In vitro selective cytotoxicity of activated parasporal proteins produced by Bacillus thuringiensis serovars kumamotoensis and tohokuensis against human cancer cell lines

The anti-cancer activity of alkali-solubilized protease-activated parasporal proteins produced by 78 local Bacillus thuringiensis strains and 14 reference B. thuringiensis strains was screened against five human cancer cell lines (CACO-2, Hep2, HepG2, K562, and MCF-7). Activated parasporal proteins were tested for their hemolytic activity against human erythrocytes. It was found that activated parasporal proteins of 25 local B. thuringiensis strains and 9 reference strains were non-hemolytic. Non-hemolytic parasporal proteins produced by 9 local B. thuringiensis strains were found to exhibit no to low cytotoxicity against human non-cancerous Hs27 cells. Out of them, activated parasporal proteins of two local B. thuringiensis strains (J61; B. thuringiensis serovar kumamotoensis and J72; B. thuringiensis serovar tohokuensis) were found to produce high to very high in vitro selective cytotoxicities, preferentially toxic to cancerous cells, against all cancer cell lines used in this study. This is the first observation of the anti-cancer activity from B. thuringiensis serovar kumamotoensis. Based on IC50 values, activated parasporal proteins of J61 strain produced the most significant cytotoxicity against all cancer cell lines. Furthermore, CACO-2 and MCF-7 cells were found to be the most sensitive. Thus, parasporal proteins produced by B. thuringiensis serovar kumamotoensis strain J61 and/or B. thuringiensis serovar tohokuensis strain J72 may be used as alternative or improving means for current cancer therapy.

lung cancer, and leukemia) was responsible for the death of over 10,000 Jordanians from 1996 until now.A total of 8,744 new cancer cases were registered by Jordan Cancer Registry (JCR) in 2013.Out of new cases of cancer recorded amongst Jordanians in 2013, colorectal cancer (12.7%) followed by lung cancer (11%) were the most common among Jordanian males.Whereas, breast cancer (36.5%) followed by colorectal cancer (9.4%) were the most common among Jordanian females.The spread of cancer is increasing over the world especially in developing countries (Plummer et al., 2016).The number of new cancer cases diagnosed among Jordanians has increased up to 51% in the past ten years with 0.9:1 male to female ratio according to JCR.
There is a great deal of controversy within the medical community over what kind of medical treatment is most efficacious in treating cancer.Most conventional medical treatments for cancer are of little help, so alternative techniques are required to be developed to target cancer.
In this research, the possibility of using Bacillus thuringiensis (Bt) as a source of anti-cancer compounds that are usually too difficult to create synthetically will be investigated.The bacterium B. thuringiensis is a Grampositive spore-forming soil bacterium.Several strains of this bacterium produce crystalline parasporal proteins during sporulation.These parasporal proteins often contain -endotoxin proteins (known as insecticidal Cry proteins) that can exhibit strong insecticidal activity against agriculturally and medically important insect pests (Saraswathy and Kumar, 2004).It has long been believed that B. thuringiensis is characterized by insecticidal activity associated with its parasporal proteins.However, several studies (Ohba et al., 1988;Ohba, 1996;Roh et al., 1996;Mizuki et al., 1999a) have demonstrated that B. thuringiensis strains producing non-insecticidal parasporal proteins are more widely distributed than insecticidal ones.In the beginning of this century, many efforts were oriented toward studying the biological activities of non-insecticidal parasporal proteins yet to be discovered.Mizuki et al. (1999b) reported cytotoxicity against human leukemic T cells and other human cancer cells for parasporal proteins produced by some noninsecticidal B. thuringiensis strains.Moreover, it was found that these non-insecticidal Cry proteins, designated parasporins, are capable of preferentially killing cancer cells upon proteolytic degradation (Mizuki et al., 1999b(Mizuki et al., , 2000;;Yamashita et al., 2000;Ito et al., 2004;Okumura et al., 2004;Katayama et al., 2005;Hayakawa et al., 2007;Jung et al., 2007;Uemori et al., 2008;Yasutake et al., 2008;Nagamatsu et al., 2010).Six parasporin families, including 19 parasporin toxins isolated from B. thuringiensis strains, were identified by the Committee of Parasporin Classification and Nomenclature (Okumura et al., 2010;Wong, 2010).For example, parasporin-1 was isolated from B. thuringiensis strain A1190 (Mizuki et al., 1999b(Mizuki et al., , 2000)).This parasporin when degraded by proteases exhibited selective cytotoxicity against cancer cells such as human leukemic T cells (Mizuki et al., 2000).It was found that parasporin-1 activates apoptotic pathway (Katayama et al., 2007).Activated parasporin-2 isolated from B. thuringiensis A1547 belonging to serovar dakota showed extremely high cytotoxicity against different kinds of cancer cells including leukemic T cells such as MOLT-4, Jurkat, and HL-60 (Kim et al., 2000;Ito et al., 2004;Kitada et al., 2006).It was found that the cytotoxic effect of parasporin-2 is non-apoptotic toward cancer cells but causes swelling of the susceptible cells (Ito et al., 2004).Moreover, another parasporin, called parasporin-3, isolated from B. thuringiensis isolate A1462 exhibited limited cytotoxicity against human leukemic T cells (Yamashita et al., 2000(Yamashita et al., , 2005)).It was hypothesized that parasporin-3 kills cancer cells by a receptormediated mechanism (Yamashita et al., 2005).An important parasporin known as parasporin-4 was obtained from B. thuringiensis strain A1470 which belongs to serovar shandongiensis induced necrosis-like cytotoxicity against human leukemic T cells (Lee et al., 2000;Okumura et al., 2005;Saitoh et al., 2006).A cytotoxic protein toward the leukemic T cell, MOLT-4, was also isolated from B. thuringiensis serovar coreanensis strain A1519 (Namba et al., 2003).Parasporin-5, purified from B. thuringiensis serovar tohokuensis strain A1100, was found to exhibit cytocidal activity against human leukemic T cells (Ekino et al., 2014).A novel parasporin isolated from B. thuringiensis strain M019 was classified into a new parasporin family (parasporin-6) preferentially kill human hepatocyte cancer HepG2 cells and uterus cervix cancer HeLa cells (Nagamatsu et al., 2010).
Efforts dedicated to screening microbial natural products for cancer therapy are very limited.Therefore, the current research is an important advance in the fight against cancer because bacterial metabolites are not well explored for their effectiveness in cancer therapy.These metabolic products may become an alternative or a supplement to current cancer therapies.Thus, this research aimed to test B. thuringiensis parasporal proteins against several cancer cells as alternative or improving means for current cancer therapy.

