Cytotoxicity of n-Butanol Extracts of Streptomyces Against Human Breast...

[Full title: Cytotoxicity of n-Butanol Extracts of Streptomyces Against Human Breast Cancer Cells]

OPEN ACCESS International Journal of Pharmacology ISSN 1811-7775 DOI: 10.3923/ijp.2017.969.979 Research Article Cytotoxicity of n-Butanol Extracts of Streptomyces Against Human Breast Cancer Cells Maher Obeidat Department of Biotechnology, Faculty of Agricultural Technology, Al-Balqa Applied University, Al-Salt 19117, Jordan Abstract Background and Objectives: Breast cancer is the second most common cancer in the world and the most frequent cancer among women This study was conducted to investigate the anticancer activities of n-butanol extracts prepared from Streptomyces isolated from soils in Jordan against breast cancer MCF 7 cells. Materials and Methods: After isolation and identification of Streptomyces isolates by conventional methods, n-butanol extracts were prepared from Streptomyces cultures. Hemolytic activity of extracts was determined The cytotoxic effect of non-hemolytic extracts on normal MCF 10 A cells was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H tetrazolium bromide (MTT) assay. The in vitro and in vivo selective cytotoxicity of extracts against breast cancer was estimated. The mechanism of action of extracts that exhibited in vivo cytotoxicity was determined. Isolates that gave in vivo cytotoxicity were identified by PCR amplification and sequencing. The IC 50 values of extracts were determined by non-linear regression analysis. For tumor volume inhibition ratio, one-way ANOVA was applied for statistical evaluation of data and significant differences were considered significant at p<0.05. Results: The white aerial mycelia and the production of Rectus-Flexibilis (RF) sporophores as well as soluble pigments were the most common among Streptomyces isolates that screened from soil samples. The non-hemolytic n-butanol crude extracts of Streptomyces isolates (48 isolates) were screened for their cytotoxicity against normal breast MCF 10 A cells. Results indicated that out of the 23 non-toxic extracts on MCF 10 A cells, extracts of 9 isolates showed selective in vitro cytotoxicity against breast cancer MCF 7 cells with IC 50 values ranged from 0.68-1.64 mg mL G 1 . It was found that in vivo inhibition of breast cancer tumor in experimental animals was significantly increased, at " = 0.05, after treatment with extracts of 3 Streptomyces isolates (S 7, S 17 and S 61). The DNA laddering (apoptosis feature) was observed in MCF 7 cells treated with extracts of isolates S 7 and S 61. Analysis of 16 S-23 S rRNA gene sequence revealed that those 3 isolates have maximal identity to the genus Streptomyces . Conclusion: The result of the current study suggests that n-butanol extracts of 3 Streptomyces isolates have selective cytotoxicity against breast cancer MCF 7 cells and 2 of the extracts induce apoptotic property in MCF 7 cells Key words: Streptomyces, n-butanol, cytotoxicity, breast cancer, 16 S rRNA Received: March 18, 2017 Accepted: July 17, 2017 Published: October 15, 2017 Citation: Maher Obeidat, 2017. Cytotoxicity of n-butanol extracts of streptomyces against human breast cancer cells. Int. J. Pharmacol., 13: 969-979 Corresponding Author: Maher Obeidat, Department of Biotechnology, Faculty of Agricultural Technology, Al-Balqa Applied University, Al-Salt 19117, Jordan Tel: 00962775609846 Copyright: © 2017 Maher Obeidat. This is an open access article distributed under the terms of the creative commons attribution License, which permitsunrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Competing Interest: The authors have declared that no competing interest exists Data Availability: All relevant data are within the paper and its supporting information files.

Int. J. Pharmacol., 13 (8): 969-979, 2017 INTRODUCTION Cancer is undoubtedly a serious and potentially life-threatening illness. It was reported that breast cancer is the second most common cancer in the world and the most frequent cancer among women with an estimated 1.67 million new cancer cases diagnosed in 2012 1 . This represents about 12% of all new cancer cases and 25% of all cancers in women. Breast cancer ranks as the fifth cause of death from cancer overall (522,000 deaths). In Jordan, cancer is the second most frequent cause of death after heart disease 2 . It was responsible for the death of more than 10,000 Jordanians from 1996 till now. A total of 8744 new cancer cases were registered by Jordan Cancer Registry (JCR) in 2013, of these, 5416 cases (61.9%) were among Jordanians 3 . Out of new cases of cancer recorded amongst Jordanians in the 2013, 2852 cases (52.6%) were females. Breast cancer was the most common among Jordanian females, with 1040 diagnoses in 2013 (36.5% of all cancers in women) 3 Natural products were and are still the main reservoir to investigate in search for remedies for various afflictions and cancer treatment is no exception 4-7 . Spread of cancer among human populations raises the demand to find an urgent and a safe therapy for this disease. However, efforts dedicated