# Optimization of the Cytotoxic Activity of ThreeStreptomyces Strains Isolated from Guaviare River Sediments (Colombia, South America)

Authors: Laura Ramirez-Rodriguez; Boghos Stepanian-Martinez; Maria Morales-Gonzalez; Luis Diaz
Journal: BioMed Research International
 (2018)

---

## Abstract

ThreeStreptomycesstrains isolated from Guaviare sediments (Colombia, South America) with cytotoxic activity against prostate cancer (PC3), breast cancer (MDA-MB-231), and lung cancer (A549) line cells were studied. The present investigation reveals the enhancement of the cytotoxic activity evaluating different values of pH, carbon sources (sucrose, glucose, xylose, maltose, and starch), and nitrogen sources (malt extract, yeast extract, meat extract, peptone, and potassium nitrate). Based on the response surface methodology, the isolatesStreptomyces aburaviensis (73) had the maximum activity for lung cancer (IC50= 25.00
±
1.86 ppm) with 4% of yeast extract, 3% of starch, and a pH value of 7.Streptomyces gramineus (386) had the maximum activity against prostate cancer (IC50= 6.14
±
2.07 ppm) with 5% of malt extract, 3% of glucose, and a pH value of 6. Finally,Streptomyces psammoticus (519) had the maximum activity against breast cancer (IC50= 35.53
±
2.71 ppm) with 1% of yeast extract, 4% of starch, and a pH 8. The results suggest that the ethyl acetate extracts from isolatesStreptomyces aburaviensis (73),Streptomyces gramineus (386), andStreptomyces psammoticus (519) have a potential for use in pharmaceuticals as cytotoxic agents.


---

## Body

## 1. Introduction

Cancer is a disease that has great relevance today because it is one of the most serious human health problems in the world [1]. In 2018, 1,735,350 new cancer cases and 609,640 cancer deaths are projected to occur in the United States [2]. The most common types of cancers in 2017 were prostate with 19% and lung and bronchus cancer with 14% for men [3]. Siegel et al. [2] state that, for women, the most commonly diagnosed cancers are breast, lung and bronchus, and colorectum; which collectively represent one-half of all cases. The lifetime probability of being diagnosed with invasive cancer is higher for men (39.7%) than for women (37.6). The American Cancer Society states that cancer occurs when there is uncontrolled cell growth and spread of abnormal cells caused by external factors, such as inherited genetic mutations, hormones, and immune conditions [3]. Cancer is treated with surgery, radiation, chemotherapy, hormone therapy, immune therapy, and targeted therapy [4]. Chemotherapy is one of the most effective antitumor treatments, but this therapy needs a new active compound as the major sources of natural products with anticancer activity are microbes [5]. Maskey et al. indicate thatActinobacteria is famous as a great source of antitumor compounds, as cited in [6], mainlyStreptomyces sp. There are several anticancer drugs derived fromStreptomyces sp. like Proximicin A, B, and C, Daunomycin, Drimentine G, Indotertine B, Bleomycin, and Benzastatins that are already in clinical use [6].Streptomyces spp. are Gram positive filamentous, sporulating bacteria with DNA rich in G+C content [7]; they have been isolated in major part from soil and aquatic habitats like rivers which have been poorly explored as a source ofStreptomyces spp. with biological activity (antibacterial, antifungal, antitumor, and antioxidant) [8]. These bacteria are the major source of bioactive compounds since they shelter different gene clusters, thus producing different compounds with diverse chemical backbones [9]. Most of these compounds are secreted in the broth and can be extracted with organic solvents [10]. The aim of this study is to investigate the cytotoxic activity (against PC3, MDA-MB-231, and A549 cancer cell lines) of extracts fromStreptomyces broths isolated from a Colombian river, establishing the best conditions of carbon source, nitrogen source, and pH.


## 2. Materials and Methods

### 2.1.Streptomyces Strains and Their Maintenance

A total of forty Actinobacterial strains were selected based on their antimicrobial activity from the previously described collection of 275 isolates collected from two different locations in the Guaviare river, Colombia (2°34′51.6′′N 72°41′34.7′′W and 2°34′51.4′′N 72°39′53.6′′W) [8]. Morphological parameters such as aerial and substrate mycelium arrangements were carried out on ISP 3 medium as described in Bergey’s Manual of Systematic Bacteriology [11] and by Shirling and Gottlieb [12]. The spore mass and mycelium of the pure isolate were stored at −80°C as glycerol suspension (40%, v/v) for further analysis.


### 2.2. Preparation of Active Organic Extracts

Streptomyces strains were inoculated in 50 mL Erlenmeyer flask containing 10 mL of ISP 2 (8.0 g of glucose, 10.0 g of malt extract, and 4.0 g of yeast extract, pH 7.2
±
2°C) for seven days at 30°C under continues shaking (200 rpm) [13], and the medium was sterilized by autoclaving at 121°C for 15 min prior to experiment [14]. Further, equal volume (1:1) of ethyl acetate was added and mixed by vigorous shaking (200 rpm). The organic phase was collected, washed with three volumes of water, and evaporated to dryness. The extract was suspended in phosphate buffered saline (PBS) at a concentration of 1000 ppm [15, 16].


### 2.3. Cytotoxicity Assay

Human A549 lung adenocarcinoma cancer cell line (ATCC® CCL-185), PC3 adenocarcinoma prostate cancer cell line (ATCC CRL-7934), and L929 mouse fibroblasts (ATCC CRL-6364) were grown as monolayer culture in DMEM medium (10% (v/v) with fetal bovine serum, 1% (v/v) penicillin, and 1% (v/v) streptomycin). The breast cancer cell line MDA-MB-231 (ATCC HTB-26) was incubated in RPMI (10% (v/v) with fetal bovine serum, 1% (v/v) penicillin, and 1% (v/v) streptomycin). The cells were maintained in a humidified atmosphere with 5% CO2 at 37°C [17]. Briefly, 100 μL of cell suspension was inoculated to 96-well plates with a density of 5 × 103 cells/well and cultured for 24 h. After that, culture medium was replaced with 100 μL serum-free medium containing various concentrations (10, 25, 50, 100, and 200 ppm) ofStreptomyces broth extracts, each extract was tested in triplicate based on Ravikumar et al. [16]. Doxorubicin (1-25 ppm) was used as the cytotoxic positive control, medium supplemented with bovine serum albumin 1% (w/v) as negative control (100% survival), and free medium containing PBS as blank [18].The cell viability was assessed by adding 10μl of MTT (5 mg/mL) per well, after 48 h of incubation, and the plates were incubated with 5% of CO2 at 37°C for 3 h. The formazan crystals formed in the cells were dissolved with 100 μL of DMSO measured at 570 nm using a microplate reader (iMarkTM Microplate Reader) [19]. Cytotoxicity of each sample was expressed as an IC50 value. The IC50 value is the concentration of the test sample that causes 50% inhibition of cell growth averaged from three replicate experiments [15].


### 2.4. Effect of Different Carbon and Nitrogen Sources on Cytotoxic Activity

To improve the cytotoxic activity of treeStreptomycesstrains with the highest cytotoxic activity (73, 386, and 519), preliminary experiments of carbon and nitrogen sources were carried out.Streptomyces strains were grown in 12 well microplates with 5 mL basal agar ((NH4)2SO4 2.64 g/L, KH2PO4 2.38 g/L, K2HPO4 4.31 g/L, MgSO4 x 7H2O 1.0 g/L) and 1.0 ml/L trace element solution (CuSO4 x 5H2O 0.64 g/L, FeSO4 x 7H2O 0.11 g/L, ZnSO4 x 7H2O 0.15 g/L, MnCl2 x 4H2O), supplemented with 0.8% (w/v) of carbon source: lactose, galactose, ribose, xylose, maltose, starch, sucrose, fructose, and carboxymethylcellulose, with glucose as positive control and no carbon source as negative control [8]. Nitrogen sources were evaluated in the same way at the same conditions at 1.4% (w/v): L-asparagine, L-glutamine, proline, ammonium sulphate, meat extract, yeast extract, potassium nitrate, peptone, and malt extract [18]. The experiments were performed for 7 days, pH 7, 200 rpm, and 30°C; the results demonstrated that the five carbon sources that enhanced the cytotoxic activity ofStreptomyces strains were glucose, xylose, maltose, starch, and sucrose. Likewise, the nitrogen sources selected were meat extract, yeast extract, potassium nitrate, peptone, and malt extract [20].