Solubilization of B. thuringiensis parasporal proteins
Bacterial strains were cultured in 75 ml of T3 broth (Travers et al., 1987).These cultures were incubated at 150 rpm for 3 days at 37°C, and then left at 4°C for 1 day.Sporulated cultures of B. thuringiensis were centrifuged at 5000 rpm for 10 min at 4°C, and washed three times with distilled water before solubilization (Saitoh et al., 1996).The crude parasporal proteins were solubilized in 5 ml of 50 mM Na2CO3 (pH 10) containing 10 mM dithiothreitol (DTT) and 1 mM EDTA for 1 h at 37°C (Mizuki et al., 1999b).After solubilization, the mixture was centrifuged at 14000 rpm for 5 min at 4°C, and the pH of the supernatant was adjusted to 8 by 1 M HCl then passed through a 0.45-m membrane filter.

Activation of B. thuringiensis parasporal proteins
Solubilized parasporal proteins were activated by proteolysis in 0.3 mg/ml proteinase-K at 37°C for 1.5 h.After that, 1 mM phenylmethylsulphonyl fluoride (PMSF) was added to stop the proteolytic reaction (Mizuki et al., 2000).The mixture was then filtered through a 0.45-m membrane filter and the filtered fluid was stored at 4°C until use.

Protein quantification
The protein concentration in the filtered fluid was determined by the method of Bradford (1976).Bradford procedure was performed by using bovine serum albumin (BSA) as standard.After assessment of protein concentration by spectrophotometry at 595 nm absorbance, the protein concentration in each sample was adjusted to 1 mg /ml.

