Anticancer Activity Assay of Nano-Fractional Compounds that Purified from Soil Actinomycetes

https://doi.org/10.36547/ae.2021.3.2.32-38 Abstract Background and objectives: Cancer remains a global problem of health, and has been recorded as one of the causes of death after heart disease Natural products from plants, the environment and microorganisms are leveraged for the purpose of fighting cancer. Actinobacteria have been recognized as main sources of bioactive natural products as early as in the 1950s, for which about half of the secondary metabolites revealed, including enzymes, antibiotics, immunosuppressive, and anti-tumor agents Materials and methods: The methods of this study included isolated and identification of bacteria from soil samples and identified by morphology characters and biochemical test. Subjected extract of actinomycetes to HPLC purification then collected purified fractions then analyzed by GC-mass. After the fractions were mixed with liposome nanoparticles which tested activity on HT29 colon cancer cell line. Results: The results of identification of bacterial isolates showed the colonies growing on a SNA medium were morphologically identified where the colonies were well-growth and had a gray color, not producing dyes in the medium. The results of the biochemical tests indicated that isolates were amylase, catalase, and gelatinase producing isolates and non-lipase producing, non H2S production and consuming urea, while the carbon consumption test indicated the isolates' ability to consume starch, glucose and sucrose respectively. While the results of preparative HPLC revealed that 4 fractions were collected with desired amounts of each compound when using fraction collector in depend on mobile phase system in analytical HPLC with (50 % HPLC-grade acetonitrile) at 254 nm and cycling up was employed to increase the separation efficiency. The chemical composition of the HPLC fractions using GC-MS showed the identification of many components example (Hexadeconic acid, Octadecanoic acid, ethyl ester and Fumaric acid). The results of In vitro antitumor cytotoxicity showed that all four nano purified fractions were applied on HT 29 colon cancer cells and exhibited significantly differences compared with control treatments of inhibition cells number, these data were used to calculate the values of IC50 (the inhibitory value of half the number for all nanofractions. the application of concentration with inhibition value and solved the equation to IC50 value were gained, which were (151.4,16.4 , 16.6 and 43.8 μg/ml ) to four nano fractions respectively. Conclusion: This study showed that the use of HPLC to purify the bacterial extract and then combine the purified fractions with the nanoparticles liposome has inhibited cancer cells with high efficacy. Archives of Ecotoxicology, Vol. 3, No. 2, pp. 32-38, 2021


Introduction
To date, cancer remains a global problem of health, and has been recorded as one of the causes of death after heart disease ( . Natural products are also an alternative solution to chemotherapy and its associated side effects such as heart failure, diarrhea, and others.Due to its high toxicity, it may lose specialization in treatment (Tan et al., 2015; Suter& Ewer, 2013). Medical chemotherapy must be a specialist to get rid of a type of cancerous cell, but it cannot distinguish between normal and cancerous cells. However, most 33 of the presently used anticancer treatments tend to destroy cancer and normal cells (Ser et al., 2015). Cancer chemoprevention is similarly essential as an interference in carcinogenesis. These can be obstructive agents that stop neoplastic process or defeating agents that inhibit the progress of cancer cells' malignant phenotype ( Tan et al., 2013; Surh,  2003). Thus, it is a continuing work to search for highly specific and potent chemotherapy agents from substitute sources for example microorganisms. Actinobacteria have been recognized as main sources of bioactive natural products as early as in the 1950s, for which about half of the secondary metabolites revealed, including enzymes, antibiotics, immunosuppressive, and anti-tumour agents, are formed by actinomycetes ( Recently the nanoparticle liposome has become important as a drug delivery vehicles. These carriers consist of a lipid bilayer that is a hollow spherical shape occupied by an aqueous phase. Therefore, any compound can be encapsulated inside the liposome in one of the two parts if it is hydrophilic inside and if it is lipophilic within the bilayer liposome (Singh et al., 2019). This formulation can encapsulate more than one drug at the same time, protecting the encapsulated compound from hydrolysis and degradation. In addition, targeting the surface proteins on the cells and ligand on lipid bilayer shell have addition functional allowing targeted entry of liposome into the cell, by ligand receptors target. These ligands attach to cell receptors that are over-expressed in convinced diseased cells, permitting entry of the drug through the cell membrane (Eloy et al., 2014). Despite all of the above, studies regarding the potential biological effectiveness of actinomycetes metabolites as anticancer drugs and the discovery of new drugs are limited . In this respect, this study was developed as an attempt to discover the anticancer property from actinomycetes against Human colon adenocarcinoma cell lines.

