In vitro Anticancer Activity Effect of Extracellular Metabolites of Some Bacterial Species on HeLa Cell Line

Background: Cancer is still one of the most serious problems that affect human health. Despite the intense efforts to develop treatments, effective agents are still not available. In some cases, conventional therapy could be harmful or fail because of emerging drug resistance. Therefore, the development of novel therapies against cancer is of utmost importance. Assessment of anticancer effects of bacterial metabolites on cancer cells may help in the process of finding new cheap, reliable, contentious and safe cancer therapy. Objective: To determine the anticancer effect of the extracellular metabolites of eight bacterial species on HeLa cell line. Methodology: Extracellular metabolites were prepared by isolating and culturing eight bacterial species (Escherichia coli, Staphylococcus aureus, Micrococcus, Pseudomonas aeruginosa, Lactic acid bacteria, Klebsiella, Proteus and E. coli with its phage) in liquid media. Tubes were incubated overnight and centrifuged. Supernatant was filtered and concentrated using Infra-Red concentrator. Different concentrations were prepared and their anticancer effect were evaluated using MTT cell proliferation assay. Results: Results showed variation among the eight bacteria concerning proliferation inhibition against HeLa cells in a time and concentration dependent manner. Pseudomonas and E. coli with its phage revealed considerable anticancer activity with 63% and 86% inhibitory effects (both at 1000 μg\ml) and IC50 of 301 and 1395 μg/dl at 24h respectively. While Proteus and Micrococcus showed low inhibitory effects and S. aureus enhanced the proliferation of HeLa cells at low concentrations. Conclusion: Among the tested bacteria, Pseudomonas and E. coli and its phage gave the best anticancer inhibitory effects against HeLa cells. Further studies using purified components of effective bacteria are recommended. 1Department of Medical Laboratory Sciences, Faculty of Health Science, Islamic University of Gaza, Gaza Strip, Palestine * Corresponding author e-mail address: elmanama1996@gmail.com


In vitro Anticancer Activity
Effect of Extracellular Metabolites of Some Bacterial Species on HeLa Cell Line.

Introduction:
Cancer is still one of the most serious problems that affect human health. Globally is considered as the second leading cause of death with nearly 8.8 million deaths in 2015. Nearly 1 in 6 deaths is due to cancer (WHO, 2017). Cancer is an uncontrolled growth of cells that in some cases can metastasize through lymph or blood (Ravuri and Kumari, 2013 immunotherapy, radiotherapy and the popular one is chemotherapy (Gillet, et al., 2007). These techniques are useful in certain cases but could be harmful, failure sometimes due to chemotherapy-induced resistance.
Despite the intense efforts to develop treatments, effective agents are still not available. There upon, natural products extracts seem to be the most promising source of new drugs for cancer (El-Gendy et al., 2008).
Some microorganisms such bacteria can be a source of bioactive natural products with the advantage of sustainable production of secondary metabolites (ER et al., 2015). Bacteria have unique capabilities that make them well-suited as 'perfect' anticancer agents.
In recent studies they showed that they could control tumor growth (Forbes, 2010). For example, Lactic acid bacteria (LAB) have been reported to possess certain anticancer properties

HeLa cells culture
HeLa cell line was used and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS, 100 units/ml penicillin and 100 µg/ml streptomycin at 37 o C in a humidified 5% CO2 incubator.

MTT Assay
HeLa cells were plated and allowed to reach approximately 70% -80% confluence where a 100 µl of cells were added into each well and incubated overnight. On the next day: Cells were treated with different concentrations of the extracts starting from 12.5 to 1000 µg/ml. Final volume should be 100 µl per well.

RESULTS AND DISCUSSION
The effect of extracellular extracts on the viability of  (Er, et al., 2015). This technique is based on the ability of cells to metabolize the yellow tetrazolium salt MTT to a blue crystalline formazan product (Er, et al., 2015). The manner in which a cell dies determines the nature of the response by the surrounding tissue.
Death by necrosis acts by inducing oxidative stress and production of numerous pro-inflammatory cytokines (Er, et al., 2015).  coli with its phage crude extract at different time points on HeLa cells was studied. It increased the inhibitory effect in dose dependent manner. The inhibitory effect increased from 20% to 85.7% as the concentration extract increased from 12.5 to 1000μg/ml for 24 h (Table 1).
Colicins A, E1 and E3 are produced by E. coli and have molecular sizes: more than 20, 57 and 9.8 kDa, respectively (Kaur, et al., 2015). Colicin E1 and A Our results showed that Proteus spp. crude extracellular metabolites enhanced the proliferation of HeLa cells at 12.5 μg/ml concentrations. In contrast, the inhibitory effect slightly increased from 11.2% to 27.1% as the concentration of the crude extract was increased from 25 to 1000 μg/ml. As shown in Table 1 sensing-dependent manner in this microorganism (Campbell, et al., 2009, Galloway, et al., 2010, Rojas et al., 2015& Dandekar et al., 2013.
In The probable reason for the observed effects is that molecules isolated from living entities such as P.
Plantaricin A is a bacteriocin of L. plantarum C11 and its molecular weight reaches 2.4 kDa. In the case of artificially synthesized plantaricin A, the cytotoxicity against the human T cell leukaemia was determined in vitro (Zhao, et al., 2006).
A study has shown that P. pentosaceus, L. plantarum, and W. confusa have the potential to inhibit the proliferation of Caco-2 cells. L. plantarum showed the strongest inhibitory effect. Although there are anticancer studies with L. plantarum , there are not adequate data regarding the anticancer effects of P. pentosaceus and W. confusa. By using the CC BY 4.0 ) / Islamic University of Gaza IUG Journal of Natural Studies ( MTT assay, Patel et al., (2010) reported that dextran isolated from P. pentosaceus has no cytotoxic effect on HeLa cells (Patel, et al., 2010 (Thomas, et al., 2011).
In another study by Adnyana et al., in 2007, they found that the ethyl acetate extract of S. galbus TP2 strain shows a potential anticancer (T47D cell line) activities (Andayani, et al., 2015).
In a study, Vijayabharathi et al., reported that Sterptomyces strain, isolated from humus soils in the Western Ghats, has an anticancer activity against HepG2 (hepatic carcinoma) and HeLa (cervical carcinoma) in vitro (Vijayabharathi, et al., 2011).
A study performed by WAHYUDI et al., in (2006) showed that the best toxicity and anticancer activity were performed by B. subtilis crude extract with LC50 and IC50 were 378 µg/ml and 132.877 µg/ml, respectively (Safari, et al., 2015).
Microbes are the large repositories of bioactive compounds with different structural and functional forms, namely; sugars, acids, esters, ethers, terpenoids, peptides, proteins, and nucleopeptides, which are meant for the immense applications in the treatment of the wide range of disease types, such as anticancer, anti-inflammatory, antimicrobial, antiviral, and antifungal (Kumavath, 2015).
This study suggests that E. coli with its phage and P. aeruginosa may considered as potential candidates for anticancer use (as illustrated in Figure 1). More work is needed in order to identify and purify the active metabolites from those crude extracts and then to be tested on HeLa cell lines and other available tumor cell lines.