New Insights into cytotoxic metabolites produced by Streptomyces griseus isolate KJ623766: Large scale production, structure elucidation and biological activities CURRENT

Natural products particularly microbial metabolites have been the mainstay of cancer chemotherapy and are likely to provide many of the lead structures and derivatives with new biological activities. In this research, the production of some potential cytotoxic metabolites from Streptomyces (S.) griseus isolate KJ623766 was carried out in 1 4 L laboratory fermenter under specified optimum conditions (28°C temperature, 200 RPM rotation speed, uncontrolled PH, 3 vvm aeration and 2 bar airflow pressure). Using 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide (MTT) assay, the cytotoxic activity of the ethyl acetate (1:1, v/v) extract of cell free culture supernatant (CFCS) against Caco2 and Hela cancer cell lines was determined with CD 50 of 14 µg/ml and 20 µg/ml, respectively. Bioassay guided fractionation of the ethyl acetate extract using different chromatographic techniques had led to the purification of the cytotoxic metabolites coded W1, R1 and R2 with reproducible amounts of 20, 5, and 1.5 mg/l, respectively. The structures of respective metabolites were determined using various spectroscopic analysis and identified as genistein, β-rhodomycinone and γ- rhodomycinone, respectively. Accordingly, S. griseus isolate KJ623766 can be used as a potential industrial strain for the large scale production of the isoflavonoid genistein, as well as for the production of β-and γ- rhodomycinone to be used for the construction of new derivatives with more potent cytotoxic activities of the anthracycline family. This is the first report about the production of the isoflavonoid genistein by S. griseus KJ623766.


Introduction
Cancer diseases are a major health problem in both developed and developing countries.
Therefore, combating against cancer requires integration between primary, secondary, and tertiary medical care in any nation [1]. Liver cancer is most prevalent cancer in the some countries such as Egypt and Saudi Arabia particularly in men while colorectal cancer is the second highest cause of malignancy and death in both sex in the USA [2,3]. On the other hand, breast cancer is the most frequently diagnosed cancer and the leading causing of cancer death in females, worldwide [4]. Moreover, cervical cancer is the third leading cause of cancer death among females in the less developed countries [5,6].
Natural products particularly microbial metabolites have been the mainstay of cancer chemotherapy and are likely to provide many of the lead structures, that can be used as models for the discovering of new derivatives with improved biological activities [7].
These chemotherapeutic agents isolated from plant, marine and bacterial sources are commonly used in chemotherapy as well as to treat lupus and juvenile rheumatoid arthritis [8]. Various members of Streptomyces have made imperative contributions to human with their capabilities to produce various important active metabolites including antimicrobial, antioxidant, anticancer agents [8][9][10]. Many drugs such as bleomycins, dactinomycin, mitomycin C, daunomycin and doxorubicin originating from Streptomycetes are currently used in the treatment of cancer ande most of them were introduced into clinic before discovering their modes of actions [7]. Therefore, this study aimed at production, purification, structural elucidation and determining the biological activities of certain metabolites produced by S. griseus KJ623766, a recovered soil isolate previously screened in our lab to have a promising cytotoxic activities against various cancer cell lines [11] Materials And Methods

Cell lines
Three cell lines including, kidney epithelial cells derived from the African green monkey (Vero cell line, ATCC No.CCL-81), colorectal adenocarcinoma derived from human colon (Caco-2 cell line) and epitheliod carcinoma derived from human cervix (HeLa cell line) were obtained from VACSERA, Egypt. The Caco-2 and HeLa cell lines were used to study the cytotoxic activities of the crude extract and isolated metabolites; stock culture of these cell lines was grown in T-75 tissue culture flasks containing 20 ml of RPMI-1640 medium with 1% antibiotic antimycotic solution and 10% fetal bovine serum. The medium was changed at 48 h intervals and cells dissociated with trypsin solution (0.25% in phosphate buffer saline). Vero cell line is a continuous non-tumourigenic when a cell passage was not prolonged [12] and was used to analyze the cytotoxic activities of isolated metabolites. It was propagated in Eagle minimum essential medium (EMEM) with Hank's balanced salt solution (HBSS), supplemented with 10% Fetal bovine serum (FBS) and antibiotics (100 IU penicillin and 100 IU streptomycin/ml) solution, and maintained in EMEM with Earlʼs balanced salt solution (EBSS) supplemented with 2% FBS and antibiotics solution [12].