Collection of Samples and Isolation of Actinomycetes
A 25 gm of agricultural soil sample was collected from a depth of 20 cm. It was placed in a sterile bag and transferred to the laboratory. For the purpose of isolating the Actinomycetes bacteria, the serial dilution method was implemented for the soil sample (1 gm of soil was diluted in 9 ml of distilled water and then 1 ml of this dilution was transferred. To 9 ml of distilled water and so on until the sixth dilution (Williams et al., 1983). 100 μl of the fourth and fifth dilutions were plotted on medium (starch nitrate agar (SNA)) prepared by (of 20 g/l starch, 1 g/L KNO3, 0.5 g/l K2HPO4, 0.5 g/l MgSO4.7H2O, 0.5 g/l NaCl, 0.01 g/l FeSO4, 15 g/l agar) and containing antifungal and bacterial agents (nystatin and naldixic acid) at 50 and 20 mg / l, respectively. The plates were then incubated at 30C for 7 days. The growth colonies were diagnosed morphologically and with some biochemical tests (Aghamirian and Ghiasian, 2009; Reddy et al., 2011).

Preparation of actinomycetes extract
Diagnosed isolates from Actinomycetes cultured on medium SNA At a degree of 28 c for a period of 7-14 days until the spors are completely formed. In a 250 ml flask containing medium ISP2 medium containing (4 g/l glucose, 4 g/l yeast extract, and 10 g/l malt extract,) was inoculated with spore suspension and incubated at 30 using a shaker incubator (150 rpm) for 15 days, the cells were separated using a centrifuge 5000 rpm and 4c and the extraction of cell biomass was with acetone and then the acetone was evaporated under the vacuum. The remaining water was extracted by acetyl acetate (Shaaban et al., 2013).

Development of separation protocol by analytical DAD-HPLC
The column temperature adjusted at 30 Cº at a flow rate of 1.0 ml/min to achieve the optimum resolution of the separation of many compounds .The injection volume was maintained at 20 μl of watery extract, the mobile phase had been employed to achieve the best separation condition was 50 % HPLC-grade acetonitrile

Fractionation protocol by preparative HPLC
The column temperature adjusted at 30 Cº at a flow rate of 10 ml/min to achieve the optimum resolution of the separation of compounds. The injection volume was maintained at 500 and 1000μl of extract.

Identification of chemical composition of the HPLC fractions using GC-MS
The collected fractions was analyzed by a coupled Varian gas chromatography/mass spectrometry (Perkin Elmer Auto XL GC, Waltham, MA, USA) equipped with a flame ionization detector to identify their chemical composition. The GC conditions were EQUITY-5 column (60 m 0.32 mm x 0.25 mm); H2 carrier gas; column head pressure 10 psi, the oven temperature was maintained initially at 70 C for 2 min, and then programmed from 70 to 250 C at a rate of 3 C/min. The ionization voltage was 70 eV and mass range m/z 39e400 amu. The identification of individual compounds was based on their retention times relative to those of authentic samples and matching spectral peaks available with the published data (Iwasa et al., 2015).

Nanoparticles-fractional mixture preparation
Nanoparticles Liposome Solution: It was ready prepared solution (according to Sigma Aldrich, Germany) and supplied in glass vial (0.4 mg) provided with nuclease free water (1ml) as a stock solution. The stock solution was diluted with adding distilled water in proportion of 100 μl of liposome: 900 μl D.W (4μg/ml). Each purified fractions at a concentration of 500 μg/0.5ml mixed with 0.5ml of Liposome. The proportion of 100 μl of liposome (Stock Solution) 900 μl D.W.(4μg/ml).

In vitro Anti-cancer Cytotoxicity
Each nano purified fractions were evaluated for their cytotoxicity using tissue culture technique. HT29 (Human colon adenocarcinoma)cell line was kindly provided by the National Cell Bank of Iran (NCBI), Pasteur Institute of Iran. Cells were maintained in RPM1medium with 10% fetal calf serum, sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C and 5% CO2 till the cytotoxicity bioassay was carried out. The potential cytotoxicity of nano purified fractions was tested using the method of Alley et al. (1988). Briefly, 100 cells/well were plated onto 96-well dishes overnight before the treatment with the tested compounds to allow the attachment of cells to the wall of the plate. Different concentrations of each tested compound (0, 15.6, 31.25, 62.5, 125, 250 and 500μg/ml) were added to the cell monolayer and triple wells were used for each individual dose. Monolayer cells were incubated with the tested agent(s) for 48 h at 37°C and 5% CO2. At the end of the incubation period, Crystal violet (C.V) assay was used to determine the optical density of the cell growth in each well of the microtiter plate, by using plate reader. After the end point of cytotoxicity assay, the maintenance medium with the test substance was discarded out and the wells washed with 100 μl of cold PBS by automatic pipette. Then the cell cultures were fixed with 10 % buffered formalin for 20 min at room temperature. Fixative solution was discarded and 100 μl of 0.1 % aqueous CV solution was added to each well. The samples were incubated at room temperature for 20 min with gentle shaking. After that the plates were washed by submersion in flowing tap water for 15 min. The plates were allowed to dry in the air and the absorbance was read at 570nm by a microplate reader The relation between surviving fraction and compound concentration was plotted to get the survival curve of each tumor cell line and the IC50. The concentration of an agent that causes a 50% growth inhibition, for each tested agent using each cell line was obtained from the survival curve (Skehan et al.,  1990).

