Iron plays an essential role in the proliferation of aggressive tumors therefore it represents an ideal target for cancer therapy. Cell free supernatants from a siderophore producing actinobacterium previously isolated from Thailand were tested against six human cancer cell lines including malignant melanoma A 375 (ATCC no.: CRL-1619), colorectal adenocarcinoma SW620 (ATCC

no.: CCL-227), gastric carcinoma Katob III (ATCC no.: HTB-103), liver hepatoblastoma HepG2 (ATCC no.: HB-8065), breast carcinoma BT474 (ATCC no.: HTB-20) and Acute T cell leukemia Jurkat (ATCC no.: CRL-2063). Following treatment of cells with the bacterial culture supernatant the cell viability of A375 cells was dramatically decreased with cell survival of less than 34 % within

48 h. The rest of the cell lines were largely unaffected. Therefore it is suggested that the actinobacterium produced a cytotoxic compound responsible for the cell death by inducing apoptotic activity. We further speculate that this compound was desferioxamine E as the bacterium is known to produce this compound under the culture conditions used

Hobbs, G

Nakouti, I

Palaga, T

Sihanonth, P

LJMU Research Online (Liverpool John Moores University)

1This article can be downloaded from http://www.ijpmbs.com/currentissue.phpInt. J. Pharm. Med. & Bio. Sc. 2013 Glyn Hobbs et al. , 2013EFFECT OF A SIDEROPHORE PRODUCER ONANIMAL CELL APOPTOSIS: A POSSIBLEROLE AS ANTI-CANCER AGENTIsmini Nakouti1, Prakitsin Sihanonth2, Tanapat Palaga2 and Glyn Hobbs1*Research PaperIron plays an essential role in the proliferation of aggressive tumors therefore it represents anideal target for cancer therapy. Cell free supernatants from a siderophore producing actinobacterspecies previously isolated from Thailand were tested against six  human cancer cell linesincluding malignant melanoma A 375 (ATCC no.: CRL-1619), colorectal adenocarcinoma SW620(ATCC no.: CCL-227), gastric carcinoma Katob! (ATCC no.: HTB-103), liver hepatoblastomaHepG2 (ATCC no.: HB-8065), breast carcinoma BT474 (ATCC no.: HTB-20) and Acute T cellleukemia Jurkat (ATCC no.: CRL-2063). Following treatment of cells with the  bacterial culturesupernatant the cell viability of A375 cells was dramatically decreased with cell survival of lessthan 34 % within 48 h. The rest of the cell lines were largely unaffected. Therefore it is suggestedthat the actinobacterium produced a cytotoxic compound  responsible for the cell death byinducing apoptotic activity. We further speculate that this compound was desferioxamine E asthe bacterium is known to produce this compound under the culture conditions used.Keywords: Siderophores, Actinobacteria, Human cell cancer lines, Apoptosis*Corresponding Author: Glyn Hobbs,g.hobbs@ljmu.ac.ukINTRODUCTIONCancer cells have an increased requirement foriron compared with ‘healthy’ cells due to their rapiddivision. They also posess a higher rate of ironuptake and storage (Elford et al., 1970; Vaughn,1987). Moreover the ribonucleotide reductase(RR) enzyme involved in DNA synthesis isupregulated in cancerous cells leading toincreased DNA replication and cell proliferation(Buss et al., 2003; Dayani et al., 2004,ISSN  2278 – 5221 www.ijpmbs.comVol. 2, No. 2, April 2013© 2013 IJPMBS. All Rights Reserved1 School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK.2 Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok, 10300, Thailand.Richardson, 1997). Iron is fundamental for theactivity of RR making iron an obvious anti-cancercandidate (Thelander and Gräslund, 1983;Whitnall et al., 2006).Siderophores are low molecular weightcompounds (600-1500 Daltons) that chelate ferriccomplexes and other ions with a high affinity. Dueto the role of iron in cellular proliferation, ironchelators, such as siderophores, are agents thatmay be beneficial for the treatment of cancer2This article can be downloaded from http://www.ijpmbs.com/currentissue.phpInt. J. Pharm. Med. & Bio. Sc. 2013 Glyn Hobbs et al. , 2013(Wandersman and Delepelaire, 2004). Indeedsome data demonstrated