Parasporin A13-2 of Bacillus thuringiensis isolate from Papaloapan region, induce a cytotoxic effect by late apoptosis against breast cancer cells


 The protein A13-2 was obtained from Bacillus thuringiensis strains isolated from the Papaloapan watershed region (Oaxaca, Mexico). The cytotoxic activity of parasporal inclusions was studied against breast cancer cell line (MCF-7) and normal cell (human peripheral blood mononuclear cells). The MTT, the formation of reactive species, nitric oxide, free cell DNA, and the type of death cellular were assessed. The protein A13-2 shows the highest cytotoxic activity against MCF-7 (13% at 6 µg/mL), the extracellular DNA increases, and it shows no stress for reactive species or nitric oxide. Besides, the A13-2 parasporin shows no toxicity to peripheral blood mononuclear cells, and it does not generate changes in nitric oxide levels or free cell DNA. According to microscopy and flow cytometry, A13-2 leads to the death of MCF-7 cell by late apoptosis. Due to that, the cytotoxic effect of A13-2 was specific for MCF-7, and it does not affect peripheral blood mononuclear cells (normal cells). When analyzed together, our results show for the first time that the A13-2 protein isolated from Mexican strains of B. thuringiensis has a high selectivity against the MCF- 7 cell line, thus representing a promising alternative for the treatment of cancer breast.


Introduction
Bacillus thuringiensis is a Gram-positive bacterium that during the sporulation stage produces parasporal crystals [1]. The parasporal crystals can be constituted of the proteins Cry, Cyt, and parasporins, each of them with particular activities, insecticidal, hemolytic and anticancer respectively [2,3]. Parasporins are a set of proteins that are divided or grouped into six families (PS1-PS6) and they have different sizes and modes of action [4]. It has been demonstrated that parasporins are cytotoxic against mammalian cancer cells [5][6][7][8]. The molecular weights of proteins are from 34 to 94 kDa. Most of B. thuringiensis parasporins are from strains isolated from the Asian continent (Japan, Vietnam, Malaysia, and India) [9]. Although, there are reports of this type of strain in America, in the Caribbean islands, Canada and México [9,10].
The parasporins are good candidates for cancer therapy due to their selective activity against cancer cells and their ubiquity [11]. Breast cancer is the second most prevalent in the world and leads to 627,000 deaths per year [12]. It is considered the most common malignancy in women; whose treatment methods depend on molecular subtype and the stage of breast cancer. The main treatments are surgery, chemotherapy, and hormonal therapies.
The principal disadvantage about these treatments is its poor specificity for cancer cells because they attack or released factors that can damage the DNA of normal cells, affecting even more so the patient and causing collateral and irreversible damage in some cases [13]. The scientific community remains in the constant struggle to generate new strategies to avoid these side effects. i.e., the creation of drugs that directly attack cancer cells without damaging other tissues implying a lower risk of negative effects and improving the quality of life of the affected people. Even though, the parasporin definition is: "Bacillus thuringiensis and related bacterial parasporal proteins that are non-hemolytic but capable of preferentially killing cancer cells" [4], a few reports are found in the literature about the effect of the parasporins against normal cells. Therefore, the aim of this study was to obtain a new parasporin produced by B. thuringiensis with high selectivity: activity against breast cancer and without toxicity against normal cells.

Selection of strains and proteins
The SDS-PAGE gels for protein purification process show that A-13 strain produced bands of approximately 26 and 30 kDa ( fig. 1). The bands for cytotoxicity assay were selected according to their size and intensity; and they were called A13-2 (26 kDa) and A13-5 (30 kDa) ( fig. 1).