to screening of microbial natural products for cancer therapy is very small compared to other therapeutic natural products Streptomyces is an interesting Gram-positive soil bacterium and it is unusual among bacteria in having a complex developmental life cycle involving several morphologically distinct cell types: Spherical spore, branching hyphae that form a mycelium and aerial structures that turn into chains of spores Streptomyces species are of great industrial importance because of their ability to produce many clinically useful antibiotics 8 and has been the subject of many studies in the quest for novel antibiotics 9 It was found that several compounds isolated from Streptomyces were able to inhibit tumor growth. For example, Borrelidin which was isolated from S. rochei serves as an efficient inhibitor of lung metastasis of B 16-BL 6 melanoma cells 10 . Furthermore, Geldanamycin compounds isolated from S. hygroscopicus PNK 1-3 showed strong cytotoxicity against human epidermoid carcinoma cell lines of nasopharynx and breast cancer cell lines 11 . Also, Vijayabharathi et al 12 reported that Sterptomyces showed anticancer activity against HepG 2 (hepatic carcinoma) and HeLa (cervical carcinoma) in vitro . In addition, Yip et al 13 isolated a purified fraction from a novel strain of Streptomyces induced an anti-proliferative effect in MCF 7 and MDA-MB-231 breast cancer cells It is observed that all conventional medical treatments for cancer until now is hopeless. Therefore, alternative techniques are needed to be developed against cancer. All the efforts are dedicated to screening microbial natural products for cancer therapy are incomplete. For this reason, this research aims to examine the anticancer activities of Streptomyces isolates for the possible use to improve means for current cancer therapy. This study could be useful for scientists, pharmacists and doctors as they work to find new alternative therapies for cancer problem. Having a clearer understanding of how microbial metabolites may help to reconsider the current conventional research methods which are used for exploring new anticancer agents from natural sources MATERIALS AND METHODS Collection of samples and isolation of Streptomyces : A total of 150 soil samples were collected in summer 2015 from 12 locations of Jordan representing various habitats; including, Red Sea shore, Shoaib Valley, Dead Sea shore, Yarmouk river margins, Jordan river margins and Jordan desert. The bacterium Streptomyces was isolated according to the methods of Taddei et al 14 and Dastager et al 15 using Starch Casein Nitrate Agar (SCNA) medium supplemented with antifungal agents (50 mg L G 1 cyclohexamide and 50 mg L G 1 Nystatin). Inoculated SCNA plates with diluted soil were incubated at 30 E C up to 3 weeks. After incubation, typically pigmented dry powdery colonies were selected from mixed plate culture and subcultured on new SCNA plates and incubated at 30 E C for 3 weeks. The mass color of mature sporulating aerial mycelium and the distinctive colors of the substrate (reverse) mycelium were determined. The production of soluble pigments and the sporophore shapes were also recorded after growth on SCNA plates Streptomyces crude extracts preparation: Streptomyces cultures, prepared in 250 mL Tryptone Soy Broth (TSB) and incubated at 30 E C for 3 weeks, were centrifuged at 13,000 rpm for 10 min. The supernatant was extracted with an equal volume of n-butanol. Then, the extracts were filtered through 0.45 µm membrane syringe filter. The filtrated extracts were evaporated at 40 E C in water bath. After evaporation, the remained residues were resuspended in Phosphate Buffer Saline (PBS) to achieve a concentration of 200 mg mL G 1 concentration and used for testing the hemolytic and anticancer activities Hemolytic activity: Human erythrocytes were freshly prepared and used to determine the hemolytic activity of 970

Int. J. Pharmacol., 13 (8): 969-979, 2017 bacterial crude extracts. Hemolytic activity was tested on blood agar medium, composed from normal human erythrocytes (5%), by inoculating 50 µL of bacterial crude extract into each well (5 mm i.d.) prepared on blood agar plates. The type of hemolysis was determined after incubation of plates at 37 E C for 48 h 16 Cells and culture conditions: Two human cell lines, including; MCF 10 A (non-tumorigenic human breast epithelial cell line) and MCF 7 (human breast adenocarcinoma cell line) were kindly supplied from Dr. Saeid Ismaeil, Faculty of Medicine/University of Jordan and used in this study. The human cell line MCF 7 was used to investigate the anticancer activities of bacterial crude extracts. Whereas, MCF 10 A cells were used to determine the selectivity of anticancer activity produced from bacterial extracts against breast cancer MCF 7 cells The adherent human breast cancer cell line MCF 7 was grown in Dulbeccoʼs Modified Eagleʼs Medium (DMEM) 17 , pH 7.4, supplemented with 10% Feotal Bovine Serum (FBS), 40 µg mL G 1 gentamicin, 50 µM 2-mercaptoethanol, 10 mM N-2-hydroxyethylpiperzine-N-2-ethane sulfonic acid (HEPES), 1 mM sodium pyruvate, 100 U mL G 