### 2.5. Media Optimization

The influence of environmental and nutritional conditions in growth culture, such as initial pH (6–8) and carbon and nitrogen sources concentrations (0,1-5% w/v) [21], on the cytotoxic activity was evaluated using a face central composite design (FCCD), evaluating 15 trials with five levels for each factor.The experimental results of FCCD were statistically studied by analysis of variance (ANOVA). All the experiments were done in triplicate, and the average of the cytotoxic activity was obtained and taken as the dependent variable or response (Y). The results were fitted via the response surface regression procedure using the following second order polynomial equation [22]:(1)
Y
=
β
0
+
∑
i
=
1
n
β
i
x
i
+
∑
i
=
1
n
-
1
∑
j
=
i
+
1
n
β
i
j
x
i
x
j
+
∑
i
=
1
n
β
i
i
x
i
2in which Y is the predicted response, β
0 is the regression coefficients, β
i is the linear coefficient, β
i
i is the quadratic coefficients, β
i
j is the interaction coefficients, and X
i is the coded levels of independent variables [20].


### 2.6. Molecular Characterization

Total genomic DNA was isolated using the phenol chloroform method [23]. PCR amplification of 16S rRNA gene locus was conducted using primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′- TACGGYTACCTTGTTACGACTT-3′). The PCR product was detected by agarose gel electrophoresis [8]. PCR thermal cycling is described elsewhere [24]. PCR products were sequenced by Macrogen (Korea). Sequences were assembled using the CLC Main Workbench 7.0 program and were aligned using SINA. The sequences were subjected to homology search using EzTaxon system (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and the BLAST program of the National Centre for Biotechnology Information (NCBI) [18].


### 2.7. LC-MS Analysis

The dry extracts were dissolved in methanol and were subjected to LC-MS analysis on LCMS-2020 (Shimadzu Corp, Japan) system equipped with a single quadrupole analyser and an electrospray ion source (ESI). The analysis was done using a Synergi RP C18 column (Phenomenex) dimension 150 × 4.4 mm and film thickness of 4 μm (0.7 mL/min flow rate; 30°C column oven temperature). 0.1% formic acid in acetonitrile and 0.1% formic acid in water were used as mobile phases A and B, respectively. The gradient elution was done as follows: 0 min, 10% A; 3 min, 10% A; 14 min, 40% A; 21 min, 70% A; 25 min, 100% A; 30 min, 10% A. ESI was simultaneously operated in positive and negative ion mode (scan 200–800 m/z; 250°C CDL temperature; 1.2 kV detector voltage; 1.3 L min−nebulizing gas flow rate; 8.0 L min−drying gas flow rate) [25].


### 2.8. Statistical Analysis

Design Experts® v7 software was used for the experimental designs and the models were statistically studied by analysis of variance (ANOVA) applied to the established regression, with a statistical significance (P<0.05). The surface plots were made with Statistica ® software.


## 2.1.Streptomyces Strains and Their Maintenance

A total of forty Actinobacterial strains were selected based on their antimicrobial activity from the previously described collection of 275 isolates collected from two different locations in the Guaviare river, Colombia (2°34′51.6′′N 72°41′34.7′′W and 2°34′51.4′′N 72°39′53.6′′W) [8]. Morphological parameters such as aerial and substrate mycelium arrangements were carried out on ISP 3 medium as described in Bergey’s Manual of Systematic Bacteriology [11] and by Shirling and Gottlieb [12]. The spore mass and mycelium of the pure isolate were stored at −80°C as glycerol suspension (40%, v/v) for further analysis.


## 2.2. Preparation of Active Organic Extracts

Streptomyces strains were inoculated in 50 mL Erlenmeyer flask containing 10 mL of ISP 2 (8.0 g of glucose, 10.0 g of malt extract, and 4.0 g of yeast extract, pH 7.2
±
2°C) for seven days at 30°C under continues shaking (200 rpm) [13], and the medium was sterilized by autoclaving at 121°C for 15 min prior to experiment [14]. Further, equal volume (1:1) of ethyl acetate was added and mixed by vigorous shaking (200 rpm). The organic phase was collected, washed with three volumes of water, and evaporated to dryness. The extract was suspended in phosphate buffered saline (PBS) at a concentration of 1000 ppm [15, 16].


## 2.3. Cytotoxicity Assay

Human A549 lung adenocarcinoma cancer cell line (ATCC® CCL-185), PC3 adenocarcinoma prostate cancer cell line (ATCC CRL-7934), and L929 mouse fibroblasts (ATCC CRL-6364) were grown as monolayer culture in DMEM medium (10% (v/v) with fetal bovine serum, 1% (v/v) penicillin, and 1% (v/v) streptomycin). The breast cancer cell line MDA-MB-231 (ATCC HTB-26) was incubated in RPMI (10% (v/v) with fetal bovine serum, 1% (v/v) penicillin, and 1% (v/v) streptomycin). The cells were maintained in a humidified atmosphere with 5% CO2 at 37°C [17]. Briefly, 100 μL of cell suspension was inoculated to 96-well plates with a density of 5 × 103 cells/well and cultured for 24 h. After that, culture medium was replaced with 100 μL serum-free medium containing various concentrations (10, 25, 50, 100, and 200 ppm) ofStreptomyces broth extracts, each extract was tested in triplicate based on Ravikumar et al. [16]. Doxorubicin (1-25 ppm) was used as the cytotoxic positive control, medium supplemented with bovine serum albumin 1% (w/v) as negative control (100% survival), and free medium containing PBS as blank [18].The cell viability was assessed by adding 10μl of MTT (5 mg/mL) per well, after 48 h of incubation, and the plates were incubated with 5% of CO2 at 37°C for 3 h. The formazan crystals formed in the cells were dissolved with 100 μL of DMSO measured at 570 nm using a microplate reader (iMarkTM Microplate Reader) [19]. Cytotoxicity of each sample was expressed as an IC50 value. The IC50 value is the concentration of the test sample that causes 50% inhibition of cell growth averaged from three replicate experiments [15].


## 2.4. Effect of Different Carbon and Nitrogen Sources on Cytotoxic Activity

To improve the cytotoxic activity of treeStreptomycesstrains with the highest cytotoxic activity (73, 386, and 519), preliminary experiments of carbon and nitrogen sources were carried out.Streptomyces strains were grown in 12 well microplates with 5 mL basal agar ((NH4)2SO4 2.64 g/L, KH2PO4 2.38 g/L, K2HPO4 4.31 g/L, MgSO4 x 7H2O 1.0 g/L) and 1.0 ml/L trace element solution (CuSO4 x 5H2O 0.64 g/L, FeSO4 x 7H2O 0.11 g/L, ZnSO4 x 7H2O 0.15 g/L, MnCl2 x 4H2O), supplemented with 0.8% (w/v) of carbon source: lactose, galactose, ribose, xylose, maltose, starch, sucrose, fructose, and carboxymethylcellulose, with glucose as positive control and no carbon source as negative control [8]. Nitrogen sources were evaluated in the same way at the same conditions at 1.4% (w/v): L-asparagine, L-glutamine, proline, ammonium sulphate, meat extract, yeast extract, potassium nitrate, peptone, and malt extract [18]. The experiments were performed for 7 days, pH 7, 200 rpm, and 30°C; the results demonstrated that the five carbon sources that enhanced the cytotoxic activity ofStreptomyces strains were glucose, xylose, maltose, starch, and sucrose. Likewise, the nitrogen sources selected were meat extract, yeast extract, potassium nitrate, peptone, and malt extract [20].


## 2.5. Media Optimization

The influence of environmental and nutritional conditions in growth culture, such as initial pH (6–8) and carbon and nitrogen sources concentrations (0,1-5% w/v) [21], on the cytotoxic activity was evaluated using a face central composite design (FCCD), evaluating 15 trials with five levels for each factor.The experimental results of FCCD were statistically studied by analysis of variance (ANOVA). All the experiments were done in triplicate, and the average of the cytotoxic activity was obtained and taken as the dependent variable or response (Y). The results were fitted via the response surface regression procedure using the following second order polynomial equation [22]:(1)
Y
=
β
0
+
∑
i
=
1
n
β
i
x
i
+
∑
i
=
1
n
-
1
∑
j
=
i
+
1
n
β
i
j
x
i
x
j
+
∑
i
=
1
n
β
i
i
x
i
2in which Y is the predicted response, β
0 is the regression coefficients, β
i is the linear coefficient, β
i
i is the quadratic coefficients, β
i
j is the interaction coefficients, and X
i is the coded levels of independent variables [20].