Cytotoxic effect of parasporal protein A13-2 in MCF-7 7 and PBMC cells
The cytotoxic activity assay results of the MCF-7 cells treated with the parasporal protein A13-2 during 48 h are shown in figure 2a. Data show that the A13-2 protein is cytotoxic against MCF-7, at 6 μg/mL cell viability has been reduced by 87%, while A13-5 protein at 8 μg/mL reduces cell viability by 80%. These results indicate that MCF-7 cells are more susceptible to A13-2 protein than normal PBMC cells. Therefore, A13-2 protein was chosen for further analysis.

Fig. 2
Interestingly, MTT assays in PBMC (normal cells) at 48 hours incubation using the A13-2 and A13-5 treatments show no cytotoxic effect at the concentrations tested ( fig. 2b), suggesting high selectivity of these parasporal proteins against cancerous cells. While the cells treated with A13-5 showed no significant difference with the control.

Role of oxidative stress in the cytotoxicity of A13-2 protein in MCF-7 and PBMC cells
In figures 3a and 3b are shown the results for NO assay of the parasporal protein against MCF-7 and PBMC, respectively. The parasporal protein treatments increase the NO levels to 24% at 4 μg/mL in MCF-7 cultures.
That increment is not enough to trigger the oxidative stress. There are no significant differences in NO levels for parasporal protein treatments against PBMC cells.  agar plates, incubated at 30 °C for 96 h, observing that at least six different parasporal proteins were produced by each isolated in different sizes and concentration. Brasseur et al. [15] reported that B. thuringiensis 4R2, cultivated at 30 °C on nutrient agar at pH 7.1, produces five parasporal proteins where the one at 37 kDa was identified as PS2Aa1. This variation in the number of parasporal proteins found is due to the growth and environmental conditions of each B. thuringiensis strain. In soil, the bacteria population is not randomly distributed because factors such as soil composition, organic matter, pH, water availability and oxygen, along with the host plant, play an essential role in the adaptation of this microflora [16].
The cytotoxicity effect of A13-2 and A13-5 (26 and 30 kDa, respectively) on MCF-7 cells was similar to that described in previous reports. Brasseur et al. [15] reported that PS2Aa1 (37 kDa) had good activity against MCF-7 at of 2.5 µg/mL reducing viability to 20%. However, the authors used chemical or enzymatic processes for protein activation. Maher [17] identified two strains J61 and J72 with cytotoxic activity against MCF-7 that reduce to 50% cell viability at 1 µg/mL and 2.79 µg/mL, respectively. Other authors tested differents parasporins observing no significant cytotoxic activity against MCF-7 [18][19][20]. It has been reported that a parasporin could affect more than one cancer line cells. Brasseur et al. [15] and Maher [17]  To mechanistically understand how parasporal proteins A13-2 reduces cell proliferation and to identify if oxidative stress is involved, NO and ROS levels were examined after exposure to different concentrations of parasporal protein A13-2. Cancer cells have a dual cellular response to oxidative stress. When the stress levels are low, the cells activate essential processes that guarantee their survival; while at high levels of stress, the cells died due to oxidative damage [23]. Moreover, cancer cells are more susceptibles to oxidative stress than normal cells, a small increment in the oxidative environment activates the death mechanisms in cancer cells but not in the normal cells [24].
The role of NO in cancer is complex, it has been reported that it is involved in multiple steps of tumor development, but it is also involved in the cell cycle arrest, i.e., in the apoptosis. [25]. NO can act as an intraand extracellular signaler participating in numerous pathological processes. It is a modulator in several essential biological processes, and it has a dubious role to cells, sometimes beneficial and sometimes harmful [26]. Also, NO is an important cytotoxic mediator of effector cells capable of destroying pathogens and tumor cells [27]. However, NO is potentially toxic, especially in situations of oxidative stress, generation of oxygen intermediates and antioxidant system deficiencies [28]. The nitric oxide synthase (iNOS) is responsible for the endogenous production of NO. iNOS is only expressed when it is induced by cytokines or endotoxins in cells such as macrophages, T lymphocytes, neutrophils, and platelets, among others [29]. Hence, for MCF-7 and PBMC cultures with a parasporal protein treatment, the NO levels measured indicate than the cells were not induced to produce proinflammatory cytokines and that the parasporal proteins were not recognized as endotoxins.
The dichlorofluorescein (DCFH-DA) is the most widely used probe for detecting oxidative stress through measurement of ROS formation due to the increment in H2O2, due to the changes in intracellular iron signaling and due to the peroxynitrite formation [30]. Probably, natural ROS scavengers in MCF-7 and PBMC cells, like the glutathione peroxidase, catalase, and superoxide dismutase, can control the increment in the ROS levels and therefore maintaining it in non-toxic levels. Likewise, the increase in PBMC proliferation observed after the A13-2 treatment might involve signaling mechanisms related to ROS levels. Previous studies described that the presence of low ROS levels in some blood cells induce its proliferation [31,32]. The most chemotherapeutic agents and radiotherapy treatment lead to apoptosis death cells by inducing intracellular ROS production [33,34]. Furthermore, it has been proposed that in peptide therapy, one possible mechanism of cell damage is associated with the increment of intracellular ROS [33,34]. Although, the results of the oxidative stress point out that the cytotoxic effect of parasporal protein A13-2 is not through this process.
Due to endogenous and exogenous factors, the DNA could be fragmented, and it could be exported out of the cells. Hence, through the detection of DNA in the medium, a genotoxic effect may be evaluated [35] and also, indirectly the formation of pores in the membrane [36]. Some authors proposed that one of the possible cytotoxic mechanisms of parasporins is through the formation of pores in the membrane of the tumor cells, which leads to an osmotic imbalance and consequently to the cell death [37][38][39]. The Picogreen assay is an easy and fast technique to detect a minimum amount of dsDNA (25 pg/mL) in the medium [40]. Therefore,

