraction of antimicrobial metabolite The marine soil sediment samples collected at the depth of 20 cm, 20 meter near the sea side in Bigeum island, South west in Korea / grid 34º 45' 02.23'' N 125º 53' 42.32''. Samples were kindly provided by Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea. The collected soil sediment samples were placed in sterile polyethylene bags, closed tightly and stored in the refrigerator at 4ºC until use. For the isolation of actinomycetes, soil sample were first mixed, suspended in sterile distilled water (1 g in 100 ml) homogenized by vortexing and finally treated 5–10 min by sonication according to Ouhdouch methodology.[18] The treated samples were serially diluted up to 10-6 and spread (0.1 ml) over the surface of starch casein agar (SCA, pH 7.2 ) medium supplemented with 25 µg/ml cycloheximide to inhibit fungal growth and 10 µg /ml nalidixic acid to inhibit the bacteria capable of swarming, without affecting the growth of actinomycetes. All of the inoculated plates were incubated at 30°C for 7–10 days.

The biochemical, physiological and molecular characterization of the selected antagonistic actinomycete was already being confirmed as Streptomyces hygroscopicus BDUS 49[19] and its 16S rRNA has been deposited in the gene bank under the accession number of GU195049.[20] Antibacterial compound was recovered from the filtrate by specific solvent extraction like ethyl acetate. Ethyl acetate was added to the filtrate in the ratio 1:1 (v/v) and shaken vigorously for 1 hour for complete extraction. The ethyl acetate phase that contains an antibiotic was separated from the aqueous phase. It was evaporated to dryness in a water bath at 80º-90ºC and the residue obtained was weighed. Thus, obtained compound was used to determine the antimicrobial activity. Filter paper disks (6mm in diameter) were impregnated with extracted broth, dried and placed onto BSM plates previously seeded with Bacillus subtilis. The plates were incubated at 37ºC for 48h and examined for zones of inhibition and verified active substance extraction.[21] The absorption spectrum of each active extract was determined in the UV region (200-400nm) by using a Perkin-Elmer Lambda 15 UV/VIS spectrophotometer, FTIR, MS and NMR analysis which were reported in our earlier studies. 2.2. Preparation of Cell line Follicular thyroid carcinoma (FTC-133) cell line was provided by Endocrinology department, Yonsei cancer research center, South Korea. Cells were cultured in DMEM:F12 medium supplemented with L-glutamine, 10% fetal calf serum (FBS), Thyroid stimulating hormone (TSH) 10mU/mL, 100 U/mL Penicillin and 100 µg/mL Streptomycin and Insulin 10 µg/mL, in a humidified incubator with 5% CO2. All the cells were passaged twice weekly and routinely examined for microbial contamination. Cells in logarithmic growth phase were used for further experiments. 2.3. Immunofluorescence staining (IF) For indirect IF, cell line (FTC-133) was fixed in 5% ice cold ethanol for 10 min at 37ºC, permealized with ice-cold Triton buffer (1% Triton-X-100) for 5 min on ice, blocked with 5% goat serum in PBS for 30 minutes on ice and incubated with primary antibodies for 2 hr at 37ºC in a moisture chamber. Sufficient washing with PBS was performed to remove unbound antibodies. Secondary antibodies labelled with FITC or biotynylated Streptavidin were incubated for 40 minutes at room dark condition and washed as with primary antibodies.[22] Glass cover slips carrying the treated cells were mounted with the Propidium iodide (PI) mounting medium onto glass slides and analyzed under an olympus BX51 fluorescence microscope equipped with a Qimaging Retiga EXi digital camera. The apoptosis related gene expression and their positions were depicted in (Figure. 2.) 2.4 Cell viability assay (MTT assay) The viability of the cells was assessed by MTT assay, which is based on the reduction of MTT (thiazolyl blue tetrazolium bromide; Sigma, St. Louis, Mo),[23] by the mitochondrial dehydrogenase of intact cells to purple formazan product.



Briefly, thyroid cancer carcinoma cell line namely follicular thyroid carcinoma (FTC-133) cells were collected, and 2×105

cells/well was dispensed within 96-well culture plates in 100ml volumes. Then different concentrations of pure compound of Streptomyces hygroscopicus BDUS 49 (0, 10, 20, 30, 40, 50, 60 and 70 µg) were put in different wells. Each of the concentrations of above was regarded as one treated group while there was no rapamycin in the control group. Each of the treated or control group contained 6 parallel wells. Before MTT assay, the cells were first incubated in serum free RPMI for 24 hours. After rapamycin was absorbed completely, culture plates were then maintained in RPMI containing 10% fetal calf serum for 24 hours prior to the addition of tetrazolium reagent. MTT working solution was prepared as follows: 5 mg MTT/mL PBS was sterile by being filtered with o.45µm filter units. Each of the above cultured wells was added 20 µL of MTT working solution and then incubated continuously for 4 h. the water insoluble formazan was formed during incubation and it was solubilized by adding solubilization agent (dimethyl sulfoxide) to each well. Amount of formazan was determined by measuring the absorbance at 540 nm using an ELISA plate reader. All experiments were performed in triplicate and repeated at least three times. Results were shown in (Figure. 3.). 2.5. Partially purified 7-demethoxy rapamycin compound treatment In studies pertaining to long term effects, cell line FTC-133 was seeded in 6 well plates one day before pure compound treatment to achieve appropriate confluency (~80 of both the cells) at the time of treatment. After aspiration of medium, cells were washed with warm PBS (10mM sodium phosphate, pH7.4 and 0.9% NaCl) trice and cultured in serum free H-5 defined media composed of DMEM:f12 media with Insulin (10 µg/mL), transferring (5 µl/mL), somatostatin (10ng/mL), hydrocortisone (0.36ng/mL). Along with serum-deprived medium, Streptomyces hygroscopicus BDUS 49 compound (70µg) treatments were given for 24 h. The compound was dissolved in DMSO. 2.6. Western blotting and antibodies Cultured cells were harvested and lysed for 20 min in cold lysis RIPA buffer (50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphate, Sigma). After centrifugation at 12,000 rpm for 30 min at 4ºC, the supernatant was harvested as the total cellular protein extracts. The protein concentrations were determined using Bradford method. Western blots were carried out according to method developed by Towbin. Aliquots of protein corresponding to 60 µg was mixed with SDS-PAGE sample buffer and heated on hot water bath at 95°C for 5 min. The samples were resolved on a 10% SDS-PAGE.

