Apoptosis on non-small lung cancer cells and DPPH radical scavenging activity of the crude ethyl acetate extract of a soil bacterial species FEAI-1

The aim of this research is to evaluate the in vitro apoptosis and free radical scavenging activities of the crude extract of a soil bacterial species FEAI-1. Apoptosis activity was investigated using annexin V-phycoerythrin staining on human non-small-cell lung carcinoma cell line (H1299), and an antioxidant study of the crude ethyl acetate extract was carried out by the diphenylpicrylhydrazyl (DPPH) assay. The crude ethyl acetate extract of the bacterial species FEAI-1 induced apoptosis on H1299 cell lines in a concentration-dependent manner. After a 96-hours treatment period, flow cytometry showed that the extract induced total apoptosis by 10.25%, 13.48%, 20.42%, 68.10%, and 68.80% at concentrations of 2.5, 5, 10, 20, and 40 μg/ml, respectively. The crude extracts which elicited antioxidant activity with the inhibition of DPPH radicals were 24.97% ± 0.288%, 26.29% ± 0.085%, 29.06% ± 0.123%, 33.07% ± 0.01%, 38.45% ± 0.03%, 45.33% ± 0.02%, and 53.21% ± 0.115% at concentrations (μg/ml) of 10, 20, 40, 80, 160, 320, and 640, respectively. The inhibition concentration 50 value of the extract was 510 μg/ml. These results suggest that the crude extract of the bacterial sp. FEAI-1 could be a useful natural source having therapeutic potential against cancer and diseases caused by oxidative stress. Furthermore, in vivo studies and compound isolation pave the way for its biomedical applications.


INTRODUCTION
Cancer is the major global health problem and the second leading cause of death after cardiovascular disease (Siegel et al., 2015). Lung cancer is the most common cancer which is responsible for the largest number of cancer-related deaths globally for both men and women. It kills more people than female breast cancers, colorectal cancers, brain cancers, and prostate cancers combined (Siegel et al., 2013). According to the American Cancer Society, in the USA, around 234,030 new victims of lung cancer would be identified and 154,050 people will die of the disease in 2018 (Siegel et al., 2018). This is just about 26% of all cancer motilities in the USA (Smith et al., 2018). The high percentage of lung cancer is due to the fact that both smokers and nonsmokers have the risk of lung cancer (Shopland et al., 1991;Zappa and Mousa, 2016). From July 2, 2010, to July 2, 2011, a case-control survey of common cancer causes and etiology in cancer hospitals of Dhaka city was conducted with 610 subjects. The outcome of the survey was that 76 patients were suffering from lung cancer among 339 male cancer patients (Shahriar et al., 2011).
Despite the significant progress achieved in anticancer therapy, typical cancer treatments (surgery, chemotherapy, immune therapy, and radiation therapy) frequently fail to attain a complete cancer recovery (Bernardes et al., 2010). Moreover, still drug side effects, drug resistance, and greater systemic toxicity are the major challenges for anticancer drugs (Ko et al., 2018;Ling, 1992;Longley and Johnston, 2005;Naidu et al., 2004). Due to the negative consequences of radiotherapy and chemotherapy, cancer sufferers are now looking for alternative medical therapy with milder side effects (Buckner et al., 2018;Rehman et al., 2016). This has played an indispensable role in the searching and developing of myriad new approaches to prevent or treat cancer. One such strategy involves the application of bacterial secondary metabolites (Bernardes et al., 2010).
The increasing rate of free radical production or reducing rate of antioxidant defense responses may increase oxidative stress (Kim et al., 2008). The increased level of reactive oxygen species (ROS) inside the cells is the major etiology of a number of diseases, including cancer, neurodegenerative, and cardiovascular diseases. ROS is responsible for several kinds of DNA damage and mutations (Devasagayam et al., 2004;Joseph et al., 2013). Antioxidants make every effort to scavenge and prevent the generation of free radicals or to promote their decomposition and hinder the progression of these diseases (Lobo et al., 2010). Metabolites of soil bacterial species having a dominant radical scavenging activity may play a crucial role in the development of therapeutic drugs . Therefore, the objective of the current study is to explore the secondary metabolites of a soil bacterial species named FEAI-1 with apoptosis and antioxidant activity.