Morphological changes and cell death mechanism induced by parasporal protein A13-2
The MTT test shows that A13-2 promotes cell death and NO-ROS results show that this protein does not trigger oxidative stress in MCF-7 ( fig. 3a-3d). Moreover, the Picogreen test shows fragmented free dsDNA in the medium associated with death by necrosis at 48 h ( fig. 3e). Additionally, cytometry test indicated that at 48 h with A13-2 most of the cells were death by necrosis, however a small percentage were at late apoptosis suggesting a rapid transition from late apoptosis to necrosis, which could point out that lower concentrations or times-course analysis perhaps will allow us to observe more apoptotic signals. Therefore, A13-2 protein could be interacting with the cell membrane, leading to the formation of pores that drives to cell death.
It has been reported different mechanisms of cell death treated with parasporins. PS1Aa1 against HeLa cells showed a rapid intracellular increment of Ca 2+ but without lactate dehydrogenase release and IP internalization, which is strong evidence of death by apoptosis [41]. For PS2Aa1 against HepG2, an increment in the membrane permeability was observed where the lactate dehydrogenase extravasation and the PI internalization are due to a similar mechanism to Clostridium perfringens epsilon toxin. PS2Aa1 has homology with epsilon toxin, that is a pore-forming when it is in contact with lipid rafts [42]. PS3Aa1 has closely resembled pore-forming insecticidal Cry proteins, where cancer cells die by increasing membrane permeability [43]. PS4 is nonspecifically bounded to the membrane forming a cholesterol-independent oligomeric pore complex and also shows some homology to α-toxin, aerolysin, and ε-toxin [44]. PS5 has no similarity to other parasporins or Cry proteins but exhibits some homology to β-pore-forming Aerolysins (β-TFPs) and to epsilon toxin, that are poreforming toxins [45] There is little information about PS6, although, it is considered as a three-domain Cry protein with 56.4% identity to Cry2 insecticidal proteins [44]. Thus, all our results show that A13-2 is a parasporin with cytotoxic effect over MCF-7 cells.