Proteins were electro transferred to supported PVDF membrane (Milli pore Company, PV H00010-Immobilon –P, PVDF Membrane 0-45, and P15000.Rev 05/03, 03-160) by a semi-dry transferor. The membrane was treated with 5% Skim

milk agar and 0.02% PBS-Tween 20 (milk-PBST) for 1 h at room temperature in order to block the non-specific sites on the membrane. The blots were probed with primary antibodies against p53 gene in milk-PBST, for overnight at 4 °C. The membrane was then washed in PBST three times for 5 min each followed by incubation with suitable secondary antibody (antimouse IgG, santa cruz) conjugated with horseradish peroxidase for 1 h at room temperature. Following three 15 minutes PBST washes, signals were detected using ECL (enhanced chemiluminescence) plus Western blotting detection reagents (Amersham). Antibodies against ß – actin (positive control) and p53 gene were purchased from Santa Cruz, California.[24] The results were shown in (Figure. 4.) 3. RESULTS AND DISCUSSIONS The antimicrobial compound in the ethyl acetate were purified and separated by column and thin layer chromatography. Based on the spectral data, the given partially purified compound was identified as 7-demethoxy rapamycin (Figure. 1.), and their molecular weight and formula is 905.12 (C50H75NO12+NA). The tumour suppressor gene (p53) and other apoptosis associated gene expression patterns were studied by immune fluorescence staining method (Figure. 2.). Figure. 1. Structure of partially purified compound 7, demethoxy rapamycin derived from S. hygroscopicus BDUS 49

N

O

O

O

O

OH

O

OOO

O

HO

OHO

Na

41

12

7

N

O

O

O

OH

O

OOO

O

HO

OHO

Na

41

12

7

-CH3OH

Rapamycin 7-Demethoxyrapamycin

C50H75NO12+NaMol. Wt.: 905.12

C51H79NO13+NaMol. Wt.: 937.16

Figure. 2. Expression pattern of apoptosis related gene expression studied by immune fluorescence staining method

p 21 gene (Nuclear) SIRT2 (Cytoplasm,

Cytoskeleton)



SIRT3 (Cytoplasm)

SIRT7 (Cytoplasm) TERT (Telomere)

BAD (Cytoplasm)

BAX (Membrane, Cytoplasm)

BCL2 (Membrane, Cytoplasm)

COX2 (Cytoplasm)

BCL-XL (Cytoplasm) p53 (Nuclear)

GADD45A (Nuclear)

To investigate the cytotoxicity of rapamycin on Follicular thyroid carcinoma cell lines, FTC-133 cells were treated with various concentrations of rapamycin (0, 10, 20, 30, 40, 50, 60 and 70 µg) for 24 h. As shown in Fig. 5.2, 7-demethoxy rapamycin (over 40 and 80 µg) had significant growth inhibition effects on Follicular thyroid carcinoma cell lines in a dose- and time-dependent manner. Cell viability was decreased remarkably, after the cells were treated with 80 µg of rapamycin for 24 h.

0

10

20

30

40

50

60

1 2 3 4 5 6 77-demethoxy Rapamycin (µg/ml)

Inhi

bita

ry r

ate

(%)

FTC-133

Figure. 3. Cell growth inhibition detected by MTT assy. After cells were treated with different concentrations of pure compound, MTT assay was used to detect cell growth inhibition. The inhibitory rate of rapamycin between 60 & 70 µg/ml is much higher than that of lower concentrations of 7-demethoxy rapamycin (p<0.01).

To clarify the mechanism of 7-demethoxy rapamycin-induced apoptosis in Follicular thyroid carcinoma cells, the expression of p53 gene were detected after the cells were treated with 70 µg of rapamycin for 24 h. The results revealed that the expression of p53 gene was up regulated concurrently after the cells were treated with rapamycin for 24 hour, which indirectly promote the apoptosis effect on the tumor cells (Figure. 3.). The given rapamycin treatment have been increased the amount of p53 gene expression level, which may



initially seem a good way to treat tumors or prevent them from spreading. In unstressed cells, p53 levels are kept low through a continuous degradation of p53. A protein called Mdm2 (also called HDM2 in humans) binds to p53, preventing its action and transports it from the nucleus to the cytosol.

Marker Control Treated (kda) cell line cell line

Figure. 4. Western blot analysis of p53 gene expression by 7-demethoxy rapamycin treated and untreated (control) cancer cell line FTC-133 4. CONCLUSIONS The present investigation clearly reveals the cytotoxic activity of partially purified compound 7-demethoxy rapamycin derived from Streptomyces hygroscopicus BDUS 49. This study also demonstrated that p53 induction by 7-demethoxy rapamycin could significantly suppresses the growth of a human, metastatic follicular thyroid cancer (FTC-133) cell line, which may initially seem a good way to treat tumors or prevent them from spreading, is in actuality not a usable method of treatment, since it can cause premature aging. To exploit these findings as alternative therapy for human welfare, it is necessary to carry out clinical trials and strategies for optimization of large-scale production of both biomass and cytotoxic compounds. Acknowledgements This work was supported by the 21C Frontier Program of Microbial Genomics and Applications from the Korean Ministry of Education, Science & Technology (MEST), and by a grant from KRIBB Research Initiative Program, Korea. I also would like to thank Dr. Chung Woong-Youn and Dr. Haengrang Ryu, Endocrinology department, Yonsei University, South Korea for their valuable support. 5. REFERENCES [1] Cocco, M., Congiu, C. and Onnis, V. (2003). Synthesis

and in vitro antitumoral activity of new N-phenyl-3- pyrrolecarbothioamides. Bioorg. Med. Chem., 11: 495–503.

[2] Cragg, G., Newman, D. and Snader, K. (1997). Natural products in drug discovery and development. J. Nat. Prod., 60: 52–60.

[3] Newman, D.J., Cragg, G.M. and Snader, K.M. (2003). Natural products as sources of new drugs over the period 1981–2002. J. Nat. Prod., 66: 1022–1037.

[4] Mendez, C. and Salas, J.A. (2001). Altering the glycosylation pattern of bioactive compounds. Trends. Biotechnol ., 19(11): 449–456.

[5] Vining, L.C. (1992). Secondary metabolism, inventive evolution and biochemical diversity - a review. Gene., 115: 135–140.

[6] Edwards, C. (1993). Isolation, properties and potential applications of thermophilic actinomycetes. Appl. Biochem Biotechnol., 42: 161–179.

[7] Demain, A.L. (1995). Why do microorganisms produce antimicrobials? In: Hunter PA, Darby GK, Russell NJ (eds) Fifty years of antimicrobials: past, prospective and future trends –Symposium 53. Society of General Microbiology., Cambridge University Press, pp: 205–228.

[8] Xu, L.H., Jiang, Y., Li, W.J., Wen, M.L., Li, M.G. and Jiang, C.L. (2005). Streptomyces roseoalbus sp. nov., an actinomycete isolated from soil in Yunnan, China. Antonie. Van. Leeuwenhoek., 87: 189–194.

[9] Miyadoh, S. (1993). Research on antibiotic screening in Japan over the last decade: a producing microorganisms approach. Actinomycetologica., 7: 100–106.

[10] Rocha, A.B., Lopes, R.M. and Schwartsmann, G. (2001). Natural products in anticancer therapy. Curr. Opin. Pharmacol., 1: 364–369.

[11] Nolan, R.D. and Cross, T. (1998). Isolation and screening of actinomycetes. In: Goodfellow M, Williams ST, Mordarski MM (eds) Actinomycetes in biotechnology., Academic Press, ISBN 0-12-289673- 4, London.

[12] Takahashi, Y. and Omura, S. (2003). Isolation of new actinomycetes strains for the screening of new bioactive compounds. J Gen Appl. Microbiol., 49: 141–154.

[13] Watve, M.G., Tickoo, R., Jog, M.M. and Bhole, B.D. (2001). How many antibiotics are produced by the genus Streptomyces? Arch. Microbiol., 176: 386–390.