Preparation of seed culture
This was carried out according to Yin et al. [13] with some modifications as follows; surface inoculation of the tested isolate onto starch nitrate agar slant was carried out.
After 7 days of incubation at 28°C, the formed spores were scalped from the agar surface and suspended in 3 ml distilled water. An aliquot (200 µl) of the resulting spore bacterial suspension was used to inoculate 10 ml of the soybean meal medium (soybean 15 gm, glucose 15 gm, NaCl 5gm, CaCO 3 1gm per 1L of distilled water in 100 ml flask) and incubated at 28 o C with shaking 200 RPM for 72 hrs in C5KC shaking incubator (New Brunswick scientific, Edison, New jersey, USA). The culture obtained was used to inoculate the main culture.

Fermentation in a laboratory fermenter
This was done according to Radwan et al. [14] with some modifications, where the fermentation process was conducted in 14 L CelliGen 310 bioreactor (New Brunswick Scientific, Edison, NJ, USA), with its Reactor Process Control (RPC) software. Fermentation process was carried out using 5 liters working volume of soybean meal medium under a condition of 28°C incubation temperature; 200 RPM agitation speed, 3 vvm i.e 15 SLPM aeration rate and 2 bar airflow pressure. The dissolved oxygen concentration was adjusted to obtain 100% saturation at the beginning of the run and DO percentage was sensed by the DO probe and monitored during the fermentation process. PH was adjusted at PH 7 at the beginning of the run, remains uncontrolled along the fermentation process (72 h).
After inoculation with 3 days age culture of the test isolate (5% v/v) under aseptic conditions, the run started under the previously stated conditions. Fermentation process was left for 72 h during which foam was suppressed using silicon oil. The culture obtained was centrifuged at 6000 RPM for 10 min using EBA20 Centrifuge (Hettrich, Germany) and the cell free culture supernatant (CFCS) obtained was collected.

Extraction of cytotoxic metabolite(s)
1 L of CFCS was extracted with ethyl acetate at the level of 1:1 (v/v) in subsequent manner [15,16] and the collected organic layers were evaporated using rotavapour (Heidolph instruments GmbH and Co. Schwabach, Germany) under vacuum at 45°C. After complete evaporation, sample of the fraction residue was redissolved in 1.25% DMSO in tissue culture medium. The cytotoxic activity of the redissolved fraction was evaluated against Caco2 and Hela cancer cell lines using MTT assay. According to the screening program of American National Cancer Institute (NCI), a crude extract is generally considered to have in vitro cytotoxic activity if the CD 50 value is ≤ 30μg/m

Isolation of the cytotoxic metabolite(s)
Medium pressure chromatographic separations were carried out with a PuriFlash 4100 system (Interchim; Montluçon, France) consisting of a mixing HPLC quaternary pump, a PDA-UV-Vis detector 190-840nm, a fraction collector, and a sample loading module. For system controlling and process monitoring, Interchim Software 5.0 was used. 360 mg sample was dissolved in 50 mL of methanol then introduced into the column via dry load using 4 gm silica. Elution started with hexane:ethyl acetate (100%:0%) followed by ethyl acetate:methanol (100%:0%) gradient to yield 117 fractions. Elution was monitored using TLC (normal phase silica gel precoated plates F254, Merck, Germany) with UV-detection and spraying with vanillin sulphuric acid spray reagent to yield 12 major fractions (SG1-SG12); these fractions were subjected to MTT assay to measure cytotoxic activity of each against Caco-2 and Hela cell lines. Final purification steps were performed using preparative HPLC (Knauer, Germany) on Kromasil ODS preparative column (10 mm× 250 mm) at flow rates 4 ml/min and UV detection at 254, yielding metabolites W1, R1 and R2.
MTT assay was carried out to measure cytotoxic activity of each against Vero, Caco-2 and Hela cell lines.