Conclusion
The main disadvantage of the actual therapies for cancer disease is that they are toxic for normal cell generating several side effects and deteriorating the patient's quality of life. The parasporin produced for B. thuringiensis A13-2 is cytotoxic effect in MCF-7 cancer cells and it does not have toxic activity against peripheral blood mononuclear cells (non-cancerous cells), i.e., it is specific for the cancer cell. Also, this parasporin leads to cell death by late apoptosis and without triggering oxidative stress. The fundamental goal of cancer treatments is the identification of molecules with high capacity to induce selective cancer cell death, locking the chance to activate the mechanism of survival of the cancer cells. In this context, late apoptosis induced by parasporal protein A13-2 together with necrosis could be the best death mechanisms both mechanisms are irreversible for cells, it has been reported that cancer cells can progressively develop acquired resistance to apoptotic cell death, thus with parasporal protein A13-2 the death is carried out quickly without allowing the triggering of the survival mechanisms. As parasporin extraction and characterization methods are well established, large-scale production by the pharmaceutical industry is plausible and perfectly executable. Although, further studies regarding the mechanisms of cell death and in vivo effects of these proteins have to be done. Moreover, about the elucidation of protein structure, the pathways of cytotoxicity and non-toxicity towards normal cells could lead, in the future, to the drug design.

Bacterial strains and culture conditions
B. thuringiensis strains used were isolated from the Papaloapan watershed region [45]. The strains were cultivated in a Gerry-Rowe medium (MCD Lab.) at a pH of 7.4 (adjusted with 40% NaOH) at 180 rpm for seven days at 30 °C (New Brunswick Benchtop Incubator Shaker I24, Eppendorf) to obtain the protein extracts.
Once the fermentation was concluded, the culture medium was centrifuged at 5500 rpm for 20 min at 5 °C and washed with distilled water, acidified water (pH 2.5; three times), distilled water, NaCl 0.85% w/v (three times) and distilled water, in that order. For every wash, the pellet was resuspended of the solution and centrifuged at 5500 rpm for 20 min at 5 °C. After washing, the pellet was resuspended in 5 mL of distilled water [10].

Parasporal inclusion isolation
For parasporal inclusions isolation, all the washed protein extracts were solubilized with 1:2 of Laemmli buffer at 100 °C for 5 min [46]. Then, the protein extracts were separated by molecular weight through sodium dodecyl

MCF-7 cells culture conditions
The human breast cancer cell line MCF-7 was acquired from American Type Culture Collection (ATCC® HTB-22 TM , Manassas, VA, USA). MCF-7 was at passage 12 when it was used in study and routinely cultured on monolayers at 80% confluence in Dulbecco's modified Eagle's High glucose medium (DMEM, Biowest), supplemented with 10% fetal bovine serum (Biowest), 100 U/mL penicillin, 100 μg/mL streptomycin (Biowest), and 2 mM L-glutamine (Biowest). All cells were maintained at 37 °C with saturated humidity and 5% CO2. To harvest MCF-7 cells, the growth medium was removed and cells were washed with phosphate buffer saline (PBS, Biowest). To produce a cellular suspension, a cell dissociation solution made of trypsin-EDTA (Biowest) was added and incubated at 37 °C for 3 min in a humidified 5% CO2 incubator. Trypsinized cells were reseeded in fresh medium at 10 5 cells/ml and incubated at 37 °C in a humidified 5% CO2 incubator.

Nitric Oxide test
Nitric Oxide (NO) production was evaluated as metabolite involved in the apoptosis induction for MCF-7 and PBMC cells [48,48]. After 72 and 48 h incubation of MCF-7 and PBMC, respectively, with the parasporal protein treatments, the culture plate was centrifuged 10 min at 2000 rpm. In a new plate, 50 μL of the supernatant and 50 μL of the Griess reagent (1% Sulfanilamide and N-1-naphthylethylenediamine-bicyclic 0.1%) were added. The plate was incubated for 15 minutes at room temperature. Subsequently, the absorbance produced was read in a TP-Reader plate reader (Thermoplate, China) at 570 nm [50,51]. 200 µL of DMEM was used as a negative control (NC). The data were expressed as a percentage of free NO in the medium concerning the negative control: with % NO free in the middle = (Absorbance of the sample * 100) / average of the negative control.