[14] Asari, R., Koperek, O., Scheuba, C., Riss, P., Kaserer, K. and Hoffmann, M., et al. (2009). Follicular thyroid carcinoma in an iodine-replete endemic goiter region: a prospectively collected, retrospectively analyzed clinical trial. Ann Surg., 249(6): 1023-31.

[15] Syed. G. Dastager, Lee, J.C., Ju, Y.J., Park, D.J. and Kim, C.J. (2008). Nocardioides islandiensis sp. nov., isolated from soil in Bigeum Island Korea. Antonie. Van. Leeuwenhoek., 93, 401-406.

[16] Syed.G. Dastager, Lee, JC., Ju, YJ., Park, DJ. and Kim, C.J. (2008). Frigoribacterium mesophilum sp. nov., a mesophilic actinobacterium isolated from Bigeum Island, Korea. Int. J. Syst. Evol. Microbiol., 58(8): 1869-1872.

[17] Syed.G.Dastager, Lee, JC., Ju, YJ., Park, DJ. & Kim, C.J. (2008). Kim Nocardioides halotolerans sp. nov., isolated from soil on Bigeum Island, Korea. Syst. Appl. Microbiol 31(3): 24-29.

[18] Ouhdouch Y, Barakate M. and Finance, C. (2001). Actinomycetes of Moroccan habitats: isolation and



screening for antifungal activities. Eur. J. Soil. Biol., 37: 69-74.

[19] Shirling, E.B., D. Gottlieb. (1966). Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol., 16: 313-340.

[20] Tamura K, J. Dudley, M. Nei. and S. Kumar S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis MEGA) software version 4.0. Mol. Biol. Evol., 24: 1596-1599.

[21] Sambamurthy K and Ellaiah, P. (1974). A new Streptomycin producing Neomycin (B and C) complex, S. marinensis (part-1). Hind. Antibiot. Bull., 17: 24-28

[22] Drenan et al., Drenan, R.M., Liu, X.Y., Bertram, P.G. and Zheng, X.F. (2004). FKBP12 Rapamying-associated

protein or mammalian target of Rapamycin (FRAP/mTOR) localization in the endoplasmic reticulum and the golgi apparatus, J. Biol. Chem., 279: 772–779.

[23] Hansen, M.B., Nielsen, S.E. and Berg, K. (1989). Re-examination and further development of precise and rapid dye methods for measuring cell growth/cell kill. J. Immunol. Methods., 119: 203–210.

[24] Gallagher, S., Winston, S. E., Fuller, S. A. and Hurrell, J. G. R. (2003). In Current Protcols in Molecular Biology, eds. Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J. & Smith, J. (Wiley, New York). pp: 10.8.1 – 10.8.24.

Disclaimer: Statements, information, inferences, observations and findings etc., stated in the International Journal of Medicobiological Research are attributed to the authors and do in no way imply to Journal. Queries related to articles should be directed to authors.

320Journal of Basic and Applied Biology, 5(1&2), 2011, pp. 320-328© 2011, by the Centre for Biological Research, Puthalam, 629 602, TN, India

Research Article

ISSN 0973 - 8207

MARINE CYMARINE CYMARINE CYMARINE CYMARINE CYANOBAANOBAANOBAANOBAANOBACTERIA - A POCTERIA - A POCTERIA - A POCTERIA - A POCTERIA - A POTENT SOURTENT SOURTENT SOURTENT SOURTENT SOURCE FCE FCE FCE FCE FOR THERAPEUTICSOR THERAPEUTICSOR THERAPEUTICSOR THERAPEUTICSOR THERAPEUTICS

AAAAA. Ganesh K. Ganesh K. Ganesh K. Ganesh K. Ganesh Kumarumarumarumarumar11111, G. Selv, G. Selv, G. Selv, G. Selv, G. Selvakakakakakumarumarumarumarumar22222 and T and T and T and T and T. Thirunalasundari. Thirunalasundari. Thirunalasundari. Thirunalasundari. Thirunalasundari22222*****

1Chennai Dental Research Foundation, Chennai – 600 004.2Department of Biotechnology, Bharathidasan University, Tiruchirappalli – 620 024.E-mail: [email protected], [email protected]

Abstract: Marine cyanobacteria are taxonomically diverse, largely productive, biologically activeand chemically unique offering a great scope for discovery of new drugs. Marine cyanobacteriahave displayed an array of pharmacological properties especially antioxidant, immunostimulatoryand antitumour activities. In spite of vast resources enriched with chemicals, the marine cyanobacteriaare largely unexplored. Hence an attempt was made in this study to explore few bioactive propertiesof selected marine cyanobacterial strains. Crude extracts and fractions of marine cyanobacterialstrains were tested for antibacterial, immunomodulatroy, cytotoxicity and anticancer activity. Outof the ten cyanobacterial strains tested in this study, Oscillatoria willei was found to have potentantibacterial and anticancer activity. The different activities of marine cyanobacteria suggestsdifferent compounds present with different polarities.

Key words: Marine cyanobacteria, antibacterial, anticancer, cytotoxicity.

Introduction

Marine environment continues to be themost prolific source of biologically active anddiverse chemotypes as it provides environmentfor many organisms like marine flora, fauna andmicrobes. It is becoming increasingly evidentthat associated microbes may often be the sourceof biologically active compounds. Marineorganisms exhibit a wide range of biologicalactivity (Brown et al., 2004) like antimicrobial,antiviral, antibiotic, antifungal and antifertilityactivities (Pan et al., 2004). The chemicaldiversity of the marine ecosystem, starting fromsimple linear peptides to complex macrocyclicpolyethers, draws us toward the discovery ofnew marine natural products in varioustherapeutic areas like cancer, inflammation,microbial infections and various other deadlydiseases. One such microbial group having richsource of bioactive potentials are marine

cyanobacteria. Cyanobacteria are the diversegroup of photosynthetic, prokaryoticorganisms found in freshwater and marineenvironments. Cyanobacteria are a remarkablegroup of photosynthetic prokaryotes,comprising more than 150 genera and 2000species, which play diverse yet significant rolesin aquatic and terrestrial ecosystems.

Cyanobacteria have also beenrecognized as candidates for the discovery ofnovel antibiotics and use as biocontrol agentsof bacterial and fungal pathogens. They areknown to produce antibacterial compounds(Carmichael, 1994), antifungal compounds(Frankmolle, et al., 1992), anticancerous,antineoplastic agents and compounds useful intreatment of HIV (Mundt et al., 2002). Inaddition they also produce best knownneurotoxins and hepatotoxins. More than 50%of the marine cyanobacteria are potentially

321

exploitable for extracting bioactive substanceswhich are effective in killing the cancer cells byinducing apoptotic death.