Spectroscopic analysis of the recovered cytotoxic metabolites
Structures of the isolated metabolites were determined based on LCESIMS analysis performed using waters®xevo-tqd® (USA) and NMR spectroscopic analysis which recorded (Bruker AVANCE HD III 400 MHz spectrometer; Switzerland). Methanol-d4 or chloroform-d3 solvent (Sigma Aldrich, Germany) was used to dissolve the NMR samples and transferred to three mm NMR tubes thereafter (Bruker).

Cytotoxicity assay using MTT method
MTT assay was performed as described by Saliba et al. [17] with some modifications as follows; about 100 μl of the tested metabolite (1mg of the metabolite dissolved in 5% DMSO and tissue culture medium) was added to the well that contain 100 μl of tissue culture medium, followed by two fold serial dilution. A total of 12 dilutions were used for each metabolite to calculate CD 50 for each. Control wells contained two aliquots of 100 μl of ethyl acetate extract of soybean meal medium (1 mg dissolved in 5% DMSO and tissue culture medium) and 100 μl of tissue culture medium, followed by two fold serial dilutions.
After 24 hrs incubation period at 37 °C in CO 2 incubator, wells were washed with PBS, followed by incubation with 100 μl MTT solution (1 mg/ml) per each well for 1 hr at 37°C. Supernatants were obtained by decantation and the cells were mixed with 100 μl DMSO per each well to dissolve formazan particles. Elutes of the 12 wells of each tested metabolite were obtained and measured spectroscopically at 540 nm using differential wave length of 630 nm in Platos R496 Microplate reader AMD diagnostics, Graz, Austria.
Control wells were in the same way handled and the % cytotoxicity determined as follows [16]. Cytotoxicity% = 1-{A 540 of test culture/A 540 of control culture} ×100. [16].

Cytotxic activities of the CFCS ethyl acetate extract
The results showed that the CFCS ethyl acetate extract has potential cytotoxic activities against both cell lines with higher activity against Caco2 (CD 50 14 µg/ml) than Hela cell line (CD 50 20 µg/ml), which indicates the presence of metabolites with higher selectivity to human colorectal adenocarcinoma.

Isolation and purification of cytotoxic metabolite(s)
As determined by TLC with UV-detection and spraying with vanillin sulphuric acid spray reagent, a total of 12 major fractions (SG1-SG12) were obtained. All the fractions were subjected to MTT assay to measure the cytotoxic activity of each. The fraction SG-3 (70% Hexane) revealed a promising cytotoxic activity against Caco-2 and Hela cell lines (CD 50 equals to 9.4 µg/ml and 12.2 µg/ml, respectively). Results of TLC indicate the presence of two major metabolites and one minor metabolite. So further purification of the active fraction SG-3 was carried out using semi-preparative HPLC, by which three pure metabolites namely, W1, R1 and R2 were recovered (Fig. 1).

Cytotoxic activities of the recovered metabolites (W1, R1 and R2)
The cytotoxic activities against of the three metabolites, W1, R1 and R2 against Caco2 and Hela cell lines were measured and lower cytotoxic activities were observed against Vero cell line (Table 1). Being cancer cell lines, Caco2 and Hela cell lines have lost their normal regulation of growth or cell death and were more sensitive to the effect of cytotoxic agents than Vero cell line.  2S). β-rhodomycinone showed potent cytotoxic activity against Caco2 and Hela cell lines, the CD 50 were 6.3 µg/ml and 9.45 µg/ml, respectively. While lower cytotoxic activity was showen against Vero cell line (CD 50 = 64.8 µg/ml).
Comparison of 1 HNMR spectrum of R2 ( Table 3) with that of known anthracyclins in the literature showed strong similarity to the known metabolite γ-rhodomycinone ( Supplementary Fig. 3S). This was confirmed by EZI-MS which revealed pseudomolecular ion peak at m/z 369.09 (M-H)with the molecular formula C 20 H 18 O 7 (Fig. 7).
While lower cytotoxic activity was shown against Vero cell line (CD 50 = 67.3 µg/ml).