## 2.6. Molecular Characterization

Total genomic DNA was isolated using the phenol chloroform method [23]. PCR amplification of 16S rRNA gene locus was conducted using primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′- TACGGYTACCTTGTTACGACTT-3′). The PCR product was detected by agarose gel electrophoresis [8]. PCR thermal cycling is described elsewhere [24]. PCR products were sequenced by Macrogen (Korea). Sequences were assembled using the CLC Main Workbench 7.0 program and were aligned using SINA. The sequences were subjected to homology search using EzTaxon system (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and the BLAST program of the National Centre for Biotechnology Information (NCBI) [18].


## 2.7. LC-MS Analysis

The dry extracts were dissolved in methanol and were subjected to LC-MS analysis on LCMS-2020 (Shimadzu Corp, Japan) system equipped with a single quadrupole analyser and an electrospray ion source (ESI). The analysis was done using a Synergi RP C18 column (Phenomenex) dimension 150 × 4.4 mm and film thickness of 4 μm (0.7 mL/min flow rate; 30°C column oven temperature). 0.1% formic acid in acetonitrile and 0.1% formic acid in water were used as mobile phases A and B, respectively. The gradient elution was done as follows: 0 min, 10% A; 3 min, 10% A; 14 min, 40% A; 21 min, 70% A; 25 min, 100% A; 30 min, 10% A. ESI was simultaneously operated in positive and negative ion mode (scan 200–800 m/z; 250°C CDL temperature; 1.2 kV detector voltage; 1.3 L min−nebulizing gas flow rate; 8.0 L min−drying gas flow rate) [25].


## 2.8. Statistical Analysis

Design Experts® v7 software was used for the experimental designs and the models were statistically studied by analysis of variance (ANOVA) applied to the established regression, with a statistical significance (P<0.05). The surface plots were made with Statistica ® software.


## 3. Results

Forty Actinobacterial extracts at 100 ppm were tested in the cytotoxic assay against prostate (PC3), lung (A549), and breast (MDA-MB-231) cancer lines (Table1).Streptomyces strains 73, 386, and 519 presented the highest cytotoxic percent so that values of IC50 (Table 2) were calculated to improve their cytotoxic activity.Table 1
Cytotoxic activity of forty Actinobacterial extracts against prostate (PC3), lung (A549), and breast (MDA-MB-231) cancer lines (Table1). NA: non-activity. The results are expressed as an average ± standard deviation (n=3).
Strain
Cytotoxic Activity
(%)
PC3
MDA-MB-231
A549
4
NA
NA
13.05±2.08
5
3.11±5.21
20.2±3.75
13.5±5.18
11
3.06±3.57
18.5±4.31
13.1±3.57
17
3.85±4.12
21.1±2.97
6.9±2.78
18
NA
16.11±5.26
1.5±2.35
20
24.24±2.78
NA
10±6.43
26
35.44±2.97
24.2±4.82
4.7±2.54
28
50.46±3.3
58.3±5.32
9.9±1.59
29
6.98±23.65
35.7±5.11
NA
30
18.78±2.26
NA
51.5±4.72
32
76.5±5.82
61±4.73
36.21±4.62
41
18.57±2.49
22.5±5.65
37.32±3.38
42
16.7±3.86
46.3±6.76
38.2±5.33
44
13.48±1.27
7.035±4.98
NA
48
15.6±5.42
5.05±3.97
NA
73
88.21 ± 3.85
64.94±1.96
75.69±2.42
83
NA
2.8±4.48
0.7±3.85
105
49.2±4.21
46.5±5.64
56.32±3.49
159
15.86±2.39
30.6±2.37
26.9±5.32
171
31.94±1.76
49.8±4.84
24.9±4.39
194
2.78±3.12
16±7.11
NA
239
30.41±6.72
57.1±3.16
10.5±4.61
245
5.74±3.21
27.1±5.96
NA
259
34.2±3.31
17.31±4.64
37.02±5.83
278
30.93±2.98
16.82±0.12
57.11±4.74
294
16.87±4.98
66.6±2.34
1.46±3.76
339
47.41±6.27
36.6±4.72
3.1±5.56
363
27.86±3.45
53.5±4.61
3.5±4.42
386
89.73±2.07
59.28±2.67
78.12±3.01
389
15.12±4.21
39.2±4.16
23±5.35
424
7.72±3.08
49.7±6.23
1.6±3.78
429
21.12±2.73
35.7±4.97
1.4±3.34
431
30.42±4.84
39.7±3.52
20.21±3.16
441
NA
NA
39.1±4.56
452
2.1±3.21
NA
0.8±3.42
461
NA
5.5±6.95
7±4.21
487
5.21±3.2
NA
13.4±5.67
491
11.12±4.37
20±1.01
9.31±3.98
519
85.12±4.76
62.17±4.36
84.71±2.86Table 2
Determination of IC50 values for the extracts of strains 73, 386, and 519 cultivated in ISP 2; against prostate (PC3), lung (A549), and breast (MDA-MB-231) tumour lines. The results are expressed as an average ± standard deviation (n=3).
IC
50
(ppm)
Isolate
PC3
A549
MDA-MB-231
73
64.84±3.85
67.69±2.42
43.24±1.96
386
6.14±2.07
33.17±2.67
70.72±4.87
519
64.58±4.76
60.54±2.86
45.89±2.36TheStreptomyces strains 73, 386, and 519 were characterized based on colony, morphology, mycelium coloration, and pigment diffusion. The light microscopy observation of isolates on ISP 3 media showed a straight chain section with no fragmentation of the aerial mycelium. The isolates are Gram positive strains and presented spores surfaced oval shaped with short compact spirals indicating that they belong to the genusStreptomyces. [26]. Consequently,Streptomyces 73 showed an aerial and substrate mycelium, white and grey, respectively.Streptomyces 386 and 519 showed a white aerial mycelium and brown substrate mycelium with a pigment production similar to melanin (Figure 1).Figure 1
Morphologic features and Gram stain ofStreptomyces strains with cytotoxic activity grown in ISP 3 agar medium.


### 3.1. Taxonomical Identification

The phylogenetic data of the 16S rRNA gene sequences of three isolates were obtained aligning the nucleotides, and the representative sequences of related type strains of the genusStreptomyceswere retrieved from the GenBank/EMBL/DDBJ databases using CLUSTAL-X software [27]. Phylogenetic trees were constructed with the Neighbor-Joining method [28] (Figures 2, 3, and 4) using MEGA version 6.0 [29]. The EzTaxon system (http://www.ezbiocloud.net/eztaxon) was used to calculate sequence similarities [30]. Considering that the most recent definition of a prokaryotic species based on 16S rRNA gene sequence similarity is determined by a minimum similarity of 98.7% [31], the isolate 386 was identified asStreptomyces gramineus[32] with 100% similarly (accession number HM748598.2); the isolate 73 had close similarity with two Actinobacterial strains:Streptomyces avellaneus [33] (accession number AB184413.1, 99.77% sequence similarity) andStreptomyces aburaviensis[34] (accession number AY999779.1, 99.77% sequence similarity), but, according to morphological studies, the isolate 73 was identified asStreptomyces aburaviensis[35]; and the isolate 519 was identified asStreptomyces psammoticus (accession number AB184554.2, 99.77% sequence similarity).Figure 2
Neighbor-Joining phylogenetic tree based on 16S rRNA gene sequence showing the relationship between strain 73 and representatives of some other related taxa.Figure 3
Neighbor-Joining phylogenetic tree based on 16S rRNA gene sequence showing the relationship between strain 386 and representatives of some other related taxa.Figure 4
Neighbor-Joining phylogenetic tree based on 16S rRNA gene sequence showing the relationship between strain 519 and representatives of some other related taxa.