Cyanobacterial metabolites are alsonow being explored as important sources ofpharmacologically active compounds useful indiagnostics or pigments as fluorescent probesand as nutraceuticals and as food/feedsupplements. In recent times, the mostsignificant discovery from cyanobacteria is theisolation of borophycin, a boron-containingmetabolite, isolated from marine cyanobacterialstrains of Nostoc linckia and Nostoc spongiaeforme(Banker and Carmeli, 1998). The compoundexhibits potent cytotoxicity against humanepidermoid carcinoma and human colorectaladenocarcinoma cell lines (Davidson, 1995).Cyanobacteria have gained much attention asa rich source of bioactive compounds and havebeen considered as one of the most promisinggroups of organisms to produce them (Bhaduryet al., 2004; Dahms et al., 2006). Theseproperties are due to the presence of secondarymetabolite in them. The ability ofcyanobacteria to synthesize numerous complexsecondary metabolites such as peptides,depsipeptides, polyketides and alkaloids etc. hasfascinated the researchers for theirpharmaceutical and biotechnologicalexploitations (Thajuddin and Subramanian,2005; Sielaff et al., 2006; Spolaore et al., 2006).Over the last 20 years marine organisms haveprovided a large proportion of the bioactivenatural products. Having known that thesefacts this study was planned to study thetherapeutic potentials particularlyantimicrobial, anticancer and cytotoxicproperty of marine cyanobacteria.

Materials and methods

Organism chosen and maintenance

A total of ten marine cyanobacteriaviz., Nostoc calcicola, Oscillatoria boryana,Oscillatoria chlorine, Oscillatoria formosa,Oscillatoria late – virens, Phormidiumvalderianum, Oscillatoria willei, Pseudoanabenaschmedii, Synechocystis pelvalikii and Spirulina

subsalsa were used in this study. The strains wereobtained from the germplasm collection of theNational Facility for Marine Cyanobacteria[NFMC]. The organisms were cultured inASN III N+ media in an auxenic condition(Rippka et al., 1979) and incubated at 25 ± 2æ%Cin 1,500 lux with 12 hrs day / night cycle inthe germplasm of the NFMC and was allowedto grow for 15-20 days.

In this study the crude extracts wereused for antibacterial activity alone. For otheranalysis various fractions were used.

Preparation of extracts and fractions

Preparation of extracts

After 15 to 20 days growthcyanobacterial wet mass collected. Wet mass waswashed many times with tap water followedby distilled water to remove salt. The weighedwet mass was ground in a pestle and mortarwith 100% alcohol (distilled). The groundmaterial was centrifuged at 10,000g for 10minutes at 4oC (Remi Cooling Centrifuge,Model: C 24) and the supernatant wasseparated and collected. This process wasrepeated till the pellet turned grey or thesupernatant turned colorless. The supernatantwas pooled and filtered through crude filterpaper, followed by the whatman No.1 filterpaper and then it was concentrated using aspeed vacuum concentrator (Speed Vac, R plusC 210 A, Savant). Weight of this crude extractwas determined using an electronic balance(Infra Tech, model: IN200). This crude extractwas used for antimicrobial study.

Preparation of Fractions

Wet mass was washed many times withtap water followed by distilled water to removesalt. The weighed wet mass was grounded in apestle and mortor initially with hexane. Theground material was centrifuged at 10,000 x gfor 10 min at 4æ%C (Remi cooling Centrifuge,C 24) and the supernatant was separated andcollected which is fraction 1. To the pellet nextsolvent benzene was added the same procedure

Ganesh Kumar et al., 2011

322

was repeated followed by other solventschloroform, ethyl acetate, methanol and waterto obtain six fractions totally. All fractions werecollected individually and evaporated.

Antibacterial Activity Test

Disc diffusion method was followed(Bauer et al., 1966) to determine theantibacterial activity. For antibacterial testcyanobacterial crude extracts were used.Petriplates containing 20 ml of Mueller HintonAgar were seeded with 4 hours old fresh cultureof clinical isolates. By making use of templatedrawn cyanobacterial extracts loaded discswere dispensed on the solidified Mueller HintonAgar with test organisms. Oxytetracyclineantibiotic disc obtained from M/s Hi-medialaboratories Ltd, Mumbai was used as a positivecontrol and the solvent loaded discs were usedas a negative control. This was incubated at37æ%C for 24 hours in an incubator (RandsSBC). The test was performed in triplicates. Thezone of inhibition was measured by makinguse of Antibiotic Zone Scale (Hi-Media).

Clinical isolates and referral strainschosen were Escherichia coli (2065), Klebsiellapneumoniae (2719), Proteus mirabilis (2300),Pseudomonas aeroginosa (2501), Salmonella typhi(2607), Streptococcus faecalis (2079) andStaphylococcus aureus (2036). All the strains wereobtained from IMTECH, Chandigarh.

Immunomodulatory and Cytotoxicity

assessment by MTT

MTT assay was done based on theearlier work done by Mosmann et al (Mosmannet al., 1983). Briefly 100µl of differentconcentrations of the fractions were added to aseries of wells in a 96 well tissue culture platecontaining 1x105 Human Peripheral BloodMononuclear Cells (PBMC) per well. PBS wasadded as the solvent control. PHA (proliferators)and LPS (cytotoxic) were added as knowncontrols. Then the cells were incubated at 37ºCin a humidified atmosphere of 5% CO

2 in a CO

2

incubator (TC2323, Shel U.S.A) for 72 hours.After that 20 µl of MTT in Phosphate BufferedSaline (PBS) was added to each well and the

plates were wraped with aluminium foil andincubated at 37°C for 4 hours. Purpleformazone product formed was dissolved by theaddition of 100 µl of acid isopropanol to eachwell (0.04N HCl in isopropanol). Theabsorbance was measured at 570nm and630nm (reference) using a micro plate reader(Biorad, USA). The assay was performed intriplicates and the respective mean values werecalculated and used for further calculation. Thepercentage inhibition was calculated from thisdata using the formula

% inhibition =

Mean Od of untreated cells (control) - Mean OD of treated cells

Mean OD of untreated cells (controlx 100

Anticancer activity

Jurkat, a T cell lymphoma and Raji, aB cell lymphoma are the two cell lines used inthis study. Both were obtained from NationalCentre for Cell Sciences (NCCS) Pune, India.The cancer cell lines were cultured in RPMI1640 Medium (HiMedia, Mumbai, India)supplemented with 10% Fetal Calf Serum(FCS) (HiMedia, Mumbai, India). 100 IU/mlof Penicillin (HiMedia, Mumbai, India) and 100µg/ml of Streptomycin (HiMedia, Mumbai,India) were also added to media as antibioticsto control the growth of contaminatingmicrooraganisms. The cells were cultured in 96well tissue culture plates (Greiner, USA), at37ºC in a humidified atmosphere of 5% CO

2 in

a CO2 incubator (TC2323, Shel lab, U.S.A). All

the experiments were performed using cell linesof 10 to 15 passage. Using both the cell linesMTT assay was done based on the methodmentioned above.

Results

Antibacterial activity

Results revealed that Phormidiumvalderianum was found to be highly activeagainst Klebsiella pneumoniae, Salmonella typhi,and Staphylococcus aureus. Oscillatoria willei wasfound to be highly active against Pseudomonasaeroginosa, Salmonella typhi, Staphylococcus aureus


323

and Streptococcus faecalis (Plate 1). Salmonellatyphi and Staphylococcus aureus were sensitive toSynechocystis pelvalikii (Table 1). Results basedon zone of inhibition showed that, Phormidiumvalderianum had a maximum zone of inhibitionof 16 mm against Salmonella typhi, followed byStaphylococcus aureus (15 mm) and Klebsiellapneumonia (14 mm). In case of Oscillatoria willeithe zone of inhibition was found to bemaximum in Streptococcus faecalis (16 mm),followed by Salmonella typhi , (15 mm),Staphylococcus aureus (14 mm) and Pseudomonasaeroginosa (13 mm). Synechocystis pelvalikii hada zone of inhibition of 15 mm againstStaphylococcus aureus and 14 mm againstSalmonella typhi (Table 2).