1 penicillin, 100 µg mL G 1 streptomycin and 2 mM L-glutamine and then they were harvested at ~70% confluence and subcultured every 48 h at 37 o C in a humidified 5% CO 2 incubator 17 . To harvest the adherent MCF 7 cells, growth medium was removed and cells were washed with PBS. To produce a cellular suspension, a cell dissociation solution made of trypsin-EDTA (1 X) was added and incubated at 37 o C for 5 min in a humidified 5% CO 2 incubator. Trypsinized cells were reseeded in fresh medium at ~10 5 cells mL G 1 and incubated at 37 o C in a humidified 5% CO 2 incubator The adherent MCF 10 A cells were grown in DMEM/F 12 media, pH 7.4, supplemented with 5% horse serum, 20 ng mL G 1 Epidermal Growth Factor (EGF), 10 µg mL G 1 insulin, 100 µg mL G 1 hydrocortisone, 10 ng mL G 1 cholera toxin, 100 U mL G 1 penicillin and 100 µg mL G 1 streptomycin. The cells were harvested, trypsinized and reseeded in the same manner as for MCF 7 cells in vitro cytotoxicity: For testing cytotoxicity against normal MCF 10 A cells, 200 µL of non-hemolytic bacterial crude extract (5 mg mL G 1 constant concentration; i.e. 2 mg well G 1 ) was added to 200 µL of harvested MCF 10 A cells in fresh medium, mixed thoroughly by pipetting and 100 µL medium was loaded into each well of 96 well micro test plate to achieve 500 µL total volume. Cells were plated at a density 4×10 4 cells well G 1 , counted by hemocytometer. Then, the 96 well micro test plates were incubated at 37 o C in a humidified 5% CO 2 incubator for 48 h. At the end of incubation time, the viability of cells was assessed by a cell colorimetric proliferation test called 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2 H tetrazolium bromide (MTT) 18-19 . The MTT assay was done 48 h after inoculation of the bacterial crude extract into cell suspension. Each well of the 96 well microtest plate received 40 µL (50 µg) of MTT and incubated at 37 E C for 2-4 h in a humidified 5% CO 2 incubator. After that, 100 µL of dimethyl sulfoxide (DMSO) was added. The optical densities were measured at 450 nm with 630 nm reference wavelength using ELISA microplate reader. Each treatment was performed in triplicate and repeated 5 times. The survival rate was determined by comparing the average of absorbance values with that in the control without extracts. The 50% inhibitory concentration (IC 50 ) was also determined To determine the selective anticancer activity of non-hemolytic crude extracts against breast cancer MCF 7 cells, the cytotoxicity assay was performed in the same manner as for MCF 10 A cells using bacterial extracts that did not exhibit cytotoxicity against MCF 10 A cells in vivo cytotoxicity: A total of 30 female rats were used for each bacterial n-butanol extract that exhibited a non-hemolytic selective cytotoxicity against MCF-7 cells (i.e. cytotoxic against MCF 7 cells but not against MCF 10 A cells). All rats were divided into 3 groups of 10 rats each. Rats in Groups I and II were induced mammary carcinogenesis by providing single subcutaneous injection in right pectoral area of 25 mg 7,12-Dimethylbenzanthracene (DMBA) in 1 mL emulsion of 0.75 mL oil and 0.25 mL normal saline to each rat 20 . During the experimental period, animals were observed daily and weighed weekly to assess their general health After DMBA administration, right pectoral area of all rats were followed up for the tumoral development. Palpation of mammary tumors began 1 month after animals received DMBA. The volume of every tumor was measured weekly using calipers. After 2 months of the breast cancer development, treatments with bacterial extract was started in group II (Treatment Group). In Group I: control animals received no treatment. In Groups II and III, animals were given extract (100 mg kg G 1 , b.wt.) daily through an oral gavage Groups III animals had been used as control group for the side effects of crude treatment. After 1 month of treatment, rats were sacrificed. The tumor volume inhibition ratio was calculated DNA extraction and measurement of apoptosis: DNA was extracted from breast cancer MCF 7 cells as well as from MCF 7 cells treated with different concentrations (100-1000 mg with 971

Int. J. Pharmacol., 13 (8): 969-979, 2017 100 unit interval) of each promising bacterial extract which had in vivo cytotoxicity after 48 h of incubation. Cells were incubated with proteinase-K for 30 min at 37 o C. Thereafter, they were washed twice with PBS and then lysed in cold lysis solution (5 m mol L G 1 of Tris, pH 7.4, 20 m mol L G 1 of EDTA, 0.5% (v/v) Triton X 100) for 20 min. Cell lysates were centrifuged at 14,000 rpm for 15 min and DNA was extracted from the aqueous phase with phenol: chloroform: isoamyl alcohol (25:24:1 (v:v:v)) as described previously 21 . DNA was precipitated with sodium acetate and 2 volumes of cold absolute ethanol. The extracted genomic DNA was electrophresized in 2% agarose gel at 60 V for 1 h using 0.5 X TBE as running buffer. After electrophoresis was completed, genomic DNA was visualized and photographed by UV Transillumination to determine the degree of apoptotic DNA fragmentation Molecular characterization