### 3.2. Effect of Carbon and Nitrogen Sources on Cytotoxic Activity

Five carbon and nitrogen sources were evaluated to increase the cytotoxic activity of extracts from three isolates; that is why the ISP 2 media was modified (8.0 g carbon source and 14 g of nitrogen source); the results for carbon source are shown in Tables3 and 4. The carbon source to which the strainStreptomyces aburaviensis (73) had the higher cytotoxic percent was starch, obtaining cytotoxic values of 91.22%, 83.9%, and 68.1% at 100 ppm for prostate, lung, and breast cancer. Likewise, the values of IC50 for each cell line were 50.26
±
2.54, 26.16
±
3.26, and 26.65
±
0.86 ppm, respectively, being more active for lung tumour cell line (A549). The strainStreptomyces gramineus (386) had the highest cytotoxic percent, with glucose as a carbon source for the PC3 tumour line and xylose for A549 and MDA-MB-231, obtaining values of 90.64%, 83.18%, and 65.63%, respectively. The strainStreptomyces gramineus (386) was more active for prostate tumour cell line (PC3) using glucose as a carbon source with an IC50 value of 11.18
±
3.85 ppm. Finally, the strainStreptomyces psammoticus (519) had the highest cytotoxic percent at 100 ppm with xylose as the carbon source, obtaining values of cytotoxic percent at 100 ppm higher than 80%, and the values of IC50 were 55.80
±
2.96, 48.58
±
2.39, and 39.98
±
3.27 ppm for prostate, lung, and breast cancer, respectively; this strain was more active for breast cancer (MDA-MB-231).Table 3
Determination of the carbon source that improved the cytotoxic percent at 100 ppm of theStreptomyces extract for prostate (PC3), lung (A549), and breast (MDA-MB-231) tumour lines. The results are expressed as an average ± standard deviation (n=3).
%
Cytotoxicity at 100 ppm
73
386
519
PC3
A549
MDA
PC3
A549
MDA
PC3
A549
MDA
Saccharose
90.07 ± 0.21
70.31± 3.15
65.96± 3.06
89.81± 0.5
75.11± 0.74
65.1± 3.61
88.71± 0.82
85.18± 0.18
59.18± 0.84
Glucose
89.39 ± 0.63
78.17± 2.54
63.27± 1.62
90.64± 0.56
80.69± 1.38
57.11± 2.03
89.10± 0.67
86.78± 2.86
66.17± 1.83
Maltose
89.05 ± 1.05
76.09± 0.89
57.86± 2.74
87.64± 1.86
72.28± 1.24
61.33± 1.07
89.70± 0.48
86.61± 4.87
59.27± 0.72
Xylose
89.97 ± 1.77
81.83± 4.76
65.69± 3.01
89.60± 0.57
83.18± 1.63
65.63± 2.32
91.12± 1.02
87.78± 1.26
74.76± 2.79
Starch
91.22 ± 0.08
83.90± 1.05
68.10± 1.24
88.93± 1.02
81.49± 0.38
64.35± 0.84
90.59± 0.22
87.79± 0.58
62.58± 0.67Table 4
Determination of the IC50 using the best carbon and nitrogen sources in Streptomyces strains for prostate (PC3), lung (A549), and breast (MDA-MB-231) tumour lines. The results are expressed as an average ± standard deviation (n=3).
IC
50
Isolate
PC3
A549
MDA-MB-231
Carbon source/ Nitrogen source
73
50.26± 2.54
26.16± 3.26
26.65± 0.86
Starch
74.36± 1.05
69.13± 1.05
45.39± 3.06
Yeast Extract
386
11.18± 3.85
31.64± 1.45
68.57± 1.08
Xylose
14.59± 2.82
16.35± 4.03
71.25± 1.72
Malt Extract
519
55.80± 2.96
48.58± 2.39
39.98± 3.27
Xylose
68.43± 2.74
50.49± 3.64
41.23± 2.84
Malt ExtractThe results of the cytotoxic activity of different nitrogen sources in the ISP 2 broth are shown in Tables4 and 5. The nitrogen sources that improved the cytotoxic activity for the isolateStreptomyces aburaviensis (73) were meat extract for the prostate tumour cell line and yeast extract for lung and breast cancer, obtaining cytotoxic percent of 77.14%, 63.45%, and 75.78% at 100 ppm, respectively. The IC50 values were measured using yeast extract as a nitrogen source, and the IC50 values were 74.36
±
1.05, 69.13
±
1.05, and 45.39
±
3.06 ppm for prostate, lung, and breast tumour cell lines. The strainStreptomyces gramineus (386) had the best cytotoxic activity with malt extract with values of cytotoxic percent higher than 50% and had the highest cytotoxic activity for the prostate tumour cell line with an IC50 value of 14.59
±
2.82 ppm. Furthermore, the strainStreptomyces psammoticus (519) had the best cytotoxic activity using malt extract as a nitrogen source with values of 49.13%, 59.73%, and 77.59% for lung, breast, and prostate cancer, respectively, and had the lowest IC50 for the breast tumour cell line with a value of 41.23
±
2.84 (Table 4).Table 5
Determination of the nitrogen source that improved the cytotoxic percent at 100 ppm of theStreptomyces extract for prostate (PC3), lung (A549), and breast (MDA-MB-231) tumour lines. The results are expressed as an average ± standard deviation (n=3).
%
Cytotoxicity at 100 ppm
73
386
519
PC3
A549
MDA
PC3
A549
MDA
PC3
A549
MDA
Yeast extract
75.86± 0.35
63.45± 0.65
75.78± 0.61
69.32± 0.28
23.18± 3.96
18.07± 2.70
70.9 ±0.92
37.86± 0.79
46.49 ±0.80
Malt extract
68.72± 0.4
22.50± 0.59
33.24± 0.03
86.76± 4.76
67.93± 5.72
54.46± 0.36
77.59± 1.2
49.13± 2.55
59.73 ±2.16
Meat extract
77.14± 1.5
33.69± 5.58
48.85± 0.02
72.93± 0.98
21.82± 0.85
29.77± 1.54
68.42± 0.56
40.91± 2.96
47.46 ±1.96
Peptone
71.12± 5.32
29.13± 5.93
37.59± 0.94
68.79± 3.92
28.56± 5.51
48.00± 0.98
64.92± 4.98
33.96± 4.81
33.96 ±2.83
Potassium nitrate
67.51± 3.75
16.07± 4.15
22.91± 1.87
66.39± 1.81
33.10± 1.93
37.28± 1.79
71.80± 3.74
31.18± 3.62
31.18 ±0.08