Immunomodulatory and Cytotoxicity

When MTT assay was performed forall the ten cyanobacterial fractions on humanPBMC, the results revealed that Nostoc calcicola,Oscillatoria boryana, Oscillatoria chlorina,Pseudoanabena schmedii and Spirulina subsalsawere cytotoxic. Oscillatoria formosa, Oscillatorialate–virens, Phormidium valderianum andSynechocystis pelvalkii were found to be nontoxic.Oscillatoria willei alone was found to enhancethe growth of Human PBMC (Fig 1 – 6).

Anticancer activity

Activity of the cyanobacterial fractionson Jurkat by MTT assay showed that thefollowing cyanobacterial strains viz., Nostoccalcicola, Oscillatoria boryana, Oscillatoria formosa,Spirulina subsalsa, Pseudoanabena schmedii,Oscillatoria chlorina, Oscillatoria late–virens,Phormidium valderianum and Synechocystispelvalikii neither down regulated nor upregulated Jukat cell lines except Oscillatoriawillei which down regulated Jurkat cell lines.On Raji cancer cell lines also exactly similarresults were obtained (Fig 1 – 6).

Discussion

Numerous types of bioactivecompounds have been isolated from marinesources. Several of them are currently in clinicaltrials or preclinical trials or undergoing further

investigation. Although marine compounds areunder-represented in current pharmacopoeia,it is anticipated that the marine environmentwill become an invaluable source of novelcompounds in the future, as it represents 95%of the biosphere (Jimeno et al., 2004).

Cyanobacteria are well known toproduce antibacterial compounds (Ostensviket al., 1998, Jaki et al., 1999, Skulberg, 2000and Martin et al., 2008). However, little workhas been done to assess the antibacterial activityof most of the cyanobacteria of marineenvironment. In this work we assessed theactivity of marine cyanobacteria on growth ofbacteria using crude extracts prepared fromequal amounts of dried cyanobacterial culturebiomass. These results indicate that the extractscontained different antibacterial substances andreflect the variety of environmental stress(Schwartz et al., 1990; Patterson et al., 1994).Oscillatoria sp. showed maximum inhibitionzone. Important factors affecting the size ofthe inhibition zone are the chemical and physicalproperties of the growth medium and the sizeand ionic charge of the antibiotic molecule(Crosby, 1991). Campbell et al. (1994) reportedthat the toxic effects of cyanobacterial extractson luminescent bacteria did not correlate withthe concentration, but appeared to be due toother compounds present in the cyanobacteria.Most of the present results are in agreementwith these observations. The antibacterialresults obtained in the present investigationwere only based on crude extracts and did notindicate any defined antibacterial substance.However, suitable bacterial bioassays havebeen established to recognize and quantifyantibacterial effects of cyanobacterial extractsby many researchers in the past. Furtherstudies have to be made on fractionation andseparation of crude extracts in order to findout the principle antibacterial compoundpresent in cyanobacterial strains chosen.

In a study conducted by Martin et al.(2008) they found that the cyanobacterialstrains and extracts that caused higherpercentage of apoptotic HL-60 cells were theones that exhibited antibacterial activities.Martin et al. (2008) also reported that marine


324

Plate 1: Antibacterial activity of Oscillatoria willei against clinical pathogens

cyanobacterial strains of the generaSynechoc ystis and Synechococcus producesubstances with inhibitory effects onprokaryotic cells and with apoptotic activityin eukaryote cell lines, which highlights theimportance of these organisms as potentialspharmacological agents. Since different activitieswere observed in extracts obtained with organicsolvents and extracts obtained with water theysuggest that compounds with differentpolarities are involved. Similar kind of resultswere obtained in this study also.

Natural derivatives play an important

role to prevent diseases as synthetic drug

formulations cause various harmful side effects

to human beings. Marine cyanobacteria are

potential source of bioactive compounds, but

they are least explored. Owing to a diverse

chemical ecology, the marine organisms,

especially marine cyanobacteria have a great

promise for providing potent, cheaper and safer

drugs, which deserves an extensive

investigation.

+ Positive control – Tetracycline- Negative control - Methanol: DMSO: Water (1:1:1)

1 - 50 µg of the extract / disc 2 - 100 µg of the extract / disc

3 - 200 µg of the extract / disc 4 - 400 µg of the extract / disc


325

Table 1: Antibacterial activity of Cyanobacterial extract on clinical pathogens in terms of

sensitivity

E. coli K. pneumoniae P. mirabilis P. aeroginosa S. typhi S. aureus S. faecalis

1 N. calcicola - - - - - + -

2 O. boryana - - - - ++ - -

3 O. chlorina - - - - - - +

4 O. formosa - - - + - - -

5 O. late-virens - - + - - + -

6 P. valderianum - +++ - - +++ +++ -

7 O. willei - - - +++ +++ +++ +++

8 Pseudo. Schmedii - + - - - + -

9 Spirulina subsalsa - - - - - + -

10 Synechocystis pelvalikii - - - - +++ +++ -

S. No CyanobacteriumBacterial pathogens and activity

+++ Highly sensitive ++ Moderately Sensitive + Sensitive - Resistant

Table 2: Antibacterial activity of cyanobacterial extracts on clinical pathogens in terms of

inhibition zone

E. coli K. pneumoniae P. mirabilis P. aeroginosa S. typhi S. aureus S. faecalis

1 N. calcicola - - - - - 4 mm -

2 O. boryana - - - - 7 mm - -

3 O. chlorina - - - - - - 6 mm

4 O. formosa - - - 5 mm - - -

5 O. late-virens - - 5 mm - - 4 mm -

6 P. valderianum - 14 mm - - 16 mm 15 mm -

7 O. willei - - - 13 mm 15mm 14 mm 16 mm

8 Pseudo. Schmedii - 6 mm - - - 4 mm -

9 Spirulina subsalsa - - - - - 6 mm -

10 Synechocystis pelvalikii - - - - 14 mm 15 mm -

S. No CyanobacteriumBacterial pathogens and activity / Zone of inhibition in mm

- Resistant


326


327

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Cyanobacterial Biodiversity and potential

application in Biotechnology. Current

Science, 89(1): 47-57.

* Author for correspondence


1Journal of Basic and Applied Biology, 5(3&4), 2011, pp. 1-10

© 2011, by the Centre for Biological Research, Puthalam, 629 602, TN, India

Review Article

ISSN 0973 - 8207

BIOBIOBIOBIOBIOAAAAACTIVE METCTIVE METCTIVE METCTIVE METCTIVE METABOLITES FRABOLITES FRABOLITES FRABOLITES FRABOLITES FROM CYOM CYOM CYOM CYOM CYANOBAANOBAANOBAANOBAANOBACTERIA OF MARINE ORIGINCTERIA OF MARINE ORIGINCTERIA OF MARINE ORIGINCTERIA OF MARINE ORIGINCTERIA OF MARINE ORIGIN

G. SelvG. SelvG. SelvG. SelvG. Selvakakakakakumar and Tumar and Tumar and Tumar and Tumar and T. Thirunalasundari. Thirunalasundari. Thirunalasundari. Thirunalasundari. Thirunalasundari

Department of Biotechnology, Bharathidasan University, Tiruchirappalli-620 024 Tamilnadu, India.