Reactive oxygen species assay
The dichlorofluorescein (DCFH-DA) was used to indirectly measure the total rate of reactive oxygen species (ROS) present. After 72 and 48 h incubation of MCF-7 and PBMC, respectively, with the parasporal treatments, the culture plate was centrifuged for 10 min at 2000 rpm, and in a dark new plate was added 100 μL of supernatant, 130 μL of Tris-HCl (10 mM, pH 7.4) and 20 μL of DCFH-DA (1 mM). 200 µL of DMEM was used as a negative control. The plate was incubated for 60 minutes in the dark at room temperature. The reading was made done on a fluorescence meter (SpectraMax ® i3x -Molecular devices) at 525 nm emission wavelength and 488 nm of excitation [52,53]. The data were expressed as a percentage of the total rate of ROS concerning the negative control with % Total rate of ROS = (Absorbance of the sample * 100) / average of the negative control.

Genotoxicity assay
The Picogreen test is fluorimetric and quantifies the double strand of DNA by binding to it and emitting fluorescence when it is released into the medium by cellular apoptosis or necrosis. After 72 and 48 h incubation of MCF-7 and PBMC, respectively, with parasporal protein treatments, the culture plate was centrifuged for 10 minutes at 2000 rpm. 10 μL of the supernatant was placed in a new dark plate, homogenized with 80 μL of TE buffer and it was added 10 μL of the Picogreen reagent diluted in TE buffer (1:200). The reaction was incubated at room temperature and in the darkness for 5 min. The reading was performed on a fluorimeter (SpectraMax® i3x -Molecular devices) at a 520 nm emission wavelength and 480 nm of excitation [54], and 200 µL of DMEM was used as a negative control. The data were expressed as a percentage of a double strand of free DNA in the medium concerning the negative control with % Dc of free DNA in the medium = (Fluorescence * 100) / mean of the negative control.

Apoptosis detection by Annexin V/PI assay
FITC Annexin V/Dead Cell Apoptosis Kit (Molecular Probes Inc., Eugene, Oregon, USA) was used according to the manufacturer's instructions. Annexin V binds to phosphatidylserine on the outer leaflet of the plasma membrane, and its presence on the outer leaflet is a unique feature of early apoptosis. Propidium Iodide (PI) binds to DNA from cells with disrupted cell membrane, as in late apoptosis and necrosis, being excluded from cells with intact membrane [55]. Cells incubated 24 h with parasporal protein were collected, washed with PBS and diluted in 1X Annexin Binding buffer (100 μL). For each sample, 5 μL of Annexin V and 2 μL of PI were added to the cell suspension and incubated for 15 min at room temperature. An additional 100 μL volume of Annexin binding buffer was added to each sample for a total of 200 μL. Samples were analyzed (5000 events) using a BD FACSAria flow cytometer, and the analysis was performed using BD FACSDiva software (Becton, Dickinson and Company, Franklin Lakes, NJ, United States).

Light microscopic observation
The possible MCF-7 cell morphological changes induced by parasporal protein A13-2 incubated during 24,48 and 72 h were analyzed by light microscopy. The cytopathic effect was monitored by an inverted microscope (Motic AE31E, Moticam 5 plus). The images were captured and analyzed for cell morphology with the Motic images plus 3.0 software.

Statistical analysis
Data were analyzed using a One-Way ANOVA (Analysis of Variance) with Graphpad Prism software version 5.0. The Dunnet test was applied to compare each treatment with the control, with a statistical significance of p <0.05.