of cytotoxic Streptomyces isolates: The DNA was extracted from Streptomyces isolates that their n-butanol extracts exhibited both in vitro and in vivo cytotoxicities according to the method of Perez-Roth et al 22 . After that, amplification of the interspacer region 16 S-23 S rDNA with GP 1 and GP 2 primers was carried out in a DNA Thermal Cycler for 35 reaction cycles as described previously 23 . The PCR products were analyzed by electrophoresis using 10 µL of each PCR sample with 2 µL of loading buffer loaded onto a 1% agarose gel. A 500 bp DNA ladder marker (Genedirex, USA) was used to estimate the approximate molecular weight of the amplified products (the predicted size is 300-400 bp). Generated bands were digitally photographed under UV light The sequences of the interspacer region 16 S-23 S rRNA gene from PCR products of Streptomyces isolates were determined with an Applied Biosystems model 373 A DNA sequencer by using the ABI PRISM cycle sequencing kit (Macrogen, Korea). The sequences were compared with those contained within GenBank 24 by using a BLAST search 25 Furthermore, the accession number for each sequence was kindly provided after submition to GenBank. The most closely related 16 S-23 S rRNA gene sequences to the isolates of this study were retrieved from the database Statistical analysis of cytotoxicity: To calculate inhibition percentage depending on the results obtained from MTT in vitro viability assay, the obtained absorbance values were corrected by subtracting the average absorbance of blank (medium + MTT) from average vehicle (medium + cells + MTT) absorbance and from average treatment (medium + cells + extract + MTT) absorbance. To calculate inhibition percentage of cells, the following formula was used: A B Inhibition (%) 100 A    where, A is the vehicle absorbance at 450 nm and B is the treatment absorbance at 450 nm 26 The 50% inhibitory concentration (IC 50 ) was determined by comparing the average of in vitro mortality values of 5 bacterial extract concentrations (1.25, 2.5, 5, 7.5 and 10 mg mL G 1 ; i.e. 0.25, 0.5, 1, 1.5 and 2 mg well G 1 , respectively) with that in the control without extract. The IC 50 values, regression equations and correlation coefficients (R 2 ) were determined by non-linear regression analysis (MS Excell, Microsoft Co., 2010). Each treatment was achieved in triplicate For in vivo study, the tumor volume inhibition ratio (%) was calculated by the following formula: A B Inhibition (%) 100 A    where, A is the average tumor volume of the control group (Group I) and B is the average tumor volume of the treated group (Group II) 26 . All data were expressed as the Mean±Standard Deviation (SD) Statistical analysis of in vivo tumor inhibition: For statistical evaluation of data for tumor volume inhibition ratio, one-way ANOVA (Tukeyʼs studentized range) was applied using the program IBM SPSS statistics 19.0 for Windows 27 . Significant differences were considered significant at p<0.05 RESULTS Phenotypic and microscopic characterization of isolates: It was found that 127 bacterial colonies isolated from the screened soil samples were belonged to the genus Streptomyces (Table 1) Streptomyces isolates were classified Table 1: Isolation of Streptomyces from Jordanian soils No. of bacterial colonies Location No. of samples (No. of Streptomyces ) Ajloun 14 44 (9) Amman 19 93 (19) Aqaba 12 47 (9) Balqa 25 106 (25) Irbid 25 113 (34) Jerash 8 32 (8) Karak 7 17 (4) Maʼan 8 18 (3) Madaba 10 29 (7) Mafraq 7 25 (2) Tafilah 8 24 (5) Zarqa 7 26 (2) Total 150 574 (127) 972

Int. J. Pharmacol., 13 (8): 969-979, 2017 Table 2: Classification of Streptomyces isolates based on morphological and cultural characteristics Color series of aerial mycelium -------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Streptomyces isolates Black Brown Gray Green Orange Pink Red White Yellow Total (%) No. of isolates 2 9 17 38 3 2 2 49 5 127 Pigment production Melanin 2 9 5 10 0 0 0 16 2 44 (34.65) Substrate 2 9 5 38 0 0 2 16 5 77 (60.63) Soluble 2 9 14 38 0 0 1 36 5 105 (82.68) Sporophore morphology Rectus-Flexibilis 2 4 11 34 2 0 2 34 5 94 (74.02) Monoverticillate 0 2 2 2 0 2 0 10 0 18 (14.17) Biverticillate 0 2 0 0 0 0 0 2 0 4 (3.15) Spiral 0 1 4 2 1 0 0 3 0 11 (8.66) Table 3: Hemolytic activity of n-butanol extracts of Streptomyces isolates against human erythrocytes Type of hemolysis a ------------------------------------------------------ Color series (Aerial Mycelium) " $ ( Black 1 0 1 Brown 2 2 5 Gray 5 7 5 Green 7 14 17 Orange 0 2 1 Pink 0 1 1 Red 0 1 1 White 11 23 15 Yellow 2 1 2 Total 28 51 48 a Type of hemolysis: $ ; complete hemolysis, " : Partial hemolysis, ( : No hemolysis according to the color of aerial mycelium into 9 color series (Table 2). The white color series were the most common among the isolates followed by the green color series. Production of melanin, substrate and soluble pigments was observed (Table 2). It was found thta 44 isolates were melanin producers, 77 isolates were able to produce substrate pigments and most of the isolates (82.68%) gave soluble pigments. The isolates that displayed orange and