Abstract: Cyanobacteria (Blue Green Algae) are photosynthetic prokaryotes which have a prolificpotential for bioactive compounds that are pharmacologically active. Marine cyanobacteria are arich source of novel bioactive secondary metabolites which have attracted much attention of chemists,pharmacologists and molecular biologists. Apart from bioactive compounds, cyanobacteria are asignificant source of fine chemicals and renewable fuel. Their role as antiviral, antiHIV, antibacterial,and antitumor, has been well established. Cyanobacteria continue to be an efficient and powerfultool to address arresting of disease perspective. In this review, cyanobacteria involved in the synthesisof novel bioactive metabolites are stressed. Among the published reviews related to bioactivemetabolites, only few of them describes its potential from ecology to molecular genetics.

Key words: Cyanobacteria, Bioactive metabolites and metabolic pathways.

Introduction

Cyanobacteria are among the mostprimitive forms of life on the earth. They areubiquitous and morphologically diverseoccurring in almost every habitat. Due to theirphotosynthetic and aquatic nature they arereferred to as “Blue Green Algae”. Mostly,cyanobacteria occur in unicellular forms,nevertheless filamentous forms are also present.The distinct nature of cyanobacteria inevolution is determined by key geochemical andbiotic transitions which are inclusive ofoxygenic photosynthesis (1). They are capableof using

carbondioxide as their sole source ofcarbon using Phosphate Pentose Pathway orCalvin cycle. Medicinal capabilities ofcyanobacteria have long been stressed since1500 BC, when Nostoc sp was used to treat gout,fistula and several cancers (2). Innovativeresearch concerned with novel bioactive

metabolites revealed that cyanobacteria are asignificant source of pharmacologically usefulnatural products (3). Screening programs allover the world have further confirmed thediversity and rich repertoire of pigmentsproduced by cyanobacteria that canrevolutionize the industrial uses of “colours”with their nutraceutical and pharmaceuticalvalue (4). Cyanobacterial toxins are a richsource of molecular tools to characterizefunctional nature of voltage gated sodiumchannels, potential analgesics and newprotectants (5).

Pharmacologically active metabolites from

cyanobacteria

Cyanobacteria produce a wide variety ofbioactive compounds, which include 40%lipopeptides, 5.6% amino acids, 4.2% fatty acids,4.2% macrolides and 9% amides. Cyanobacteriasuch as Microc ystis, Anabaena, Nostoc andOscillatoria produce a great variety ofsecondary metabolites. The only comparable

2

group is actinomycetes, which has yielded atremendous number of metabolites (6).Bioactive metabolites from marinecyanobacteria range widely in the arena ofantiviral, antiHIV, antibacterial, antifungal,antiprotozoaic, antiparasitic, antihelminthes,antitumor, anticancer, antineoplastic andantiinflammatory. Jenson and Fenical, (7)showed that the blue green algae Aphanizomenon

flos-aquae stimulate the mobilization of T andB cells and proves that cyanobacteria act asimmune modulator.

Antimicrobial

Cyanobacteria have been identified asa rich source of biologically active compoundswith antiviral, antibacterial, antifungal andanticancer activities (8). In addition to thesecyanobacteria are also used in aquaculture,waste water treatment, food, fertilizers,production of secondary metabolites includingexopolysaccharides, vitamins, toxins, enzymesand other pharmaceuticals (9).

The potential contribution of marinecyanobacteria to the discovery of new bioactivemolecules is increasingly challenging (10).Natural products have been isolated from awide variety of taxa and tested for variousbiological activities with applications inagriculture & industry in general (11&12) andpharmaceuticals in particular (13). Althoughcyanobacteria are still primarily viewed as anenvironmental nuisance or a source of toxins,hazardous to man and aquatic livestock, thereare many potential benefits to research onchemicals produced by these organismsparticularly as an antimicrobial agent.Antibacterial, antiviral, antifungal, algicide andcytotoxic activities have been reported (14 to19). The role of bioactive molecules in theproducer organism itself is poorly understoodbut, considering the wide spectrum of biologicaladaptations and tolerance to environmentalstress revealed by cyanobacteria, some of thesecompounds can be produced in an attempt toconfer advantages for their survival. Thedetection of extracellular compounds ofcyanobacterial origin that inhibit either bacteria

or cyanobacteria (20) as well as fungi (21) hasbeen known for some time.

The cyanobacterium Lyngbya majusculahas alsobeen shown to be a source of diverse bioactivecompounds, some of which possessantimicrobial properties (22). Accordingly 75%of the 113 biogenic compounds isolated from L.

majuscula exhibit some type of biologicalactivity, and more than 10% have been reportedto have antifungal and antimicrobial properties(23). Compounds like majusculamide A-D,malyngolide and laxaphycin A-B isolatedfrom L. majuscula have shown promisingresults as antifungal and antibacterial agentswhilst a cyclic peptide provided wideantimicrobial activity (22). Thecyanobacterium Scytonema hofmanni inhibits thegrowth of a common microfouling diatom (24).

Antiviral

Several cyanobacterial extracts werefound to exhibit antiviral activity againstHerpes Simplex Virus II (HSV-2) (25). In ascreening test, 10% of the cyanobacterialextracts showed anti HSV-2 activity, while 2.5%of them showed activity against respiratorysyncytial virus. Lipophilic and hydrophilicextracts from approximately 600 strains ofcultured cyanobacteria, representing some 300species, significantly reduced the cytopathiceffect normally associated with viral infections(26).

Lyngbya lagerheimeii and Phormidium

tenue have been found to protect humanlymphoblastoid T cells from the cytopathiceffect of HIV infection (27). A new class of HIVinhibitors called sulfonic acid containingglycolipid, were isolated from the extract ofblue-green algae and the compounds were foundto be active against the HIV virus.Cyanoviridin-N, isolated from blue-green algaeinactivates the strains of HIV virus and inhibitscell to cell and virus to cell fusion (28). Calciumspirulan (Ca-SP), a novel sulphatedpolysaccharide is an antiviral agent. Thiscompound selectively inhibits the entry ofenveloped virus (Herpes simplex, human

Selvakumar and Thirunalasundari, 2011

3

cytomegalovirus, measles virus) into the cell(29).

An aqueous extract of thecyanobacterium Arthrosspira plantesis blockedRaucher Murine Leukemia Virus (RLV)induced plaque (27). Similarly a water solubleextract of Spirulina showed a dose-dependentinhibition of the replication of Herplex simplexVirus type 1 (HSV – 1) in HeLa cells withinthe concentration range of 0.08-50 mg/ml.However, this extract did not have any virucidalactivity and did not interface with its adsorptionto host cells (30).