Discussion
In this study, we aimed at identifying the major active metabolites produced by S. griseus KJ623766, a local isolate previously recovered from a soil sample in our lab and showed a promising cytotoxic activities against various cancer cell lines [11]. The production was carried out in 14 L laboratory fermentor under a previously determined optimum conditions [11] in order to recover sufficient pure quantities of the active metabolites as an attempt for structural elucidation. Bioassay guided fractionation using different chromatographic techniques had led to the purification of cytotoxic metabolites coded W1, R1 and R2 in amounts enough to identify the chemical structures of the respective metabolites using various techniques such as mass, 1D and 2D NMR spectroscopic analysis. According, to the obtained results, the structures of respective metabolites were determined and identified as genistein, β-rhodomycinone and γ-rhodomycinone, respectively.
For the metabolite W1, the 1HMNR, ESI-MS and APT spectra were identical to those of the isoflavonoid aglycone genistein in the literature [18], thus metabolite W1 was unambiguously identified as genistein. However, till now it was not reported in literature that genistein is produced by Streptomyces sp. including S. griseus. Accordingly, this is the first report of the production of genistein by our studied isolate S. griseus KJ623766.
Genistein is an isoflavone metabolite found in a number of plants such as lupin, soybeans, kudzu, fava beans and psoralea being the principal food source [19,20] as well as in some medicinal plants, including Flemingia vestita [21] and F. macrophylla [22,23], coffee [24] and in Maackia amurensis cell culture [25]. Genistein showed potent cytotoxic activity against µg/ml and 43µg/ml respectively. These results matched with that reported by Ganai and H. Farooqi [18] and Russo et al. [26] which showed the anticancer activity of genistein against human colon cancer.
The cytotoxic activity of genistein was previously determined by inhibiting the intracellular signal transduction pathway that support and potentiate cell proliferation and growth as well as promoting the angiogenesis process [27]. The activity of genistein against Hela cell line may be due to inhibition of the expression of VEGF and VEGF receptors and Down-regulation of the expression of miR-27a which lead to induction of cell cycle arrest [26].
Both β-rhodomycinone (R1) and γ-rhodomycinone (R2) showed potent cytotoxic activity against Caco2 and Hela cell lines, the CD 50 were 7.11 µg/ml and 9.35 µg/ml respectively. While lower cytotoxic activity was shown against Vero cell line (CD 50 = 67.3 µg/ml) confirming its applicability to be used as a potential anticancer drug in human.
These results matched with that of Tsuji et al. [35] which showed that γ-rhodomycinone, had no differentiation inducing activity, but was cytotoxic however, this cytotoxicity activity against normal cell lines was much lower than cancer cell lines. Supong and his co works recently showed that ε-rhodomycinone produced by a rare actinomycete Nonomuraea rhodomycinica NR4-ASC07 has a potential antimalarial activity [36]. Further studies should be conducted to explore the biosynthetic pathways of the respective metabolites as well as to produce new derivatives of the anthracycline family with higher antitumor activities and lower side effects. The later can be achieved by structural modifications or microbial transformation of the recovered metabolites.

Conclusions
This study targeting large scale production of cytotoxic metabolites by S. griseus isolate KJ623766 on 14L fermentor and studying different recovery methods for downstream processing. The structures of isolated metabolites were determined based on the mass, 1D and 2D NMR spectroscopic analysis and identified as genistein, β-rhodomycinone and γrhodomycinone, respectively. The respective metabolites were biologically analyzed using various cancer cell lines for their cytotoxic actives and exhibiting promising cytotoxic activities. Accordingly, S. griseus isolate KJ623766 can be used as an industrial strain for the commercial production of the isoflavonoid genistein, as well as the production of βrhodomycinone and γ-rhodomycinone to be used for production of different derivatives of antitumor antibiotics of the anthracycline family.

Declarations
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Availability of data and materials
All data generated or analyzed during this study are included in this published article in the main manuscript and additional supporting file.  1HMNR spectra of isolated compound R1. Figure 6 1HMNR spectra of isolated compound R2.

Supplementary Files
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