pink aerial mycelia did not produce substrate and soluble pigments. According to the shape of sporophores, the isolates were grouped into four groups (Rectus-Flexibilis (RF), Monoverticillate (MV), Biverticillate (BIV) and Spiral (S)) (Table 2). About three fourths of isolates (74%) produced RF sporophores. Whereas, it was found that only 2 isolates produced BIV sporophores Hemolytic activities: The Streptomyces crude extracts were tested for their hemolytic activity against human erythrocytes (Table 3). About one-third of the isolates (48 isolates; 37.8%) were non-hemolytic ( ( -type). The remaining 79 isolates exhibited either partial hemolysis ( " -type) or complete hemolysis ( $ -type) In vitro cytotoxicity: Non-hemolytic Streptomyces isolates (48 isolates) were screened for their cytotoxicity against normal MCF 10 A cell line (Table 4). To determine the degree of cytotoxicity of Streptomyces extracts, MTT assay was used. As illustrated in Table 4, 23 non-hemolytic extracts exhibited no (-) to low (+) cytotoxic activity against normal MCF 10 A cells. Whereas, the remaining non-hemolytic extracts were found to exhibit cytotoxicity against MCF 10 A cells ranging from moderate (++) to very high (++++). All extracts of isolates that displayed black, brown and pink aerial mycelia color were cytotoxic to MCF 10 A cells The 23 Streptomyces extracts that showed no to low cytotoxicities against MCF 10 A cells were screened for their selective cytotoxicity against breast cancer MCF 7 cells (Table 5). It was found that 14 extracts produced no to low cytotoxicity against MCF 7 cells. Whereas, 9 extracts, which had no to low cytotoxic effects against MCF 10 A cells, showed high to very high selective cytotoxicity against MCF 7 cells. These selective isolates distributed in three color series including gray (3 isolates), green (2 isolates) and white (4 isolates) color series (Table 5). Seven of them produced RF sporophores (Table 6), whereas isolates S 20 and S 46 produced MV and BIV sporophores, respectively As shown in Table 6, the IC 50 values of the 9 cytotoxic Streptomyces crude extracts against breast cancer MCF 7 cells ranged from 0.68-1.64 mg mL G 1 . It was observed that isolate S 46, which belonged to white color series and produced BIV sporophores, showed the highest significant cytotoxic effect against MCF 7 cells. According to the IC 50 values, MCF 7 cells were more susceptible to the cytotoxic crude extracts of Streptomyces isolates that produced white aerial mycelia in vivo cytotoxicity: When the tumor volume reached to about 200 mm 3 , treatment with nine Streptomyces crude extracts, which had in vitro selective cytotoxic activities against breast cancer MCF 7 cells, started in Group II of rats. Control group (Group I) received no treatment. The tumor volume inhibition ratio was calculated for each crude. As shown in Table 7, only crude extracts of three Streptomyces isolates (S 7, S 17 and S 61) appeared to induce significant 973

Int. J. Pharmacol., 13 (8): 969-979, 2017 Table 4: In vitro cytotoxicity of non-hemolytic n-butanol extracts of Streptomyces isolates against normal human breast MCF 10 A cells Cytotoxicity degree against MCF 10 A a ---------------------------------------------------------------------------------------------------------------------------------------------------------------------- Color series (Aerial Mycelium) - ± + ++ +++ ++++ Black 0 0 0 1 0 0 Brown 0 0 0 3 1 1 Gray 1 1 1 2 0 0 Green 1 3 6 0 2 5 Orange 0 0 1 0 0 0 Pink 0 0 0 0 0 1 Red 0 0 1 0 0 0 White 5 0 2 0 1 7 Yellow 1 0 0 0 1 0 Total 8 4 11 6 5 14 a Degree of cytotoxicity was graded on the basis of the relative value of absorbance to the vehicle: ++++, very high (<0.1), +++: High (0.1-<0.4), ++: Moderate (0.4-<0.7), +: Low (0.7-<0.9), ±: Very low (0.9-<0.95), -: Non-toxic (>0.95) Table 5: In vitro selective cytotoxicity of non-hemolytic n-butanol extracts of Streptomyces isolates against human breast cancer MCF 7 cells Cytotoxicity Degree against MCF 7 a Color series ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ (Aerial Mycelium) - ± + ++ +++ ++++ Black 0 0 0 0 0 0 Brown 0 0 0 0 0 0 Gray 0 0 0 0 3 0 Green 0 2 6 0 2 0 Orange 0 0 1 0 0 0 Pink 0 0 0 0 0 0 Red 0 1 0 0 0 0 White 1 1 1 0 1 3 Yellow 0 0 1 0 0 0 Total 1 4 9 0 6 3 a Isolates that exhibited no to low cytotoxicity against normal breast MCF 10 A cells were examined for their cytotoxicity against breast cancer MCF 7 cells. The degree of cytotoxicity was graded on the basis of the relative value of absorbance to the vehicle: ++++: Very high (<0.1); +++: High (0.1-<0.4); ++: Moderate (0.4-<0.7); +: Low (0.7-<0.9); ±: Very low (0.9-<0.95), -: Non-toxic (>0.95) Table 6: Median inhibitory concentration of selectively cytotoxic n-butanol extracts of Streptomyces isolates against breast cancer MCF 7 cells pigments ------------------------------------------- Isolate Color series Substrate Soluble Sporophore morphology a IC 50 b (mg mL G 1 ) S 7 Gray - Brown RF-Straight 1.21 (1.03-1.36) S 9 Gray - Brown RF-Fascicled 1.06 (0.94-1.23) S 10 Gray - Yellow RF-Straight 1.64 (1.38-1.90) S 17 Green Brown Brown RF-Flexous 