Hemolytic activity
Hemolytic activity was tested on blood agar medium using fresh human erythrocytes (5%).50 µl of activated parasporal proteins was added into each well (5 mm i.d.) prepared on blood agar plates.The type of hemolysis was determined after incubation of plates at 37°C for 48 h (Carillo et al., 1996).

Cells and culture conditions
Six human cell lines were used in this study, including noncancerous normal cells (Hs27; human foreskin fibroblast cell line) that was used as a non-transformed control and five cancerous cells (CACO-2; human colorectal adenocarcinoma cell line, Hep2; human larynx epidermoid carcinoma cell line, HepG2; human liver hepatocellular carcinoma cell line, K562; human leukemic T cells, and MCF-7; human breast cancer cell line) that were kindly supplied by Dr. Saeid Ismaeil (Faculty of Medicine, University of Jordan).

Screening of anti-cancer activity
For screening assay, 20 µl of non-hemolytic proteinase K-activated parasporal proteins (1 mg/ml constant concentration, that is, 20 µg/well) was added to 200 µl of harvested Hs27 cells in fresh DMEM, mixed thoroughly by pipetting and 180 µl medium were loaded into each well of 96 well micro test plate to bring the total volume to 500 µl/well.Cells were plated at a density 4×10 4 cells/well, and counted by hemocytometer.Then, the 96 well micro test plates were incubated at 37°C in a humidified 5% CO2 incubator for 48 h.At the end of incubation time, the viability of cells was assessed by 3-(4,5-dimethythiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983;Heiss et al., 1997).In the assay, each well of the 96 well microtest plate received 40 µl (50 µg) of MTT and incubated at 37°C for 4 h in a humidified 5% CO2 incubator.After that, 100 µl of dimethyl sulfoxide (DMSO) was added.The optical densities were measured at 450 nm with 630 nm reference wavelength using enzyme-linked immunosorbent assay (ELISA) microplate reader.Each treatment was performed in triplicate and repeated five times.The survival rate was determined by comparing the average of absorbance values with that in the control without parasporal proteins.
To determine the anticancer activities, the non-hemolytic parasporal proteins that exhibited no cytotoxicity against normal Hs27 cells were tested against cancer cell lines (CACO-2, Hep2, HepG2, K562, and MCF-7).The cytotoxicity assay was performed for adherent cell lines in the same manner as for Hs27 cells.Likewise, the cytotoxicity was tested on the non-adherent K562 cells in the same manner as for adherent cells but using RPMI 1640 medium instead of DMEM medium.

In vitro selective cytotoxicity
The cytotoxicity of non-hemolytic protease-activated parasporal proteins that exhibited selective cytotoxicity (that is, non-toxic to non-cancerous Hs27 cell line but toxic to other cancerous cell lines) was performed in the same fashion as in the anticancer screening assay but using six concentrations (20, 10, 5, 1, 0.5, and 0.1 µg/ml) of activated parasporal proteins.The mortality of cells was monitored after 48 h by MTT assay.

Statistical analysis
After treatment with MTT assay, the obtained absorbance values were corrected by subtracting the average absorbance of blank (contains the growth medium) from the average absorbance of vehicle (contains the growth medium and cells) and average absorbance of treatment (contains the growth medium, cells, and activated parasporal proteins).To calculate inhibition percentage of cells, the formula "Inhibition% = ((A-B)/A)×100" was used, where A is the vehicle absorbance and B is the treatment absorbance.
The median inhibitory concentration (IC50) was determined by comparing the average mortality values of the six concentrations of activated parasporal proteins with that in the control without activated parasporal proteins.Each treatment was achieved in triplicate.The IC50 values, regression equations, and correlation coefficients (R 2 ) were determined by non-linear regression analysis (MS Excell, Microsoft Co., 2010).The 95% confidence limits (CLs) were also calculated using MS Excel (2010).The statistical outliers in data were excluded using Q-Test.