Identification of Actinomycetes isolates
The colonies growing on a SNA medium were morphologically identified where the colonies were well-growth and had a gray color, not producing dyes in the medium. The results of the biochemical tests indicated that isolates were amylase, catalase, and gelatinase producing isolates and non-lipase producing, non H2S production and consuming urea, while the carbon consumption test indicated the isolates' ability to consume starch, glucose and sucrose (Table 1).

Characters
Results Growth on SNA medium Gray color colony, no dyes production Amylase production + Catalase production + Gelatinase production + Lipase production -H2S production -Urea decomposition + Starch utilization + Glucose utilization + Sucrose utilization +

Fractionation by preparative HPLC
The results of preparative HPLC revealed that 4 fractions were collected with desired amounts of each compound when using fraction collector in depend on mobile phase system in analytical HPLC with (50 % HPLC-grade acetonitrile ) at 254 nm and cycling up was employed to increase the separation efficiency, each fraction were collected at specific retention time (min) ( Table 2).

Chemical composition of the HPLC fractions using GC-MS
The chemical composition of four fractions which analysis by GC/MS showed the identification of many components, representing the major components in the each fractions arranged based on the retention time and area were showed in (Table 3) and (Figure 1).

In vitro Anti-tumor Cytotoxicity
The results in ( figure 2) showed that all four nano purified fractions were applied on HT 29 colon cancer cells and exhibited significantly differences compared with control treatments of inhibition cells number. Where the highest rate of inhibition of cancer cells was about 81.5, 89, 93.8 and 90.1%) for the nanofractions 1, 2, 3 and 4, respectively, which subjected with concentration 500 mg/ml. When using the half dilution series, the inhibition percentage decreased gradually with dose dependent response decreasing, And these data were used to calculate the values of IC50 (the inhibitory value of half the number for all nanofractions. the application of concentration with inhibition value and solved the equation to IC50 value were gained, which were (151.4, 16.4, 16.6 and 43.8 µg/ml) to four nano fractions respectively (Figure 2 and 3).

Discussion
Actinomycetes is a Gram-positive, aerobic bacterium that is belonging to the order actinomycetales characterized by having an aerial mycelium. It is the most common filamentous organisms in the soil, and it is responsible for the smell of the earth, which indicates the vitality of the soil. It has a major role in the recycling of organic matter (Bhatti and Bhat, 2017).Actinomycetes is widespread in various habitats and participates in important processes, as it not only can live in harsh soil conditions such as lack of moisture and high salinity, but it stimulates plant growth. (Hamdali et al., 2008).
In Georgia in the United States I refer to an example of filamentous bacteria prevalent in Pasture and cultivated soils (Lauber et al., 2009).In addition, Burck et al. (2003) indicated that Actinomycosis is the most common bacterial community in agricultural soils compared with forest soils when these soils were analyzed and compared in different countries. Moreover, he determined that actinomycetes increases after the transfer of lands from forest to agricultural ( IR% D as increased production leads to apoptosis of cancer cell (McIlwain et al., 2015).
A recent study demonstrates that heptadecanoic acid can exert anti-cancer effects on lung carcinoma cell line, emphasizing the efficacy of fatty acids in targeting human lung cancer cells (Xu et  al., 2019).
Also some researches showed that Fumaric acid used for inhibiting the solid growth of Ehrlich tumor in mice, was found to reduce markedly the growth and viability of Ehrlich, MH134, and L1210 mouse tumor cells in culture at concentration of 0.3 approximately 1.2 mg/ml. ( Kuroda and Akao, 1981) .
There is no doubt that the use of nanocomposites as a catalyst in increasing target identification and ensuring intracellular access to drugs has been referred to in many studies one important example Nanoscale drug delivery systems using liposomes and nanoparticles are emerging technologies for the rational delivery of chemotherapeutic drugs in the treatment of cancer. Their use offers improved pharmacokinetic properties, controlled and sustained release of drugs and, more importantly, lower systemic toxicity (Malam et al., 2009).

Conclusion
This study showed that the actinobacteria extract has very high efficacy against cancer cell lines of the type of colon cancer, as the compounds purified from the extract by HPLC, the chemical analysis of them by GC-mass showed they contain compounds that act to inhibit the cancer cells in addition to the increase in the effectiveness of these compounds. From its combination with liposomes nanoparticles that served to deliver the active substance into the cancer cell and destroy it.

Declaration of interest
The authors report no conflicts of interest.