that desferrioxamines(iron-free siderophores) can significantly reducethe growth of aggresive tumours in patientssuffering from neuroblastom (NB) or leukemia(Buss et al., 2003; Lovejoy and Richardson,2003). Clinical studies have demonstrated thatfollowing a 4 h incubation desferrioxamine caninhibit DNA replication of NB cells (Blatt et al.,1988). In addition a 72 h exposure of NB cells to60 Figure 1(a)M of desferrioxamine reduces thecells’ viability by 80 % (Blatt and Stitely, 1987).An actinobacterium (GenBank accessionnumber EF585403), previously isolated from Takprovince in Thailand, was tested positive forsiderophore production and its main chelatingagent released was identified as desferrioxamineE by HPLC analysis (Nakoutiet al., 2012). Theaim of this work was to screen the culturesupernatant for anti-cancer activity using a rangeof cancerous cell lines and to investigate if aposssible response mechanism was due toapoptosis.MATERIALS AND METHODSGrowth MediaStarch casein media: Casein (0.4 g), starch (1.0g), KNO3 (0.5 g), K2HPO4 (0.2 g), MgPO4 (0.1 g),CaCO3 (0.1 g) and bacteriological agar (20 g)were dissolved in 1 litre of distilled H2O andsterilised by autoclaving at 1210C for 15 min (solidmedia). Liquid media were prepared by omittingthe bacteriological agar.Culture ConditionsSpores of the strain were stored in a bacterialpreservation system (Technical Service Consul-tants Ltd, Lancashire) at -800C and 10 l werespread onto starch casein agar plates andincubated at 300 C for a week until sporulationoccurred. Fresh spores were collectedaseptically (in 20 ml of sterile distilled H2O) andan aliquot (100 l) was introduced into a 250 mlconical flask containing 50 ml of sterile starchcasein media (liquid). The cultures weresupplemented with 5 g/L lysine prior to incubationon a rotary shaker (200 rpm) at 300 C for 4 days.The conditions were previously optimised formaximum desferrioxamine E production (Nakoutiand Hobbs, 2012). Following incubation themedium was centrifuged for 20 min at 8000 g toseparate the mycelia. The supernatant was filter-sterilised by using a 0.20 Figure 1(a)m syringefilter (Corning) and stored at 40C.Biological Assay of Anticancer ActivityThe effect of the culture supernatant on the cellviability was assessed using the MTT assay(Palaga et al., 1996) against 6 human cancer celllines including malignant melanoma A375 (ATCCno. CRL-1619), colorectal adenocarcinomaSW620 (ATCC no. CCL-227), gastric carcinomaKatob! (ATCC no. HTB-103), liver hepato-blastoma HepG2 (ATCC no. HB-8065), breastcarcinoma BT474 (ATCC no. HTB-20) and AcuteT cell leukemia Jurkat (ATCC no. CRL-2063). Cellsuspensions in PRMI-1640 medium supplementedwith 10% (v/v) fetal bovine serum and 100 mg/mlgentamicin were seeded into flat-bottomed 96-well plates at a density of 2-3 x 104 cells/well andincubated at 37°C in a 5 % CO2 atmosphere for24 h. An aliquot (50 l) of the culture supernatantwas added to the well and incubated (48 h contacttime) under the same conditions.Cell viability was calculated as (Asample-Ablank)/(Acontrol-Ablank) x 100 % while absorbance (540 nm)3This article can be downloaded from http://www.ijpmbs.com/currentissue.phpInt. J. Pharm. Med. & Bio. Sc. 2013 Glyn Hobbs et al. , 2013obtained from cells incubated with sterile PRMI-1640 medium was used as the control (positive)and cell free medium as the blank (negative).Ferrichrome (iron-free) from Ustilagosphaerogena (5l of 1 mg/ml concentration) wasalso tested against the cell lines for anti-canceractivity. The reason behind this was thatdesferrioxamine E was not available in themarket; therefore ferrichrome, also ahydroxamate, was purchased from Sigma-Aldrich.Apoptosis TestsA cell culture dish (35 x 10 mm) containing asterile glass coverslip in RPMI-1640 medium wasseeded with a cell suspention of A375 cells (finalconcentration of 1 x 106 cells/ml). The cells wereallowed to attach for 24 h, followed by treatmentfor 48 h. They were then washed with phosphatebuffer saline (PBS), f ixed with 1% (v/v)glutaraldehyde