RESULTS
The current study was performed to screen the anticancer activity of proteinase K-activated parasporal proteins produced by 78 local B. thuringiensis strains and 14 reference B. thuringiensis strains against five human cancer cell lines, including, colorectal adenocarcinoma CACO-2 cells, larynx epidermoid carcinoma Hep2 cells, liver hepatocellular carcinoma HepG2 cells, leukemic K562 cells, and breast cancer MCF-7 cells.
Proteinase K-activated parasporal proteins of the 34 B. thuringiensis strains (25 local strains and 9 reference strains) that exhibited no hemolysis against human erythrocytes were screened for their cytotoxicity against normal non-cancerous human Hs27 cells (Table 2).The colorimetric MTT assay was used to determine the degree of cytotoxicity of activated parasporal proteins against Hs27 cells.It was found that activated parasporal
proteins produced by nine local B. thuringiensis strains of seven serovars (sooncheon, kumamotoensis, tohokuensis, thuringiensis, morrisoni, kenyae, and kurstaki) exhibited no to low cytotoxicity against Hs27 cells (Table 2).Whereas, the non-hemolytic parasporal proteins of the remaining B. thuringiensis strains (16 local strains and 9 reference strains) were found to exhibit moderate to very high cytotoxicity against Hs27 cells.
The activated parasporal proteins of B. thuringiensis strains (9 strains) that exhibited no hemolysis against human erythrocytes and no (-) to low (+) cytotoxicities against normal non-cancerous human Hs27 cells were examined for their selective cytotoxicity against colorectal adenocarcinoma CACO-2 cells, larynx epidermoid carcinoma Hep2 cells, liver hepatocellular carcinoma HepG2 cells, leukemic K562 cells, and breast cancer MCF-7 cells (Table 3).It was found that activated parasporal proteins of two B. thuringiensis strains (J61; B. thuringiensis serovar kumamotoensis and J72; B. thuringiensis serovar tohokuensis) produced high to very high (degree of cytotoxicity is greater than 90%) selective cytotoxicities against cancer cells (Table 3).Activated parasporal proteins of both B. thuringiensis strains J61 and J72 were non-toxic (the degree of cytotoxicity is less than 5%) to Hs27 cells.However, activated parasporal proteins of J61 strain exhibited high selective cytotoxicity against MCF-7 cells and very high selective cytotoxicity against CACO-2, K562, Hep2, and HepG2 cells (Table 3).Whereas, activated parasporal proteins of J72 strain exhibited high selective cytotoxicity against K562 and Hep2 cells and very high selective cytotoxicity against CACO-2, MCF-7, and HepG2 cells.It was observed that the shape of parasporal proteins of strains J61 and J72 was spherical (Figure 1).As shown in Table 4, the IC 50 values of the cytotoxic parasporal proteins against cancer cells were determined.Based on IC 50 values, it was clearly observed that all cancer cells investigated in this study were more susceptible to activated parasporal proteins of serovar kumamotoensis strain J61 than that of serovar tohokuensis strain J72.Furthermore, CACO-2 and MCF-7 cells were the most sensitive to activated parasporal proteins of strains J61 and J72.

DISCUSSION
Cancer is a leading cause of death worldwide (nearly 1 in 6 deaths is due to cancer), accounting for 8.8 million deaths in 2015 according to the World Health Organization (WHO, 2017).The most common causes of cancer death and the deadliest are cancers of the lung (19.20% of deaths), liver (8.95%), colorectal (8.80%), stomach (8.57%), and breast (6.49%).About 70% of cancer deaths occur in low-and middle-income countries (WHO, 2017).As reported by the Ministry of Health (MOH, 2013), the cancer incidence in Jordan is in proportional increase.In addition, colorectal cancers and breast cancer are ranked first among Jordanian males and females, respectively.Therefore, the current study was established to investigate the anti-cancer activity of parasporal proteins produced by local B. thuringiensis strains against various human cancer cell types, in particular colorectal adenocarcinoma colorectal CACO-2 cells and breast cancer MCF-7 cells.Furthermore, the anticancer activity of B. thuringiensis parasporal proteins toward larynx epidermoid carcinoma Hep2 cells, liver hepatocellular carcinoma HepG2 cells, and leukemic K562T cells was examined.Alkali solubilized and proteolytic processed parasporal proteins of the 92 B. thuringiensis strains used in this study were screened for their hemolytic activity against human erythrocytes.The hemolytic activity of parasporal proteins was measured before anti-cancer screening assay to ensure that the anti-cancer activity was not induced by hemolysins such as Cyt proteins that exhibited cytolytic activity against human