1.40 (1.27-1.56) S 20 Green Brown Brown MV 1.34 (1.24-1.64) S 44 White - Pink RF-Flexous 0.94 (0.83-1.14) S 46 White - Pink BIV 0.68 (0.53-0.80) S 47 White - Pink RF-Fascicled 1.48 (1.25-1.73) S 61 White - Orange RF-Fascicled 0.94 (0.82-1.11) a RF: Rectus-Flexibilis, MV: Monoverticillate no spirals, BIV: Biverticillate no spirals, b IC 50 : Median inhibitory concentration. Confidence limits for the mean are given in parentheses and they are expressed in terms of a confidence coefficient with 95% interval Table 7: Tumor volume inhibition ratio for selectively cytotoxic n-butanol extracts of Streptomyces isolates Tumor volume inhibition ratio a (%) after (weeks) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Isolate 0 1 2 3 4 S 7 0 a 18.3±2.1 b 26.1±4.3 c 32.4±3.7 c 43.5±5.1 d S 9 0 a 6.7±2.9 b 8.1±3.2 b 8.5±3.0 b 9.3±3.2 b S 10 0 a 0 a 2.1±1.6 b 4.3±2.2 bc 6.2±1.9 c S 17 0 a 16.7±1.8 b 31.3±3.9 c 40.3±4.6 d 61.2±6.8 e S 20 0 a 0 a 0 a 2.1±1.7 b 5.5±3.5 b S 44 0 a 1.3±0.4 b 3.8±2.1 bc 7.2±4.1 cd 9.9±5.1 d S 46 0 a 0 a 0 a 2.9±1.1 b 5.6±2.9 b S 47 0 a 0 a 0 a 3.2±0.9 b 4.1±1.7 b S 61 0 a 14.3±2.6 b 20.1±5.3 bc 27.9±3.9 c 39.8±4.2 d a Inhibtion ratio was represented as Means±SD. Means±SD within column followed by the same letter are not significantly different (Tukeyʼs studentized range test: " = 0.05) 974

Int. J. Pharmacol., 13 (8): 969-979, 2017 Table 8: Molecular characterization of in vivo cytotoxic Streptomyces isolates Sequence --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Isolate GenBank Accession No No. of nucleotides a Closest phylogenetic relative b Score E-value Gaps (%) Identity (%) c S 7 KY 711432 430 S. fulvissimus DSM 40593 (CP 005080) 278 6 e-118 1 80 S 17 KY 711433 428 Streptomyces sp. SirexAA-E (CP 002993) 268 6 e-68 1 80 S 61 KY 711434 451 S. griseus subsp. griseus NBRC 13350 (AP 009493) 435 1 e-70 2 95 a Number of 16 S-23 S rRNA gene nucleotides used for the alignment, b GenBank acession number was provided between parentheses, c Percentage identity with the 16 S-23 S rRNA gene sequence of the closest phylogenetic relative of Streptomyces inhibition of breast cancer tumor in experimental animals. After 4 weeks of treatment, the mean tumor volume inhibition ratio of crudes of isolates S 7, S 17 and S 61 in treated group (Group II) was 43.5±5.1, 61.2±6.8 and 39.8±4.2% in that order compared with the untreated animals in Group I (Table 7). Whereas, extracts of the remaining isolates showed weak inhibitory effect (less than 10% mean tumor volume inhibition). No signs of toxicity (weight loss, ruffled fur and behavioral changes) were observed in any of the treated animals in Group II compared with that in Group III Mechanism of action: Agarose gel electrophoresis was performed to detect if the cytotoxic Streptomyces crude extracts induced DNA laddering (apoptotic feature) or DNA smearing (necrotic feature). DNA laddering was observed in response to the treatment of MCF 7 cells with n-butanol extract prepared from Streptomyces isolates S 7 and S 61 (Fig. 1) suggesting that these extacts induced apoptosis in breast cancer MCF 7 cells. Whereas, the cytotoxic extracts of the remaining Streptomyces isolate (S 17) at different concentrations did not induce apoptotic effect Molecular characterization of isolates: Based on conventional classification, it was found that isolate S 7 produced gray aerial mycelia, RF-Straight sporophores and brown soluble pigment, isolate S 17 produced green aerial mycelia, RF-Flexous sporophores and brown substrate and soluble pigments and isolate S 61 produced white aerial mycelia, RF-Fascicled sporophores and orange soluble pigment (Table 6). To confirm the classification of Streptomyces isolates that exhibited in vitro and in vivo cytotoxicities against MCF 7 cells, genomic DNA was extracted from S 7, S 17 and S 61 isolates. The 16 S-23 S rRNA gene sequence was analyzed by amplification with GP 1 and GP 2 primers. The amplified genomic DNA of the isolates produced a single PCR band of about 500 bp in size (Fig. 2). The obtained 16 S-23 S rRNA gene sequences were aligned by BLAST alignment of GenBank sequences. Based on BLAST alignment, the three isolates were allocated to the phylum Actinobacteria that contains the genus Streptomyces with 80-95% identity (Table 8). Moreover, the Fig. 1: Agarose gel (2%) electrophoresis of DNA fragmentation after exposure of MCF 7 cells to n-butanol extracts of Streptomyces isolates S 7, S 17 and S 61. A slight fragmentation of DNA was demonstrated after treatment of MCF 7 cells with S 7 and S 61 extracts Fig. 2: Agarose gel (1%) electrophoresis of PCR amplification of 16 S-23 S rRNA gene fragments with forward GP 1 and reverse GP 2 primers of Streptomyces isolates S 7, S 17 and S 61. Lane M: 50 bp DNA ladder marker (Genedirex, USA). Lanes of isolates S 7, S 17 and S 61 show amplicons (about 500 bp) derived from 16 S-23 S rRNA gene 975 MCF 7 MCF 7+ S 7 MCF 7+ S 17 MCF 7+ S 61 M S 7 S 17 S 61 500 bp