Isolation of soil bacterial species
During the isolation process, we focused on isolating actinomycetes from the soil, as their secondary metabolites are well known for antibacterial, anticancer, antitumor, antifungal, antiviral, antioxidant, and immunosuppressive activities (Ajijur Rahman et al., 2010;Hong et al., 2009;Kawahara et al., 2012;Kumar et al., 2014;Qin et al., 2011;Tan et al., 2015). A total of 11 soil samples were collected from the Mirzapur region of Rajshahi city area in Bangladesh. At 60°C-65°C, soil samples were dried in an oven for 3 hours to decrease the number of other bacterial species, except for actinomycetes, and were then preserved at 4°C before screening. 1 gm of preserved soil was weighed and tenfold serial dilution was carried out in sterile distilled water. 0.1 ml of each solution, at dilutions of 10 −4 -10 −5 , was then spread on selective media (per 1 litre: 0.4 g casein, 1.0 g starch, 0.5 g KNO 3 , 0.1 g CaCO 3 , 0.2 g K 2 HPO 4 , 0.1 g Mg 3 (PO 4 ) 2 , and 15 g agar). After that, the Petri dishes were incubated at 30°C for 7-14 days. Starch and casein of the isolation media (Pharm, 2008) are the only sources of carbon and energy of that media. Only organisms having the ability to degrade starch and casein (mostly Streptomyces and molds) can survive and multiply. 50 µg/ml nystatin was added to the culture media to prevent the growth of fungi. Typical Streptomyces colonies were picked and purified by streaking onto an inorganic salt-starch agar media (per 1 litre: 10 g starch, 2 g (NH 4 ) 2 SO 4 , 2 g CaCO 3 , 1 g K 2 HPO 4 , 1 g MgSO 4 .7H 2 O, 1 g NaCl, and 20 g agar), and then the pure cultures were placed into the incubator at 30°C for 7-14 days. A total of 27 bacteria were isolated from these soil samples. Among them, FEAI-1 had good antibacterial and cytotoxic activity (Khatun et al., 2017). The isolated species FEAI-1 was preserved on an inorganic salt-starch agar media (Küster, 1959) at 4°C.

Morphological identification
Visual observation was performed with the help of optical microscopy to determine the morphological and microscopic characteristics. Morphological features (size, shape, and arrangements) play an important role to distinguish and characterize bacterial species. Morphological characteristics of the bacterial species FEAI-1 were assessed according to the process provided by Shirling and Gottlieb (1966). Microscopic observation was carried out using the coverslip culture method. Firstly, using sterile forceps, two sterile coverslips were introduced into the sterile solid media of the Petri dish at an angle of 45°-50°. Then, at the free space of the plate, the strain was inoculated by the streaking method. After that, the plate was placed into the incubator for 10 days at 30°C. During the incubation period, the hyphae of FEAI-1 grew and spread on the coverslip and produced spores. After this period, coverslips were pulled out smoothly and mounted on slides. Lastly, the slides were examined under a microscope. The observed characteristics were shape, size, aerial mass color, and substrate mycelium. By direct microscopic examination of the culture surface, the characteristics of hyphae and spore chains were observed. Using 100x-700x magnifications, the chain of spores was observed. On the basis of Bergey's manual of systematic bacteriology, the aerial mass color was also determined (Locci, 1989). The plates of the bacterial species FEAI-1 were examined for Gram staining.

Biochemical characterization
Physiological characteristics, for instance, the capability of the bacterial species FEAI-1 to degrade casein, tyrosine, and xanthine as substrates, were used for genus confirmation.

Optimization of cultural conditions
Optimum nutritional conditions for the growth and formation of secondary metabolites of the bacterial species FEAI-1 were determined by culturing the bacteria in different culture media, such as ISP-1 agar, ISP-2 agar, ISP-4 agar, ISP-5 agar, nutrient agar, yeast-extract glucose agar, and starch-casein agar media. To determine optimum growth conditions and to ensure maximum production of secondary metabolites, the bacterial strain FEAI-1 was grown at different incubation temperatures (25°C, 30°C, 35°C, and 40°C) and different incubation periods (3, 4, 5, 7, 10, and 15 days).