### 3.3. Media Optimization Using Central Composite Design

After establishing the carbon and nitrogen sources, the effects of the concentration of nitrogen source (A), carbon source (B), and pH (C) were studied using a central composite to determine the optimum level of these parameters leading to the maximum cytotoxic percent. The experimental design was tested carrying out 15 experiments for each isolate in the cell line that had the best cytotoxic effect; results are presented in Table6.Table 6
Experimental design central composite design representing the cytotoxic percent byStreptomycesstrains influenced by four variables and five levels of each on A: carbon source concentration, B: nitrogen source concentration, and C: pH.
Run
Independent variables
%
Cytotoxicity (100 ppm)
Observed value
Predicted Value
A
B
C
73
386
519
73
386
519
(
%
w/v)
(
%
w/v)
Against PC3
Against A549
Against MDA
Against PC3
Against A549
Against MDA
1
2.5
2
6
78.05
83.482
73.204
77.319
84.139
71.740
2
2.5
0.4
8
75.075
74.138
72.774
73.985
76.455
69.707
3
0.5
2
8
71.182
82.695
87.936
70.728
84.274
85.239
4
0.5
0.4
6
75.075
75.54
74.374
75.060
75.090
75.003
5
0.1
0.8
7
74.44
81.127
72.715
73.952
80.956
74.620
6
5
0.8
7
76.138
81.127
72.715
76.401
80.439
74.271
7
1.4
0.1
7
70.717
81.705
72.834
70.258
81.766
70.462
8
1.4
4
7
72.547
77.078
75.952
72.753
76.486
76.688
9
1.4
0.8
5
72.547
77.078
75.952
72.044
77.487
75.054
10
1.4
0.8
9
74.824
83.369
72.434
74.720
81.934
74.001
11
1.4
0.8
7
73.085
80.37
73.929
73.382
80.819
74.528
12
1.4
0.8
7
70.717
80.078
72.709
73.382
80.819
74.528
13
1.4
0.8
7
72.767
81.494
74.946
73.382
80.819
74.528
14
1.4
0.8
7
72.767
81.568
72.918
73.382
80.819
74.528
15
1.4
0.8
7
74.198
82.272
74.033
73.382
80.819
74.528One model was obtained from analysis of variance for each extract. F-test for the models showed statistical significance (P<0,05), indicating that all models fit and can adequately explain the variations observed in the cytotoxic percent. Moreover, the lack of fit was not significant in all models at the 5% level (P<0.05), indicating that the experimental data obtained fit well with the model [21]. Coefficients of correlation R2 for the isolatesStreptomyces aburaviensis(73),Streptomyces gramineus (386), andStreptomyces psammoticus (519) were 0.8089, 0.8677, and 0.8020, respectively.Table7 shows the statistics analysis of cytotoxic activity from the isolateStreptomyces gramineus (386). As can be observed, the variables A, BC, and C2 were significant (P value<0,05). The regression equation for the model in terms of cytotoxic percent (Y) as a function of three independent variables (A: %w/v malt extract, B: %w/v glucose, C: pH) is given below. (2)
Y
386
=
5
,
861
+
36
,
996
A
+
2
,
260
B
+
12
,
823
C
-
5
,
3
A
C
-
0,516
B
C
-
0
,
277
C
2The response surface plot for the cytotoxic activity ofStreptomyces gramineus (386) against lung cancer shows the interactive effect of different variables (%w/v glucose, %w/v malt extract, and pH) presented in Figure 5(b). The highest theoretical value to maximize the cytotoxic percent to 100% was with 5% of malt extract, 3% of glucose, and a pH value of 6.Table 7
Analysis of variance for the fit second order polynomial model and lack of fit for the cytotoxic percent by composite central design for the strainStreptomyces gramineus (386). SS: sum of squares, DF: degrees of freedom, MSS: mean sum of squares.
Source
Sum of squares
DF
Mean square
F value
p-value
Model
101.406
6
16.901
8.746
0.0037
A
0.162
1
0.162
0.084
0.7790
B
18.572
1
18.572
9.611
0.0147
C
32.920
1
32.920
17.037
0.0033
AC
90.358
1
90.358
46.762
0.0001
BC
0.644
1
0.644
0.333
0.5795
C∧2
1.987
1
1.987
1.028
0.3402
Residual
15.458
8
1.932
Lack of Fit
12.147
4
3.037
3.669
0.1180
Pure Error
3.310
4
0.827
Cor Total
116.865
14Figure 5
Surface plots for interactions between nitrogen source concentration and carbon source concentration at the best pH predicted for (a)Streptomyces aburaviensis (73) pH 6 and (b)Streptomyces gramineus (386) pH 7. The surface plot for (c) Streptomyces psammoticus (519) represents the interaction between carbon source concentration and pH with best nitrogen source concentration (1% w/v) predicted.
(a)
(b)
(c)The regression equation for the cytotoxic activity from theStreptomyces aburaviensis(73) is shown in (3), where Y is the response in terms of cytotoxic percent, and the variables were %w/v yeast extract (A), %w/v starch (B), and pH (C). The significant terms (P value<0,05) for this model are A, B, C, AB, AC y B2. In the response surface plot presented in Figure 5(a), it can be observed that the highest point to obtain 100% of cytotoxicity appeared at 4% of yeast extract, 3% of starch, and a pH value of 7. (3)
Y
73
=
95
,
859
-
22
,
383
A
+
0
,
546
B
-
3
,
566
C
+
3.5660
A
B
+
2
,
671
A
C
+
0
,
260
A
2
-
1
,
194
B
2Finally, the model for the cytotoxic activity from theStreptomyces psammoticus(519) is given below as (4), showing the interactive effect between the variables %w/v malt extract (A), %w/v xylose (B), and pH (C). The significant terms (P value<0,05) for this model were B, C, BC y B2. The response surface plot presented in Figure 5(c) shows that the highest point for the isolate 519 against breast cancer is at 1% of yeast extract, 4% of starch, and a pH value of 8. (4)
Y
519
=
104
,
110
-
0
,
071
A
-
33
,
498
B
-
4
,
890
C
+
5
,
784
B
C
-
1
,
316
B
2The best conditions of the tested variables obtained from the experimental design CCD had been verified experimentally (in triplicate) and compared with the predicted data (Table 8). The measured cytotoxic activity at 100 ppm was 89.29% ±4.05, 89.03% ±6.47, and 66.18% ±2.83 forStreptomyces aburaviensis(73),Streptomyces gramineus (386), andStreptomyces psammoticus (519), respectively. The predicted values from the model were 97.47%, 97.96%, and 95.89%. The verification revealed a high degree of accuracy of the model forStreptomyces aburaviensis(73) andStreptomyces gramineus (386), indicating the model’s validation under the tested conditions. Moreover, the values of IC50 were measured at the best growth and nutritional conditions showing an increase compared with the initial results (Table 2). These assays were tested in L929 cell line without significative cytotoxic activity at low concentrations of extract (Figure 6).Table 8
Verification of the accuracy of the models obtained with CCP and de IC50 at the best environmental and growth conditions. Experimental results are expressed as an average ± standard deviation (n=3).
Isolate
%
Cytotoxicity at 100 ppm
Predicted
%Error
IC
50
(ppm)
Cell line
73
89.29% ±4.05
97.47
8.40%
25.00±1.86
A549
386
89.03%± 6.47
95.45
6.73%
7.93±0.98
PC3
519
66.18%±2.83
94.96
30.31%
35.53±2.71
MDA-MB-231Figure 6
Cytotoxic activity tested in L929 cell line forStreptomyces aburaviensis(73),Streptomyces gramineus (386), andStreptomyces psammoticus (519). Standard deviation (n=3).


### 3.4. Dry Biomass Growth Curve

The growth curves (Figure7) obtained for this study were constructed from the average dry biomass obtained from three experiments after 10 days. The average of the dry biomass with standard error bars was plotted with the help of the statistical program GraphPad Prism® version 6.01. The Gompertz model (Table 9) was used to describe the sigmoidal microbial growth curves shown in Figure 7; all models exhibit a high correlations (R2) with values of 0.99 forStreptomyces aburaviensis(73), 0.98 forStreptomyces gramineus(386), and 0.98 forStreptomyces psammoticus(519).Table 9
Results of Gompertz model growth rate (μMax), the lag time (
λ
), and the coefficients of correlation (R2).
Isolate
μ
M
a
x
(
d
a
y
)
λ
(day)
R
2
73
1.18
1.71
0.99
386
0.98
2.64
0.95
519
0.50
1.26
0.98Figure 7
Growth kinetics ofStreptomyces aburaviensis (73) against lung tumour cell line (a),Streptomyces gramineus (386) against prostate tumour cell line (b), andStreptomyces psammoticus(519) against breast tumour cell line (c) and dependent variables growth (mg; (blue filled circles)) and %cytotoxicity (%; (black filled squares)). Standard deviation (n=3).
(a)
(b)
(c)


### 3.5. LC-MS Analysis

The chromatograms obtained for the extracts tested showed defined signal patterns characteristic of each type of extract as indicated by arrows (Figure8). Organic extracts showed the majority of signals between 11 and 28 min retention time (RT) and signals with mass/charge (m/z) relationship values from 160 to 820. The extract ofStreptomyces gramineus (386) differs from the extracts ofStreptomyces aburaviensis (73) andStreptomyces psammoticus(519) in the signals corresponding to retention time of 11.67 min, m/z= 408.6 and 26.44 min, m/z= 709.4. The strainStreptomyces psammoticus(519) presented characteristic signals at 13.62 min, m/z= 207.25 and 17.01 min, m/z= 453.2 andStreptomyces aburaviensis (73) at 27.25 min, m/z= 817.7.Figure 8
Liquid Chromatography comparison spectrum for organic extracts fromStreptomyces gramineus (386),Streptomyces aburaviensis (73), andStreptomyces psammoticus(519) and control extract from ISP 2 media without biomass (the arrows indicate the peaks that differ from the other extracts).


## 3.1. Taxonomical Identification

The phylogenetic data of the 16S rRNA gene sequences of three isolates were obtained aligning the nucleotides, and the representative sequences of related type strains of the genusStreptomyceswere retrieved from the GenBank/EMBL/DDBJ databases using CLUSTAL-X software [27]. Phylogenetic trees were constructed with the Neighbor-Joining method [28] (Figures 2, 3, and 4) using MEGA version 6.0 [29]. The EzTaxon system (http://www.ezbiocloud.net/eztaxon) was used to calculate sequence similarities [30]. Considering that the most recent definition of a prokaryotic species based on 16S rRNA gene sequence similarity is determined by a minimum similarity of 98.7% [31], the isolate 386 was identified asStreptomyces gramineus[32] with 100% similarly (accession number HM748598.2); the isolate 73 had close similarity with two Actinobacterial strains:Streptomyces avellaneus [33] (accession number AB184413.1, 99.77% sequence similarity) andStreptomyces aburaviensis[34] (accession number AY999779.1, 99.77% sequence similarity), but, according to morphological studies, the isolate 73 was identified asStreptomyces aburaviensis[35]; and the isolate 519 was identified asStreptomyces psammoticus (accession number AB184554.2, 99.77% sequence similarity).Figure 2
Neighbor-Joining phylogenetic tree based on 16S rRNA gene sequence showing the relationship between strain 73 and representatives of some other related taxa.Figure 3
Neighbor-Joining phylogenetic tree based on 16S rRNA gene sequence showing the relationship between strain 386 and representatives of some other related taxa.Figure 4
Neighbor-Joining phylogenetic tree based on 16S rRNA gene sequence showing the relationship between strain 519 and representatives of some other related taxa.