J61 (B. thuringiensis serovar kumamotoensis) J72 (B. thuringiensis serovar tohokuensis)
erythrocytes (Crickmore et al., 1998(Crickmore et al., , 2016 The activity in these strains was not attributable to the broad-spectrum Cyt proteins in view of the fact that they showed no hemolytic activity against human erythrocytes.Furthermore, the parasporal proteins of these B. thuringiensis strains (J61 and J72) were previously reported (Obeidat et al., 2012) to exhibit no insecticidal activity against diptera and lepidoptera.This is in agreement with previous studies (Mizuki et al., 1999b(Mizuki et al., , 2000;;Lee et al., 2000;Yamashita et al., 2000;Ito et al., 2004;Okumura et al., 2004;Katayama et al., 2005;Hayakawa et al., 2007;Jung et al., 2007;Uemori et al., 2008;Yasutake et al., 2008;Nagamatsu et al., 2010) that clearly demonstrated that only non-insecticidal parasporal proteins can exhibit discriminating cytotoxicity against different cancer cell types.Unfortunately, activated parasporal proteins from all reference B. thuringiensis strains screened in this study did not exhibit selective cytotoxicity against target cells.Mizuki et al. (1999b) found that the non-hemolytic leukemia cytotoxic strains belonged to serovars dakota, neoleonensis, shandongiensis, coreanensis and other unidentified serogroups.In a recent study, Ekino et al. (2014) reported that parasporal proteins produced from B. thuringiensis serovar tohokuensis were also cytotoxic against human leukemic T cells.Similarly, in a preceding study, Obeidat (2008) demonstrated that activated parasporal proteins of B. thuringiensis serovar tohokuensis exhibited selective cytotoxicity against larynx epidermoid carcinoma Hep2 cells and leukemic T cells, Jurkat.In this study, the non-hemolytic selectively cytotoxic B. thuringiensis strains were found to belong to serovar kumamotoensis and serovar tohokuensis.This observation demonstrated that cytotoxic proteins which exhibited specific cytotoxicity against cancer cells can be found in a variety of B. thuringiensis serovars.To our knowledge, no previous study has revealed the production of selective anti-cancer activity from processed parasporal proteins produced by B. thuringiensis serovar kumamotoensis.
According to IC 50 values, protease-activated parasporal Obeidat 2187 proteins of the cytotoxic strain J61 showed higher significant toxicity against cancer cells compared to that of strain J72.The 95% confidence limits of the IC 50 values determined from MTT assay were not overlapped suggesting that J61 and J72 cytotoxicities are significantly different.Therefore, the cancer cells were more sensitive to activated parasporal proteins produced by strain J61.It also appeared from the results that there is a marked variation among parasporal proteins in the level of cellkilling activity.Of particular interest is the fact that the activated parasporal proteins from strains J61 and J72 were able to discriminate between cancerous cells and noncancerous Hs27 cells, killing the former cells specifically.This finding strongly suggests the possible occurrence of B. thuringiensis strains in local habitats of Jordan which naturally produce parasporal proteins highly selective for cancer cells.This may lead to the use of these B. thuringiensis parasporal proteins for medical and pharmaceutical purposes including the treatment of particular types of cancer cells.Further studies regarding the identification of anti-cancer protein structure found in promising B. thuringiensis crudes are in process.

Table 1 .
In vitro hemolytic activity of proteinase K-activated parasporal proteins produced by B. thuringiensis strains. B.

Table 2 .
In vitro cytotoxicity of non-hemolytic proteinase K-activated parasporal proteins produced by B. thuringiensis strains against normal human Hs27 cells.

Table 3 .
In vitro cytotoxicity of non-hemolytic proteinase K-activated parasporal proteins produced by B. thuringiensis strains against five cancer cell lines.

Table 4 .
The inhibitory concentration (IC05) of non-hemolytic proteinase K-activated parasporal proteins produced by Bacillus thuringiensis strains against five cancer cell lines. B.