Hot water extract from Spirulina

plantensis yielded a novel sulphatedpolysaccharide named Calcium spirulan (Ca-SP) with antiviral activity. This polysaccharidecomposed of rhamnose, ribose, mannose,fructose, galactose, xylose, glucose, glucuronicacid, galacturonic acid, sulfate and calcium. Ca-SP selectively inhibited replication of severalenveloped viruses, including HSV-1, humancytomegalo virus (CMV), measles virus, mumpsvirus, influenza A virus and HIV-1. Retentionof molecular conformation by chelation ofcalcium ion with sulphate groups wassuggested to be the responsible for its antiviraleffect (31).

Antibacterial

Four fractions from Spirulina subsalsa

with unknown chemical identities, showed totalinhibition of the growth of a clinical isolate ofPseudomonas aeroginosa, a tough pathogen totreat (30). Nostoc mucorum produced anantibacterial agent with a broad-spectrumactivity (32). Similarly chloroform-methanolextracts of several Oscillatoria species inhibitedthe gram-positive bacterial pathogen likeStaphylococcus aureus. While Spirulina subsalsa

possessed antibacterial compounds againstgram-negative organisms (31). Similarly, thecrude extract of Phormidium angustissimum andLyngbya exhibited potential of new broad-spectrum antibacterial compounds.

Reflecting the fact that the developmentof resistance toward current antibioticscontinues to be a significant problem in the

treatment of infectious diseases. During 2001–2002 thirteen studies contributed to theantibacterial products of marine naturalproducts & it is a marked increase from 1998–2000 (33 & 34). These two studies reportedon the mechanism of action of two novel marineantibiotics. Torres et al., (35) investigated thearenosclerins and haliclonacyclamine E, noveltetracyclic alkylpiperidine alkaloids isolatedfrom the marine sponge Arenosclera brasiliensis.The investigators reported that differences inthe stereochemistry at the bis-piperidine ringsystem played a significant role in the potentantibiotic activity of these compounds againstantibiotic-resistant Staphylococcus aureus strains.Linington et al., (36) developed a highthroughput assay to screen marine compoundsfor their ability to inhibit a type III secretorysystem which is an essential component of thepathogenicity of enteropathogenic andenterohermorragic E. coli (36).

Antifungal

Large number of compounds obtainedfrom cyanobacteria showed antifungal activity.The scytophycin compounds isolated fromTolypothrix tjipanasensis, exhibited appreciablefungicidal activity in tests againstphytopathogenic fungi (37). The laxaphycinsfound in Anabena laxa and hormothamninsisolated from the Hormothamnion

enteromorphoides are cyclic peptides showingantifungal activity. Extracts of Synechocystis

elongatous, Phormidium corium and Dichothrix

baueriana were active against Candida species(31). A compound, presumably a methoxylateddisaccharide, purified from Oscillatoria late-

virens showed anticandida, antitumour andantibacterial activity (38). Thecyanobacterium Lyngbya majuscula has alsobeen shown to be a source of diverse bioactivecompounds with antifungal properties (22).Compounds like majusculamide A-D,malyngolide and laxaphycin A-B isolatedfrom L. majuscula have shown promisingresults as antifungal agents (22). Antifungalcompounds include fisherellin A, hapalindole,carazostatin, hytoalexin, tolytoxin, scytophycin,toyocamycin, tjipanazole, nostocyclamide and


4

nostodione produced by cyanobacteriabelonging to Stigonematales, Nostocales andOscillatoriales (39).

Anti algal

Cyanobacteria produce a broadspectrum of antialgal compounds, which maybe used to control algal blooms. Cyanobacteriaprobably use these compounds in order to outcompete other microorganisms. Antialgalcompounds produced by cyanobacteria inhibitgrowth of algae, their photosynthesis,respiration, carbon uptake, enzymatic activityand induce oxidative stress (40).

It may even use the same activity togain dominance over other organisms, orinfluence the type of conspecifics and successors(41). In a study by Schlegel et al., (18) out of198 new cyanobacterial isolates tested, 20produced bioactive substances that causedinhibition of the growth of green algae (18).One of the most investigated cyanobacterialtoxins, cyanobacterin, is not only an effectiveinhibitor of other cyanobacteria, but also ofeukaryotic algae (42) and higher plants (43). Itis noteworthy that bioactivity against green algaewas found only in Fischerella, Nostoc and Calothrix

species in an extensive study by Schlegel et al.

(18).

A recent review summarised theallelochemicals produced by cyanobacteria thataffect photosynthesis (44). Cyanobacterinfrom Scytonema hofmannii (45) inhibits the PSIIat the oxidising site of the quinone-B electronacceptor, but not at the site where DCMU (3-(3,4-dichlorphenyl)-1, 1-dimethylurea)interacts with the PSII. Electron microscopestudies revealed that cyanobacterin alsospecifically disrupted the thylakoid membranestructure in the flagellate Euglena gracilis (46).These findings show that allelopathically activecompounds may have multiple modes of action.

Nostoc linckia produced and released anallelochemical named cyanobacterin LU-1 thatinhibited the growth of many cyanobacteriaand eukaryotic algae but not heterotrophicbacteria and fungi (47). Nostoc strain 31

produces cyclic heptapeptides, nostocyclamideand nostocyclamide M, which areallelopathically active against cyanobacteriaand algae (48 & 49).

Antiprotozoal and antitubercular

Broniatowska et al., (50) reported intheir study that two cyanobacteria Nostoc

commune collected when desiccated and wet andRivularia biasolettiana found to haveantiprotozoaz and antitubercular potentials.The cytotoxic potential of the extracts was alsoevaluated against primary L6 cells. Except forT. cruzi and M. tuberculosis, the crude extractswere active against all the organisms tested andshowed no toxicity. The chloroform sub-extractsof both N. commune samples showed significantactivity against T. b. rhodesiense and P. falciparum

with low toxicity. This trend was also true forR. biasolettiana extracts and its chloroform sub-extract showed notable activity against allparasitic protozoa. There were differences in thebiological activity profiles of extracts derivedfrom desiccated and hydrated forms of N.

commune. These results warrant further studyof cyanobacteria as a valuable source of newnatural product leads for the treatment ofparasitic protozoal infections.

Cyanobacterial toxins

The identification of marine toxins hasbeen one of the most challenging areas ofmarine natural products chemistry. A varietyof organic compounds containing nitrogen aspart of a heterocyclic system have been isolatedfrom marine animals (51). Microcystis aeruginosa

and Nodularia spumigena synthesize toxinsdestructive to liver cells. These two speciesproduce seven amino acid peptide ie.,microcystin and five amino acid peptidenodularin, respectively (52). To date over 50different variants of microcystins have beenisolated from the species of Anabaena,

Hapalosiphon, Microcystis, Nostoc and Oscillatoria.Microcystin-LR inhibits serine-threonineprotein phosphatases 1 (PP1) and 2A (PP2A).Lyngbya, a filamentous cyanobacteria isresponsible for the synthesis of cytotoxiccompounds such as antillatoxin, aplysiatoxin,


5

debromoaplysiatoxin and lyngbyatoxin A, Band C. Curacin A isolated from Lyngbya

majuscula is a potent inhibitor of cell growthand mitosis, binding rapidly and tightly at thecolchicine site of tubulin. The following aresome of the toxins produced by cyanobacteria.