## 3.2. Effect of Carbon and Nitrogen Sources on Cytotoxic Activity

Five carbon and nitrogen sources were evaluated to increase the cytotoxic activity of extracts from three isolates; that is why the ISP 2 media was modified (8.0 g carbon source and 14 g of nitrogen source); the results for carbon source are shown in Tables3 and 4. The carbon source to which the strainStreptomyces aburaviensis (73) had the higher cytotoxic percent was starch, obtaining cytotoxic values of 91.22%, 83.9%, and 68.1% at 100 ppm for prostate, lung, and breast cancer. Likewise, the values of IC50 for each cell line were 50.26
±
2.54, 26.16
±
3.26, and 26.65
±
0.86 ppm, respectively, being more active for lung tumour cell line (A549). The strainStreptomyces gramineus (386) had the highest cytotoxic percent, with glucose as a carbon source for the PC3 tumour line and xylose for A549 and MDA-MB-231, obtaining values of 90.64%, 83.18%, and 65.63%, respectively. The strainStreptomyces gramineus (386) was more active for prostate tumour cell line (PC3) using glucose as a carbon source with an IC50 value of 11.18
±
3.85 ppm. Finally, the strainStreptomyces psammoticus (519) had the highest cytotoxic percent at 100 ppm with xylose as the carbon source, obtaining values of cytotoxic percent at 100 ppm higher than 80%, and the values of IC50 were 55.80
±
2.96, 48.58
±
2.39, and 39.98
±
3.27 ppm for prostate, lung, and breast cancer, respectively; this strain was more active for breast cancer (MDA-MB-231).Table 3
Determination of the carbon source that improved the cytotoxic percent at 100 ppm of theStreptomyces extract for prostate (PC3), lung (A549), and breast (MDA-MB-231) tumour lines. The results are expressed as an average ± standard deviation (n=3).
%
Cytotoxicity at 100 ppm
73
386
519
PC3
A549
MDA
PC3
A549
MDA
PC3
A549
MDA
Saccharose
90.07 ± 0.21
70.31± 3.15
65.96± 3.06
89.81± 0.5
75.11± 0.74
65.1± 3.61
88.71± 0.82
85.18± 0.18
59.18± 0.84
Glucose
89.39 ± 0.63
78.17± 2.54
63.27± 1.62
90.64± 0.56
80.69± 1.38
57.11± 2.03
89.10± 0.67
86.78± 2.86
66.17± 1.83
Maltose
89.05 ± 1.05
76.09± 0.89
57.86± 2.74
87.64± 1.86
72.28± 1.24
61.33± 1.07
89.70± 0.48
86.61± 4.87
59.27± 0.72
Xylose
89.97 ± 1.77
81.83± 4.76
65.69± 3.01
89.60± 0.57
83.18± 1.63
65.63± 2.32
91.12± 1.02
87.78± 1.26
74.76± 2.79
Starch
91.22 ± 0.08
83.90± 1.05
68.10± 1.24
88.93± 1.02
81.49± 0.38
64.35± 0.84
90.59± 0.22
87.79± 0.58
62.58± 0.67Table 4
Determination of the IC50 using the best carbon and nitrogen sources in Streptomyces strains for prostate (PC3), lung (A549), and breast (MDA-MB-231) tumour lines. The results are expressed as an average ± standard deviation (n=3).
IC
50
Isolate
PC3
A549
MDA-MB-231
Carbon source/ Nitrogen source
73
50.26± 2.54
26.16± 3.26
26.65± 0.86
Starch
74.36± 1.05
69.13± 1.05
45.39± 3.06
Yeast Extract
386
11.18± 3.85
31.64± 1.45
68.57± 1.08
Xylose
14.59± 2.82
16.35± 4.03
71.25± 1.72
Malt Extract
519
55.80± 2.96
48.58± 2.39
39.98± 3.27
Xylose
68.43± 2.74
50.49± 3.64
41.23± 2.84
Malt ExtractThe results of the cytotoxic activity of different nitrogen sources in the ISP 2 broth are shown in Tables4 and 5. The nitrogen sources that improved the cytotoxic activity for the isolateStreptomyces aburaviensis (73) were meat extract for the prostate tumour cell line and yeast extract for lung and breast cancer, obtaining cytotoxic percent of 77.14%, 63.45%, and 75.78% at 100 ppm, respectively. The IC50 values were measured using yeast extract as a nitrogen source, and the IC50 values were 74.36
±
1.05, 69.13
±
1.05, and 45.39
±
3.06 ppm for prostate, lung, and breast tumour cell lines. The strainStreptomyces gramineus (386) had the best cytotoxic activity with malt extract with values of cytotoxic percent higher than 50% and had the highest cytotoxic activity for the prostate tumour cell line with an IC50 value of 14.59
±
2.82 ppm. Furthermore, the strainStreptomyces psammoticus (519) had the best cytotoxic activity using malt extract as a nitrogen source with values of 49.13%, 59.73%, and 77.59% for lung, breast, and prostate cancer, respectively, and had the lowest IC50 for the breast tumour cell line with a value of 41.23
±
2.84 (Table 4).Table 5
Determination of the nitrogen source that improved the cytotoxic percent at 100 ppm of theStreptomyces extract for prostate (PC3), lung (A549), and breast (MDA-MB-231) tumour lines. The results are expressed as an average ± standard deviation (n=3).
%
Cytotoxicity at 100 ppm
73
386
519
PC3
A549
MDA
PC3
A549
MDA
PC3
A549
MDA
Yeast extract
75.86± 0.35
63.45± 0.65
75.78± 0.61
69.32± 0.28
23.18± 3.96
18.07± 2.70
70.9 ±0.92
37.86± 0.79
46.49 ±0.80
Malt extract
68.72± 0.4
22.50± 0.59
33.24± 0.03
86.76± 4.76
67.93± 5.72
54.46± 0.36
77.59± 1.2
49.13± 2.55
59.73 ±2.16
Meat extract
77.14± 1.5
33.69± 5.58
48.85± 0.02
72.93± 0.98
21.82± 0.85
29.77± 1.54
68.42± 0.56
40.91± 2.96
47.46 ±1.96
Peptone
71.12± 5.32
29.13± 5.93
37.59± 0.94
68.79± 3.92
28.56± 5.51
48.00± 0.98
64.92± 4.98
33.96± 4.81
33.96 ±2.83
Potassium nitrate
67.51± 3.75
16.07± 4.15
22.91± 1.87
66.39± 1.81
33.10± 1.93
37.28± 1.79
71.80± 3.74
31.18± 3.62
31.18 ±0.08