Int. J. Pharmacol., 13 (8): 969-979, 2017 sequences were closely related to the 16 S-23 S rRNA gene sequence of Streptomyces retrieved from GenBank database The sequences of the 3 isolates were submitted to GenBank and the GenBank accession numbers KY 711432, KY 711433 and KY 711434 were assigned to the obtained sequences of Streptomyces isolates S 7, S 17 and S 61, respectively DISCUSSION The current study was initiated to determine the anticancer activity of n-butanol extracts of Actinomycetes, in particular local Streptomyces isolates, against breast cancer MCF 7 cells in attempt to find isolates with novel or promising anticancer activities. Actinomycetes are well known and successfully exploited as a source of secondary metabolites. There are over 23,000 known microbial secondary metabolites, 42% of which are produced by Actinobacteria, 42% by fungi and 16% by other bacteria 28 . Since the discovery of actinomycin, actinobacteria have been found to produce many commercially important bioactive compounds and antitumor agents 10 in addition to enzymes of industrial interest 29-30 . It has been estimated that approximately twothird of the thousands of naturally occurring antibiotics have been isolated from actinomycetes 31-32 In the present study, the occurrence of local Streptomyces was investigated in the collected soil samples and 127 Streptomyces isolates were recovered. The isolates were classified based on the aerial mycelium color into 9 color series; isolates with white color series appeared the most common (49 isolates). This finding is in agreement with Msameh 33 . It was observed that majority of Streptomyces isolates were able to produce RF sporophores. This is in agreement with previous studies 34-36 which demonstrated that sporophores of RF-type were the most common among Jordanian isolates In earlier studies, several metabolites isolated from Streptomyces species exhibited anticancer activity. For example, retamycin assists in the treatment of human leukemia 37 , migrastatin inhibits metastasis of several types of tumor cells 38-39 , geldanamycin has strong cytotoxicity against human epidermoid carcinoma of nasopharynx and breast cancer 11 and borrelidin has an inhibitory effect on lung cancer metastasis 10 . Furthermore, chromomycin SA and 1-(1 H-indol- 3-yl)-propane-1,2,3-triol 40 as well as kosinostatin 41 , isolated recently from Streptomyces , were found to show anticancer activity against MCF 7 cells. In this study, it was found that certain non-hemolytic n-butanol extracts of Streptomyces isolates had selective cytotoxic activity against human breast cancer MCF 7 cells. Out of the screened Streptomyces isolates in this work, non-hemolytic n-butanol extracts of nine Streptomyces isolates exhibited selective in vitro cytotoxicity against MCF 7 cells. It was demonstrated that the cytotoxic extracts were obtained from isolates belonging to different phenotypic and microscopic characters. So, nonhemolytic and selective cytotoxic compounds can be found in a variety of Streptomyces species. The anticancer activity in cytotoxic isolates was not attributable to the induced hemolysis in view of the fact that they showed no hemolytic activity against human erythrocytes It was found that only 3 isolates (S 7, S 17 and S 61) exhibited in vivo antitumor activity. Tumor volume inhibition ratio differences between the treatment and control groups were all significant and no signs of toxicity were observed in any of the treated animals. Moreover, it was observed that isolates S 7 and S 61 induced apoptosis in treated breast cancer MCF 7 cells. Whereas, S 17 isolate did not induce apoptosis in treated cells. Therefore, these 3 n-butanol extracts of Streptomyces isolates which exhibited selective in vitro cytotoxicity against breast cancer cells by discrimination between MCF 7 cancer cells and normal MCF 10 A cells, killing the former cells specifically and produced a promising in vivo cytotoxicity might be used in the future in therapies of breast cancer Abraham 42 demonstrated that n-butanol extracts of Streptomyces gave cytotoxic activity on PC 12 and HeLa cancer cell lines. Furthermore, it was revealed that n-butanol extracts of Streptomyces exhibited the highest antibacterial activity 43 . Therefore, n-butanol was chosen as the most efficient solvent for extracting the desired active compounds. However in this study, none of the n-butanol extracts of Streptomyces isolates that exhibited in vitro cytotoxicity against MCF 7 cells gave antimicrobial activity against 11 test bacteria and 7 test fungi (data not shown). Therefore, the inhibitory effect of n-butanol extracts on MCF 7 cells might be due to novel antitumor agents contained in the prepared extracts but not due to antimicrobial agents The preliminary identification of the 3 isolates that exhibited in vivo cytotoxicity against breast cancer MCF 7 cells by conventional methods was further identified by comparing the 16 S-23 S rRNA gene sequences of the isolates with those in the GenBank. As a result, the sequences of the 3 isolates (S 7, S 17 and S 61) showed sequence identity to the phylum Actinobacteria and close relatedness to the genus Streptomyces with 80-95% identity (Table 8). In general, identity below 97% 16 S rRNA gene sequence of unknown isolate with nearest relative is indicative of novel species or new type strain. Nevertheless, the globally adapted cut-off value for genus Streptomyces is 83.5% 16 S rRNA gene 976