in the dark for 2 h and washedwith PBS. Cells which had attached to the glasscoverslips, were stained with DNA dye Hoechst33342. After being left for 5 min in the dark,apoptotic cell death was determined by observingfragmented nuclei cells under the fluorescentmicroscope (modified from [14]).RESULTS AND DISCUSSIONAnticancer ActivityThe effect of the strain’s supernatant on cellularviability was assessed using the MTT assay. Theisolate was found to produce strong cytotoxicsubstances specific to malignant melanoma cells(A375). Following treatment of cells with theculture supernatant (50 l) the percentage cellviability of A375 cells was dramatically decreasedwith cell survival of less than 34 % (33.193 %)within 48 h (Table 1); whereas the remainingcancer cell lines exhibited similar cell viability. Theexperiment was an average of 12 replicates.Therefore it was suggested that the organismexhibited selective activity against the skin cancercell line. Ferrichrome produced by Ustilagosphaerogena had little effect on any of the cancercell lines (cell viability after treatement wasmeasured to be 94 %).Induction of Apoptosis by Streptomycessp. Strain 23FThe supernatant (50 l) of strain 23F (GenBankaccession number EF585403) was tested forinduction of apoptosis with malignant melanomacells (A375). Addition of the supernatant to A375cell culture resulted in the induction of apoptoticSamples 1 2 3 4 5 6 7 8 9 10 11 12Mean Viabi- STDlity (%) (%)Blank 0.038 0.035 0.035 0.035 0.035 0.038 0.038 0.035 0.035 0.035 0.038 0.038 0.036 0 N/AControl 1.178 1.226 1.293 1.190 1.198 1.227 1.210 1.293 1.193 1.224 1.194 1.290 1.226 100 N/ATreated 0.421 0.444 0.454 0.422 0.440 0.424 0.422 0.443 0.423 0.423 0.441 0.421 0.431 33.19 +/–0.957withSuper-natantTreated 1.166 1.101 1.109 1.190 1.192 1.105 1.170 1.163 1.199 1.169 1.196 1.166 1.160 94 +/–2.877withferrichromeTable 1: MTT Assay Data Demonstrating the Anticancer Activityof the Siderophore Producer Supernatant4This article can be downloaded from http://www.ijpmbs.com/currentissue.phpInt. J. Pharm. Med. & Bio. Sc. 2013 Glyn Hobbs et al. , 2013Figure 1: A375 Cancer Cells Stained with Hoechst, the Apoptotic Nucleiwere Visualised Under the Fluorescent Microscope (X 40 Magnification)Figure 2: A375 Cells Treated with Starch Casein Media (Negative Control),Cell Division is Evident (x 40 Magnification)activity. Cells from the culture were stained withHoechst dye and apoptotic nuclei were visualisedunder the fluorescent microscope (Figure 1). Thecancer cells (A375) became smaller throughshrinking and the chromatin became agglutinatedresulting in nuclear condensation followed bybreakdown of the nuclear membrane. Theremaining ‘attached’ cells appeared very similarto the untreated ones. Cells treated with sterilestarch casein media (negative control) did notdisplay any DNA fragmentation and appearedsimilar to untreated cells (Figure 2). The resultssuggested that the cytotoxic compound hasinduced apoptosis in the A375 cell line.5This article can be downloaded from http://www.ijpmbs.com/currentissue.phpInt. J. Pharm. Med. & Bio. Sc. 2013 Glyn Hobbs et al. , 2013CONCLUSIONThe actinobacterium (GenBank accessionnumber EF585403) released a cytotoxicsubstance that induced apoptosis to malignantmelanoma A375 (ATCC no. CRL-1619).AKNOWLEDGMENTI would like to that the Society For GeneralMicrobiology for awarding me the President’sFund. I would also like to thank Prof PrakitsinSihanoth and Dr. Tanapat Palaga for allowing meto conduct my research in their laboratories.REFERENCES1. Blatt J, Taylor SR and Stitely S (1988),“Mechanism of antineuroblastoma activityof deferoxamine in vitro”, J. Lab. Clin. Med.,vol. 112, pp. 433-436.2. Blatt J and Stitely S (19870, “Antineuro-blastoma activity of desferoxamine inhuman cell lines”, Cancer Res., Vol. 47, pp.1749-1750.3. 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Effect of a siderophore producer on animal cell apoptosis: a possible role as anti-cancer agent.

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