Kalkitoxin

Kalkitoxin is a natural product from themarine cyanobacterium Lyngbya majuscula. Thisneurotoxin blocks sodium channels averting thenerves from firing off their electrical signals.Kalkitoxin is a beneficial pharmaceuticalcompound and a valuable tool to understandthe working of sodium channels and the effectof disease on them (53).

Saxitoxin

Saxitoxin is a neurotoxin naturallyproduced by certain species of marinedinoflagellates like Alexandrium, Gymnodinium,

Pyrodinium and certain cyanobacteria likeAnabaena. Saxitoxins blocks neuronaltransmission by binding to the voltage gatedNa+ channels in nerve cells, thus causing aneurotoxic effect, which are known as paralyticshellfish poisons. Saxitoxin is 1000 times moretoxic than the potent nerve gas sarin (53).

Anatoxin

Two neurotoxic alkaloids, anatoxin-a

and its homologue homoanatoxin-a, were

purified from the filamentous cyanobacteria

Oscillatoria. Anatoxin-a and homoanatoxin-a

are secondary amines and are postsynaptic

depolarizing neuromuscular blocking agents

that bind strongly to the nicotinic acetylcholine

receptor. These compounds are potent

neurotoxins (54).

Brevitoxins

Brevitoxins are neurotoxins produced

by Ptychodiscus brevis (55). These are lipophilic

compounds with a molecular weight of

approximately 900 Da. There are two classes

of Brevitoxins; type I Brevitoxin and type II

Brevitoxin. The first contains eight 6-memberedring (type I brevitoxin) and the second class ofbrevitoxin has only 10 rings (type IIbrevitoxin). Brevitoxin depolarizes the openvoltage gated sodium (Na+) ion channel in thecell wall, leading to the uncontrolled Na+ influxinto the cell. Brevitoxin binds to the ion channelof nerve and muscle tissue that selectivelyallows sodium to pass into the cell. The sodiumchannel opens during an action potential inresponse to the change in electrical potentialacross the cell membrane.

Lipopeptides

Cyanobacterial lipopeptides includedifferent compounds like cytotoxic (41%),antitumor (13%), antiviral (4%), antibiotics(12%) and the remaining 18% activities includeantimalarial, antimycotics, multi-drug resistancereversers, antifeedant, herbicides andimmunosuppressive agents (52). The naturalproducts of many marine cyanobacteria containan aminoacid derived fragment linked to fattyacid derived portion, forming compoundsknown as lipopeptides. Lipopeptides areinteresting and biochemically active, and havecytotoxic, anticancer, antibiotic, enzymeinhibitor, antiviral and antifungal activities.Hapalosin, a cyclic desipeptide isolated from thecyanobacteria, Hapalosiphon welwitschii, has areversing activity against MDR (multi drugresistance) derived from P-glycoprotein.

Polyketide-derived moieties occurringas â-hydroxy or amino acid residues are thesource of non-proteinogenic units in theconstruction of lipopeptides, especially cyclicdepsipeptides. These macrocyclic moleculesusually exhibit potent biological activities andthis could in part due to the presence of â-aminoor hydroxy acid moieties which are resistant toproteolysis as well as conferring uniquestructural properties in the molecules (56). Two2-alkypyridine alkaloids, phormidinines A andB, were reported from the marinecyanobacterium, Phormidium sp. (57)

Other important bioactive metabolitesfrom marine cyanobacteria includesomocystinamide from Lyngbya majuscula/


6

Schizothrix mixed assemblage (58).Anandamide-like derivatives, thesemiplenamides isolated from Lyngbya semiplena

Malyngamides (59). Symplostatin 3 from themarine cyanobacterium, Symploca sp. VP452[36], lyngbyapeptins B isolated from Lyngbya

sp. (60). The marine cyanobacterial strain, L.

majuscula possess seven cyclic lipopeptides,named the guineamides (61). The ulongamidesare also cyclic depsipeptides lyngbyastatin fromL. majuscula, lyngbyabellin from Lyngbya sp.

Sensational discoveries from marine

cyanobacteria

Protease inhibitors reported from toxicgenera of cyanobacteria are micropeptins,aerugenosins, microginins, anabaenopeptinsand microverdins. Cyanovirin-N (CV-N) is a101 amino acid long, 11 kDa protein discoveredfrom Nostoc ellipsosporum (62). Borophycin is aboron rich metabolite from Nostoc linckia andNostoc spongiaeforme var. tenue. It exhibits potentcytotoxicity against human epidermoidcarcinoma and human colorectaladenocarcinoma cell lines and has been foundto exhibit antimicrobial activity. Cryptophycinfrom Nostoc sp. ATCC 53789 is a powerfulfungicide. It shows good activity against a broadspectrum drug-sensitive and drug-resistantmurine and human solid tumors. Until now,none of the cryptophycin analogues haveentered clinical trials. Cryptophycin-309, theglycinate of the chlorohydrin analogcryptophycin-296, emerged as superior overothers. The mechanism of cytotoxicity of thecryptophycins is tubulin-interaction, with adisruption of tubulin- dynamics, resulting inapoptosis of tumor cells. Scyptolin are cyclicdesipeptides with elastase inhibiting activityfrom Scytonema hofmanni PCC 7110.Broniatowska et al., (50) reported in their studythat two cyanobacteria Nostoc commune andRivularia biasolettiana were found to havecytotoxic potential against primary L6 cells.

Secondary metabolite biosynthetic pathways

In the last 50 years, microbialmetabolites have been isolated and elucidated

for chemical structures. Cyanobacteria areprolific and potential producers but areunderexploited. Biological activities of themarine cyanobacteria rely largely on metabolicpathways. Core pathways involved in bioactivemetabolite production include glycolysis,

pentose phosphate pathway, shikimate

pathway and purine/pyrimidine biosynthesis

(63).

An emerging pharmacological theme

among the secondary metabolites of marine

cyanobacteria is the production of a variety of

neurotoxic substances, many of which appear

to target the Voltage Gated Sodium Channel

(VGSC). For example, a new class of

cyanobacterial metabolites named the

‘hoiamides’ was discovered from several mixed

collections of Symploca and Oscillatoria from

Papua New Guinea (64). A number of

secondary metabolites from marine life forms

have shown potent and mechanistically

intriguing anti-inflammatory activity, and

marine cyanobacteria have contributed to this

recognition (64).

Natural selection noted in L.majuscula

prove that the above mentioned pathways are

involved due to the fact that novel metabolite

ypoamide share structural similarities between

malyngamides, pukeleimides, majusculamide

and microcolin. Furthermore several genetic

and biochemical methods have to be employed

for analyzing the biochemical pathway

elucidation for affirming the bioactive potentials

of marine cyanobacteria.

Conclusion

Cyanobacteria are very essential source

of novel bioactive natural compounds. Several

cyanobacterial secondary metabolites have been

shown to have significant pharmaceutical

potentials ranging from antiviral, antiHIV,

antibacterial, antifungal and antitumor

activities. Cyanobacteria in the role of toxins

plays a vital role for producing bioactive

compounds. Hence cyanobacteria prove to a

boon for medical research.


7

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