Extraction of secondary metabolite
At first, 500 ml inorganic salt-starch broth culture medium was prepared in a conical flask. Then, a small amount of FEAI-1 bacteria was transferred into the conical flask aseptically and the bacteria were incubated at 35°C for 15 days. After incubation, cell biomass was separated by filtration. Then, the filtrate was partitioned using the organic solvent ethyl acetate because the solubility of secondary metabolites was higher in ethyl acetate than other organic solvents. A rotary vacuum evaporator was used to evaporate ethyl acetate fraction until a semisolid mass was obtained. The semisolid mass was then air-dried to get a solid mass.

Cell lines collection and maintenance
For evaluation of apoptosis activity, a non-small lung cancer cell line (H1299) was used. H1299 cells were collected from the MD Anderson Cancer Center. The cell line was grown in the DMEM/F12 (1:1) medium supplemented with 10% heatinactivated FBS at 37°C in a humidified incubator containing 5% CO 2 and 95% air. The authenticity of this cell line was determined by the genomic short tandem repeat (STR) profile. STR was performed in the Research Animal Diagnostic Laboratory, University of Missouri (Columbia, MO), and by the Emory University Integrated Genomics Core.

Sample preparation
The extract of FEAI-1 was weighted appropriately and 100 µg/ml stock solution was prepared using dimethyl sulfoxide prior to assay.

Annexin V-phycoerythrin staining for screening apoptosis
Firstly, H1299 cells were treated with the extract (μg/ ml) for 96 hours. Afterward, the cells were trypsinized and washed with cold 1 × PBS. Finally, the cells were suspended in 1 × annexin binding buffer (BD Pharmingen) and stained with annexin V-phycoerythrin (annexin V-PE; BD Pharmingen) and 7-amino-actinomycin D (7-AAD; BD Pharmingen) for 15 minutes at 25°C. The stained samples were measured with a fluorescenceactivated cell sorting caliber bench-top flow cytometer. FlowJo software was used for apoptosis analysis.

Diphenylpicrylhydrazyl (DPPH) radical scavenging assay
DPPH radical scavenging assay is a frequently used and rapid method for testing the antioxidant potential of natural compounds (Swarnalatha et al., 2015). This test of FEAI-1 extract was performed according to a previous method (Swarnalatha et al., 2015) with minor changes (Elmastas et al., 2007). Different concentrations of 1 ml of the sample as well as standard compound (ascorbic acid) were added to 2 ml of freshly prepared 0.004% DPPH in 95% methanol. A 2 ml methanol solution of DPPH was added to 1 ml 95% methanol for control reading. All the mixtures were kept at 25°C in a dark place for 30 minutes. After that, the absorbance was measured at 517 nm on UV-visible spectrometer. The percentage of DPPH radical scavenging capacity was measured with the following formula: Percent inhibition = (a-b) / a × 100 where a = absorbance of control reaction and b = absorbance of test or standard samples.

Optimization of culture condition and identification of the selected bacterial strain
Biochemical characterizations and optimum culture conditions of the bacteria FEAI-1 are presented in Table 1 and cultural characterizations are presented in Table 2. The selected bacterium FEAI-1 belonged to the genus Streptomyces since it was Gram-positive and aerobic and produced well-developed substrate mycelium. Aerial hyphae were long, containing extended filamentous chains and cylindrical smooth spores. The colonies were glabrous, heaped, and folded, and the growth of the colonies was slow. The aerial and substrate mycelia possessed different colors on different culture media. In addition, it had an earthy odor. The bacterial species also had the ability to degrade casein, tyrosine, and xanthine. The strain FEAI-1 produced yellow pigment into the culture media.