## 3.3. Media Optimization Using Central Composite Design

After establishing the carbon and nitrogen sources, the effects of the concentration of nitrogen source (A), carbon source (B), and pH (C) were studied using a central composite to determine the optimum level of these parameters leading to the maximum cytotoxic percent. The experimental design was tested carrying out 15 experiments for each isolate in the cell line that had the best cytotoxic effect; results are presented in Table6.Table 6
Experimental design central composite design representing the cytotoxic percent byStreptomycesstrains influenced by four variables and five levels of each on A: carbon source concentration, B: nitrogen source concentration, and C: pH.
Run
Independent variables
%
Cytotoxicity (100 ppm)
Observed value
Predicted Value
A
B
C
73
386
519
73
386
519
(
%
w/v)
(
%
w/v)
Against PC3
Against A549
Against MDA
Against PC3
Against A549
Against MDA
1
2.5
2
6
78.05
83.482
73.204
77.319
84.139
71.740
2
2.5
0.4
8
75.075
74.138
72.774
73.985
76.455
69.707
3
0.5
2
8
71.182
82.695
87.936
70.728
84.274
85.239
4
0.5
0.4
6
75.075
75.54
74.374
75.060
75.090
75.003
5
0.1
0.8
7
74.44
81.127
72.715
73.952
80.956
74.620
6
5
0.8
7
76.138
81.127
72.715
76.401
80.439
74.271
7
1.4
0.1
7
70.717
81.705
72.834
70.258
81.766
70.462
8
1.4
4
7
72.547
77.078
75.952
72.753
76.486
76.688
9
1.4
0.8
5
72.547
77.078
75.952
72.044
77.487
75.054
10
1.4
0.8
9
74.824
83.369
72.434
74.720
81.934
74.001
11
1.4
0.8
7
73.085
80.37
73.929
73.382
80.819
74.528
12
1.4
0.8
7
70.717
80.078
72.709
73.382
80.819
74.528
13
1.4
0.8
7
72.767
81.494
74.946
73.382
80.819
74.528
14
1.4
0.8
7
72.767
81.568
72.918
73.382
80.819
74.528
15
1.4
0.8
7
74.198
82.272
74.033
73.382
80.819
74.528One model was obtained from analysis of variance for each extract. F-test for the models showed statistical significance (P<0,05), indicating that all models fit and can adequately explain the variations observed in the cytotoxic percent. Moreover, the lack of fit was not significant in all models at the 5% level (P<0.05), indicating that the experimental data obtained fit well with the model [21]. Coefficients of correlation R2 for the isolatesStreptomyces aburaviensis(73),Streptomyces gramineus (386), andStreptomyces psammoticus (519) were 0.8089, 0.8677, and 0.8020, respectively.Table7 shows the statistics analysis of cytotoxic activity from the isolateStreptomyces gramineus (386). As can be observed, the variables A, BC, and C2 were significant (P value<0,05). The regression equation for the model in terms of cytotoxic percent (Y) as a function of three independent variables (A: %w/v malt extract, B: %w/v glucose, C: pH) is given below. (2)
Y
386
=
5
,
861
+
36
,
996
A
+
2
,
260
B
+
12
,
823
C
-
5
,
3
A
C
-
0,516
B
C
-
0
,
277
C
2The response surface plot for the cytotoxic activity ofStreptomyces gramineus (386) against lung cancer shows the interactive effect of different variables (%w/v glucose, %w/v malt extract, and pH) presented in Figure 5(b). The highest theoretical value to maximize the cytotoxic percent to 100% was with 5% of malt extract, 3% of glucose, and a pH value of 6.Table 7
Analysis of variance for the fit second order polynomial model and lack of fit for the cytotoxic percent by composite central design for the strainStreptomyces gramineus (386). SS: sum of squares, DF: degrees of freedom, MSS: mean sum of squares.
Source
Sum of squares
DF
Mean square
F value
p-value
Model
101.406
6
16.901
8.746
0.0037
A
0.162
1
0.162
0.084
0.7790
B
18.572
1
18.572
9.611
0.0147
C
32.920
1
32.920
17.037
0.0033
AC
90.358
1
90.358
46.762
0.0001
BC
0.644
1
0.644
0.333
0.5795
C∧2
1.987
1
1.987
1.028
0.3402
Residual
15.458
8
1.932
Lack of Fit
12.147
4
3.037
3.669
0.1180
Pure Error
3.310
4
0.827
Cor Total
116.865
14Figure 5
Surface plots for interactions between nitrogen source concentration and carbon source concentration at the best pH predicted for (a)Streptomyces aburaviensis (73) pH 6 and (b)Streptomyces gramineus (386) pH 7. The surface plot for (c) Streptomyces psammoticus (519) represents the interaction between carbon source concentration and pH with best nitrogen source concentration (1% w/v) predicted.
(a)
(b)
(c)The regression equation for the cytotoxic activity from theStreptomyces aburaviensis(73) is shown in (3), where Y is the response in terms of cytotoxic percent, and the variables were %w/v yeast extract (A), %w/v starch (B), and pH (C). The significant terms (P value<0,05) for this model are A, B, C, AB, AC y B2. In the response surface plot presented in Figure 5(a), it can be observed that the highest point to obtain 100% of cytotoxicity appeared at 4% of yeast extract, 3% of starch, and a pH value of 7. (3)
Y
73
=
95
,
859
-
22
,
383
A
+
0
,
546
B
-
3
,
566
C
+
3.5660
A
B
+
2
,
671
A
C
+
0
,
260
A
2
-
1
,
194
B
2Finally, the model for the cytotoxic activity from theStreptomyces psammoticus(519) is given below as (4), showing the interactive effect between the variables %w/v malt extract (A), %w/v xylose (B), and pH (C). The significant terms (P value<0,05) for this model were B, C, BC y B2. The response surface plot presented in Figure 5(c) shows that the highest point for the isolate 519 against breast cancer is at 1% of yeast extract, 4% of starch, and a pH value of 8. (4)
Y
519
=
104
,
110
-
0
,
071
A
-
33
,
498
B
-
4
,
890
C
+
5
,
784
B
C
-
1
,
316
B
2The best conditions of the tested variables obtained from the experimental design CCD had been verified experimentally (in triplicate) and compared with the predicted data (Table 8). The measured cytotoxic activity at 100 ppm was 89.29% ±4.05, 89.03% ±6.47, and 66.18% ±2.83 forStreptomyces aburaviensis(73),Streptomyces gramineus (386), andStreptomyces psammoticus (519), respectively. The predicted values from the model were 97.47%, 97.96%, and 95.89%. The verification revealed a high degree of accuracy of the model forStreptomyces aburaviensis(73) andStreptomyces gramineus (386), indicating the model’s validation under the tested conditions. Moreover, the values of IC50 were measured at the best growth and nutritional conditions showing an increase compared with the initial results (Table 2). These assays were tested in L929 cell line without significative cytotoxic activity at low concentrations of extract (Figure 6).Table 8
Verification of the accuracy of the models obtained with CCP and de IC50 at the best environmental and growth conditions. Experimental results are expressed as an average ± standard deviation (n=3).
Isolate
%
Cytotoxicity at 100 ppm
Predicted
%Error
IC
50
(ppm)
Cell line
73
89.29% ±4.05
97.47
8.40%
25.00±1.86
A549
386
89.03%± 6.47
95.45
6.73%
7.93±0.98
PC3
519
66.18%±2.83
94.96
30.31%
35.53±2.71
MDA-MB-231Figure 6
Cytotoxic activity tested in L929 cell line forStreptomyces aburaviensis(73),Streptomyces gramineus (386), andStreptomyces psammoticus (519). Standard deviation (n=3).


## 3.4. Dry Biomass Growth Curve

The growth curves (Figure7) obtained for this study were constructed from the average dry biomass obtained from three experiments after 10 days. The average of the dry biomass with standard error bars was plotted with the help of the statistical program GraphPad Prism® version 6.01. The Gompertz model (Table 9) was used to describe the sigmoidal microbial growth curves shown in Figure 7; all models exhibit a high correlations (R2) with values of 0.99 forStreptomyces aburaviensis(73), 0.98 forStreptomyces gramineus(386), and 0.98 forStreptomyces psammoticus(519).Table 9
Results of Gompertz model growth rate (μMax), the lag time (
λ
), and the coefficients of correlation (R2).
Isolate
μ
M
a
x
(
d
a
y
)
λ
(day)
R
2
73
1.18
1.71
0.99
386
0.98
2.64
0.95
519
0.50
1.26
0.98Figure 7
Growth kinetics ofStreptomyces aburaviensis (73) against lung tumour cell line (a),Streptomyces gramineus (386) against prostate tumour cell line (b), andStreptomyces psammoticus(519) against breast tumour cell line (c) and dependent variables growth (mg; (blue filled circles)) and %cytotoxicity (%; (black filled squares)). Standard deviation (n=3).
(a)
(b)
(c)


## 3.5. LC-MS Analysis

The chromatograms obtained for the extracts tested showed defined signal patterns characteristic of each type of extract as indicated by arrows (Figure8). Organic extracts showed the majority of signals between 11 and 28 min retention time (RT) and signals with mass/charge (m/z) relationship values from 160 to 820. The extract ofStreptomyces gramineus (386) differs from the extracts ofStreptomyces aburaviensis (73) andStreptomyces psammoticus(519) in the signals corresponding to retention time of 11.67 min, m/z= 408.6 and 26.44 min, m/z= 709.4. The strainStreptomyces psammoticus(519) presented characteristic signals at 13.62 min, m/z= 207.25 and 17.01 min, m/z= 453.2 andStreptomyces aburaviensis (73) at 27.25 min, m/z= 817.7.Figure 8
Liquid Chromatography comparison spectrum for organic extracts fromStreptomyces gramineus (386),Streptomyces aburaviensis (73), andStreptomyces psammoticus(519) and control extract from ISP 2 media without biomass (the arrows indicate the peaks that differ from the other extracts).