Int. J. Pharmacol., 13 (8): 969-979, 2017 sequence identity 44 . However, it was reported that the lowest interspecies 16 S rRNA gene sequence similarity to the genus Streptomyces is 78% 45 . Therefore, these isolates can be assigned to the genus Streptomyces . Considering sequence analysis using 16 S rRNA gene, it was revealed that the isolates could be allocated into different Streptomyces species However, this needs to be further confirmed by fatty acid analysis, protein profiling, DNA hybridization and other techniques because in Streptomyces systematic sequences of only about 180 species (out of more than 570 species) are available and deposited in public databases 46 It was reported that yellow soluble pigment produced from Streptomyces sp. SFA 5 has inhibitory activity against breast cancer MCF 7 cells 47 . It was observed that all in vitro cytotoxic Streptomyces isolates produced soluble pigments with various colors; brown from isolates S 7, S 9, S 17 and S 20, yellow from isolate S 10, pink from isolates S 44, S 46 and S 47 and orange from isolate S 61 (Table 6). Therefore, in vitro cytotoxicity produced from n-butanol extracts of these isolates on MCF 7 breast cancer cell line could be attributed to the produced soluble pigments The finding of this study strongly suggests the possible occurrence of some Streptomyces isolates in soils that naturally produce selectively cytotoxic agents against breast cancer cells. This may lead to the use of these agents for medicinal and pharmaceutical purposes including treatment of breast cancer and possibly other types of cancer. Although n-butanol extracts from Streptomyces bacterium exhibit potent in vitro and in vivo anticancer activity against breast cancer MCF 7 cell line, further analysis using chromatography techniques such as GC, HPLC and HPTLC is required to determine the chemical structure of biologically active constituent found in crude extracts CONCLUSION This study demonstrated that there is a potent anticancer effect of n-butanol extracts prepared from Streptomyces on the growth of human breast cancer MCF 7 cells. Moreover, significant tumor volume inhibition ratios of 3 Streptomyces extracts (2 of them have apoptotic effects) were revealed in the experimental animals. The results of this study illustrated that anticancer agents can be screened from natural microbial products and they be used in medical and pharmaceutical therapies of cancer SIGNIFICANCE STATEMENT This study showed that natural microbial byproducts can be used as an alternative source for screening of anticancer agents. Moreover, the results of this study will be useful in medical and pharmaceutical applications including development and/or improvement of current therapies used in treatment of breast cancer ACKNOWLEDGEMENTS The author is grateful to “Abdul Hameed Shoman Fund for Supporting Scientific Research, grant no. AHSF-10/2011” for the financial support. A lot of thanks to Mr. Ismail Otri, Department of Biotechnology/Al-Balqa Applied University, for technical help REFERENCES 1 WHO., 2012. Breast cancer: Estimated incidence, mortality and prevalence worldwide in 2012. World Health Organization (WHO), International Agency for Research on Cancer. http://globocan.iarc.fr/old/FactSheets/cancers/ breast-new.asp 2 Al-Tarawneh, M., S. Khatib and K. Arqub, 2010. Cancer incidence in Jordan, 1996-2005. Eastern Mediterr. Health J., 16: 837-845 3 Ministry of Health, 2013. Jordan cancer registry. The 18 th Annual Report 2013, Statistic Summary for Cancer Incidence in Jordan, Non-Communicable Diseases Directorate, pp: 3-11 4 Itokawa, H., S.L. Morris-Natschke, T. Akiyama and K.H. Lee, 2008. Plant-derived natural product research aimed at new drug discovery. J. Nat. Med., 62: 263-280 5 Newman, D.J. and G.M. Cragg, 2012. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J. Nat. Prod., 75: 311-335 6 Njuguna, N.M., C. Masimirembwa and K. Chibale, 2012. Identification and characterization of reactive metabolites in natural products-driven drug discovery. J. Nat. Prod., 75: 507-513 7 Veeresham, C., 2012. Natural products derived from plants as a source of drugs. J. Adv. Pharm. Technol. Res., 3: 200-201 8 Valli, S., S.S. Suvathi, O.S. Aysha, P. Nirmala, K.P. Vinoth and A. Reena, 2012. Antimicrobial potential of Actinomycetes species isolated from marine environment. Asian Pac. J. Trop. Biomed., 2: 469-473 9 Hopwood, D.A., 2007. Streptomyces in Nature and Medicine: The Antibiotic Makers. Oxford University Press, New York, ISBN: 9780199722280, Pages: 272 10. Vino, S. and K.R. Lokesh, 2008. Borrelidin: A promising anticancer agent from Streptomyces species. Adv. Biotech., 6: 22-26 11. Jongrungruangchok, S., S. Tanasupawat, P. Kittakoop, R. Bavovada and H. Kobayashi et al ., 2006. Identification of Streptomyces and Kitasatospora strains from Thai soils with geldanamycin production strain. Actinomycetologica, 20: 10-14 977

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