Analysis of apoptosis by flow cytometer
One of the major features of apoptotic cells is the exposure of the phospholipid phosphatidylserine (PS) to the external environment. This happened by the translocation of the PS membrane from the inner to the outer leaflet of the plasma membrane. Annexin V-phycoerythrin (PE) complex has a higher affinity for PS. Annexin V-PE binds with exposed PS in apoptosis cells (Herault et al., 1999). 7-AAD has a high affinity to bind with DNA. So it is used to detect nuclear damage. Viable cells with intact cytoplasmic membranes exclude both annexin V-PE and 7-AAD, representing the lower left quadrants (Fig. 1). Lower right quadrants represented the early apoptotic cells. Here, annexin V was positive and 7-AAD was negative, which indicated that the nuclear membrane was intact but the cytoplasmic membrane was damaged. Both annexin V-PE and 7-AAD binding were positive in the upper right quadrant, which represented the late apoptotic cells or necrotic cells.
It was observed that the percentage of viable cells was decreased and apoptosis cells were increased by the increase in the crude extract treatment concentration. The total apoptotic H1299 cell populations were 10.25%, 13.48%, 20.42%, 68.10%,   (Table 3). Most of the H1299 cells were viable (88.8%) in the untreated (control) sample, whereas only 29.1% of viable cells were observed when cancer cells were treated with 40 µg/ml of the crude ethyl acetate extracts of FEAI-1 for 96 hours. Moreover, FEAI-1 extract induced early apoptosis and late apoptosis in a concentration-dependent manner (Table 3).

DISCUSSION
Cancer progression has been associated with an increased amount of free radicals (Reuter et al., 2010). Free radicals can induce DNA damage and mutations, which subsequently increase cancer risk (Dreher and Junod, 1996). Here, antioxidants can play a vital role to prevent the harmful effects of free radicals. Typical cancer therapies cause significant side effects (Bernardes et al., 2010). Due to these reasons, nowadays, considerable attention has been paid to the identification of new compounds having anticancer and antioxidant activities (Devi, 2011;Jemimah et al., 2011;Lee et al., 2014;Sharma et al., 2010). Bacteria and its secondary metabolites could be a promising source of anticancer and antioxidant compounds (Bernardes et al., 2010;Ser et al., 2016). In this research, we isolated secondary metabolites producing bacterial strain FEAI-1 from a soil sample. The bacteria were characterized on the basis of morphological and biochemical features. The isolated strain was identified as Streptomyces species. Then, apoptosis and free radical scavenging assay were performed.
Apoptosis is a normal genetically programmed process, which plays a crucial role in regulating the growth and development of a fetus (Biosciences, 2011;Fan et al., 2005). In order to destroy cancer cells without giving inflammatory effects, this apoptosis program becomes important (Alabsi et al., 2012). An apoptosis test was carried out by annexin V/7-AAD double-staining procedure. Data (Table 3) showed a concentration-dependent increase in early apoptosis and necrotic cells, with a decrease in the viable cell populations. Total apoptosis (10.25%-68.8%) was increased, respectively, with increasing concentrations (2.5-40 µg/ml) of crude extract after the 96-hours pretreatment (Table 3). A recent study reported that, after the 18-hours treatment of cancer cells HL60 with the extract (25-100 µg/ml) of Erythrina suberosa stem bark, the cells were stained with annexin V/PI dye. The total apoptosis was 6.60% in control cells, which increased to 31.09% after 75 µg/ml and to 30.35% after 100 µg/ml of extract treatment .   In our research, ascorbic acid and crude extract showed 50% radical scavenging activity at concentrations of 6.5 and 510 µg/ml, respectively, although crude extract had free radical scavenging ability but lesser than that of ascorbic acid. This may be due to the fact that ascorbic acid is a single compound but crude extract is a mixture of compounds. Some previous studies showed that the IC 50 value of Streptomyces sp. MJM-10778 (Lee et al., 2014) and PO-01 (Kekuda et al., 2015) extracts on DPPH radicals were 92.8 and 591.69 μg/ ml, respectively. Results of this research demonstrated the significant antioxidant potential of the crude extract of FEAI-1. Therefore, the results of this research indicated that the bioactive secondary metabolites of FEAI-1 could be used for cancer treatment due to its apoptosis activity and also could potentially reduce cancer risk by preventing the free radicalmediated DNA mutation.

CONCLUSION
Soil could be a good source of bioactive secondary metabolite producing bacteria having anticancer activity. The isolated bacteria of FEAI-1 showed significant apoptosis (68.80% total apoptosis was at 40 µg/ml) and antioxidant (IC 50 value = 510 µg/ml) activity gives a possibility of being used as a therapeutic agent for cancer. Figure 2. Comparison between antioxidant activity of the extract from Streptomyces sp. FEAI-1 and standard ascorbic acid in the DPPH method. The crude extract IC 50 value was 510 µg/ml compared with the standard ascorbic acid IC 50 value of 6.5 µg/ml.