## 4. Discussion

Cancer is the second leading cause of death in the world behind cardiovascular disease [5]. Despite this, chemotherapy is still one of the most used cancer treatments even with its range of serious side effects. To decrease the side effects of chemotherapy, new biologically active metabolites are needed to be explored, and natural product extracts continue to be the most promising source of new drugs for cancer [14]. Through this research, we found threeStreptomyces strains isolated from Guaviare river sediments (Colombia, South America) with cytotoxic activity against prostate, lung, and breast cancer cell lines. The isolates were identified morphologically and molecularly asStreptomyces aburaviensis(73),Streptomyces gramineus (386), andStreptomyces psammoticus (519). Some studies describeStreptomyces aburaviensis(73) [35, 36] andStreptomyces psammoticus (519) [37, 38] with antibacterial and antifungal activity. In fact onlyStreptomyces gramineus (386) has been reported with cytotoxic activity; two compounds have been isolated from this strain with a moderate cytotoxic activity against SKOV-3 (ovarian cancer cell line), Meso-1 (human malignant mesothelioma cell line), and Jurkat (leukemia cell line) cells with IC50 values of 2.3, 2.5, and 1.0 μM [39].Three organic extracts were obtained from strainsStreptomyces aburaviensis(73),Streptomyces gramineus (386), andStreptomyces psammoticus (519), which presented cytotoxic activity against lung, prostate, and breast cancer, respectively (Table 2). Furthermore, the organic extracts showed chemical differences analysed in LC-MS chromatogram (Figure 8).Streptomyces gramineus (386) spectrum showed a signal related to the compound diacarnoxide-A (RT=11.67, m/z= 408.6), which has been reported with cytotoxic activity against prostate cancer (PC3) and breast cancer (MDA-MB231) by Jingqiu et al. [40]. LC-MS analysis showed the possible presence of Streptophenazine I (RT=17.01; m/z= 453.2 (M+H)+) in the organic extract ofStreptomyces psammoticus (519), which has been reported by Bunbamrung et al. with cytotoxic activity against breast (MCF-7) and lung cancer (NCI-H187) [41].In order to optimize the cytotoxic activity of the threeStreptomyces strains, five carbon sources were evaluated. Several reports take into consideration the role of the carbon source to enhance the biological activity, thereforeStreptomyces aburaviensis(73) improved 2.58 times the cytotoxic activity against the lung tumour cell line using starch instead of glucose as a carbon source. Thumar et al. [42] demonstrated that the use of starch as a carbon source inStreptomyces aburaviensis(73) enhances growth and antibiotic production. Moreover,Streptomyces gramineus (386) had the best cytotoxic activity against the prostate cancer cell line using glucose as a carbon source instead of xylose because glucose demonstrated a higher cytotoxic activity at lower concentrations of extract. Similarly, Lee et al. [32] reported that glucose favours the growth of this strain as in this investigation.Streptomyces psammoticus (519) had the higher cytotoxic activity using xylose as a carbon source as Hamed et al. [43] reported that this strain is able to metabolize xylose to grow in culture, and Sujatha et al. [44] reported antibiotic production using this carbon source.Subsequently, the nitrogen sources that increased the cytotoxic activity forStreptomycesstrains were malt extract and yeast extract, which means that the cytotoxic activity is related to the synergic interaction between these nitrogen sources. It is why the cytotoxic screen was made using ISP 2 and the production of the cytotoxic compounds must be stimulated for these nitrogen sources. Likewise, some studies confirm that the nature of the nitrogen sources strongly affects bioactive compounds production [17] in different organisms. Raytapadar and Paul [45] found that the yeast extract supports the growth inStreptomyces aburaviensis(73). Similarly, the antibiotic [43] and laccase [46] production was enhanced using yeast extract as a nitrogen source inStreptomyces psammoticus (519), and the cytotoxic production was increased by yeast extract inStreptomyces sp. VITPSA [47].The cytotoxic activity of the threeStreptomyces strains was affected by different environmental and nutritional growth conditions (Figure 5). Firstly, the pH was the most influential variable in all models, the range 7–9 of pH constitutes the optimal values forStreptomyces aburaviensis(73) andStreptomyces psammoticus (519). Researchers confirm thatStreptomyces aburaviensis(73) grow well at pH of 9 [42] and enhance its antibiotic production at 7.4 [45]. Moreover,Streptomyces psammoticus (519) increases the production of extracellular bioactive metabolites in a pH of 6.5 [43]. At lastStreptomyces gramineus (386) has an optimal pH value of 6, and some studies report that this strain grows well at pH 4–8 [45]. Similarly, the interaction between starch with pH and yeast was significant to increase the cytotoxic activity ofStreptomyces aburaviensis(73). The most important conditions besides pH forStreptomyces gramineus (386) andStreptomyces psammoticus (519) were the nitrogen and carbon sources, respectively.Balachandran et al. [15] showed the relationship between cytotoxic activity and cell growth; this behaviour was observed in the present investigation forStreptomyces aburaviensis(73), which had a growth rate (μ
M
a
x) of 1.18 day−1 (Table 9) and had the maximum cytotoxic activity on the seventh day according to the inflection point of the growth curve (Figure 7(a)). Also, some studies ofStreptomyces aburaviensishave reported that this strain secreted antibiotic in the culture after seven days of fermentation [45]. The strainStreptomyces gramineus (386) also had the highest cytotoxic activity in the seventh day, and it kept stable until the tenth day with a growth rate (μ
M
a
x) of 0.98 day−1 (Table 9). In addition, the strainStreptomyces psammoticus (519) had a growth rate (μ
M
a
x) of 0.50
day-1 (Table 9) and presented the cytotoxic activity in the exponential phase. The cytotoxic activity (Figure 7(c)) ofStreptomyces psammoticus(519) decreases when the stationary phase begins (fifth day); this behaviour is similar in comparison with Hamed-Mohamedin et al., who set the fermentation day until the fifth day for the production of bioactive metabolites usingStreptomyces psammoticus [43].Consequently,Streptomyces aburaviensis(73) andStreptomyces gramineus (386) at the best nutritional and growth conditions showed a high cytotoxic activity, andStreptomyces psammoticus (519) had a moderate cytotoxic activity. Despite this, the National Cancer Institute of United States estimated that the value of IC50 has to be lower than 30 ppm to have a high cytotoxic activity for a crude extract [48]. Hence, the value of IC50 forStreptomyces aburaviensis (73)against lung cancer (A549) was 25.00
±
1.86 ppm, which was much greater than otherActinobacteria’s cytotoxic activities. For instance,Nocardia dassonvillei produced a novel anticancer phenazine that had a IC50 of 38.53 ppm against A549 [49]. Also, the Actinobacterial strainsERIA-31 andERIA-33 produced a protease enzyme that was tested in A549 cell line and presented IC50 values of 57.04 ppm and 55.07 ppm, respectively [50].Streptomyces psammoticus (519) had a IC50 value of 35.53
±
2.7 ppm against breast cancer (MDA-MB-231). Similarly, Actinobacterial strains ACT01, ACT02, and ACT03 showed IC50 values of 32.79
±
6.94, 69.84
±
19.54, and 84.09
±
18.93 ppm against MDA-MB-231 [16]. However, recently Actinobacterial strains EGY2 and EGY39 showed a high cytotoxic activity for the crude extracts with IC50 values of 19.50
±
0.03 ppm and 29.6
±
0.43 mg/mL, respectively [
53
]. Finally, the strainStreptomyces gramineus (386) showed the highest IC50 value among all threeStreptomyces strains studied in this investigation, with a value of 7.93
±
0.98 ppm against prostate cancer (PC3) with the growth and environmental conditions of the experimental design, but it does not have a significant difference with the first value of 6.14
±
2.07 ppm (Table 2).The results indicate thatStreptomyces aburaviensis (73),Streptomyces gramineus (386), andStreptomyces psammoticus (519) showed a promising cytotoxic activity against lung, prostate, and breast cancer, respectively. Furthermore, the extracts did not show a significative toxic activity against L929 (mouse fibroblasts) at values lower than 50 ppm of extract (Figure 6). The optimizing nutritional and growing factors improve the cytotoxic activity ofStreptomyces aburaviensis (73) in 61.35% against lung cancer and 22.57% forStreptomyces psammoticus (519) against breast cancer.


## 5. Conclusions

The study revealed that the optimization of the environmental and nutritional conditions in growth culture improves the cytotoxic activity of extracts fromStreptomyces aburaviensis(73) andStreptomyces psammoticus (519). However,Streptomyces gramineus (386) had a high cytotoxic activity (less than 30 ppm) with the initial conditions because the medium propitiated the stimulation of the cytotoxic activity without being able to improve it more.This study reveals that the strategy of optimizing the environmental and nutritional conditions in growth culture had a positive impact in the cytotoxic activity, leading the investigation group to a new strategy. Also, the incorporation of techniques like HPLC-MS and MR will be used to identify the compounds responsible for the cytotoxic activity. In future studies, flow cytometry and Western blot will be carried out to identify the cellular and biochemical mechanisms that lead to the cytotoxic activity of extracts.



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*Source: 2839356-2018-07-19.xml*