Determination of the cytotoxic effects of parasporal proteins in native Bacillus thuringiensis isolates on various cancer cell lines and investigation of the apoptotic, synergistic, and angiogenic potency of the isolate Bt 5.4.

Introduction
Cancer, a complex disease affecting cells and tissues, is one of the most common causes of death worldwide. In 2020, there were an estimated 20 million newly reported cases of cancer, resulting in a loss of 9.7 million lives. In terms of new case rates in 2022, lung cancer (12.4%) was the most common cancer, followed by female breast cancer (11.6%), colorectal cancer (9.6%), and prostate cancer (7.3%). The number of cases has been increasing over the years, and many people are expected to suffer from this problem in the coming years [1].
Today, traditional treatments such as radiotherapy and chemotherapy are continually used in the fight against cancer, but owing to several side effects and disadvantages, such as toxicity to healthy cells and the development of resistance to anticancer agents [2], researchers have focused on alternative methods. The cytotoxic effects of biological agents on cancer cells are being investigated, and studies on the development of effective drugs with fewer side effects are gaining momentum. In fact, microbial compounds are important sources in drug production. Compared with other natural resources, they have many advantages, such as providing rapid production due to their high rate of reproduction, being able to use very different carbon sources as energy sources, not being dependent on seasonal changes in biomass production, and being able to be produced in high quantities and good quality in a laboratory environment [3].

B. thuringiensis (Bt) is a gram-positive, spore-forming bacterium belonging to the Bacillaceae family. It is known as a soil bacterium; however, it has a wide range of ecological niches, such as water, stored product dust, insect habitats, leaf surfaces, and coniferous trees [4]. This bacterium is a highly functional microorganism with a variety of proteins and secondary metabolites produced against insects, nematodes, mites, protozoa, and mammalian cells [5]. To date, Bt has been noted, especially for its entomopathogenic features, and it has been used as a successful biopesticide in biological control. Insecticidal crystal proteins (ICPs), also called delta-endotoxins (Cry and Cyt proteins), are produced simultaneously during the stationary phase of bacterial growth and are highly toxic to various insect species from many orders, especially Lepidoptera, Coleoptera, and Diptera [6]. However, studies conducted on the insecticidal activities of Bt parasporal proteins have demonstrated that the distribution of non-insecticidal proteins is greater than that of insecticidal proteins [7]. This revealed the need to investigate other biological activities of Cry proteins. Mizuki et al. [8] reported the cytotoxic activities of non-insecticidal proteins against cancer cells and identified them as a new crystal protein family called parasporins (PSs) [7]. PS was first identified as a non-insecticidal and non-hemolytic parasporal protein. Research has shown that insecticidal toxins may also have anticancer effects. Therefore, the definition was updated as ‘B. thuringiensis and related bacterial parasporal proteins that are nonhemolytic but capable of preferentially killing cancer cells.’ The ability to differentiate cancer cells from noncancer cells via specific receptor recognition is an important advantage [9]. PS is present in protoxin forms such as Cry proteins and needs to be activated by proteases under alkaline conditions [5]. Parasporins are named and classified according to amino acid sequence similarity, as in Cry proteins. To date, 19 parasporins have been classified into 6 PS families (PS1-PS6) by the Committee of Parasporin Classification and Nomenclature (http://parasporin.fitc.pref.fukuoka.jp/index.html). The cytotoxic effects of these compounds have been examined in different cancer cell lines [10].
In this study, we aimed to determine the cytotoxic effects of 6 native and nonhemolytic Bt isolates known to carry parasporin genes. First, the pH of the solubilization buffer and the proteinase K concentration were optimized for the proteolytic activity of the toxins. The activated toxins were used to treat HeLa, A549, MCF-7, PC-3, and Burkitt cancer cell lines and the healthy cell line HEK-293 to demonstrate their cytotoxicity. One isolate, Bt 5.4, which has high cytotoxicity against cancer cells but not normal cells, was selected and further examined to reveal its anticancer potential. The apoptotic, synergistic, and antiangiogenic effects of this parasporal protein on A549 and PC-3 cells were investigated.

Materials and methods

Bacterial strains and isolates
In this study, Bt isolates from the Bt collection of the Molecular Biology Laboratory of the Biology Department at Muğla Sıtkı Koçman University were used. They were isolated from soil samples collected from cherry orchards in Izmir (Türkiye). For this research, 6 native Bt isolates (5.4, 5.7, 5.10, 7.1, 2.1, and 40.4) out of 250 isolates that were previously screened for cry and parasporin (ps) genes and identified as ps1, ps2, or ps6 positive isolates [11] were used to determine their biological activities. The parasporin gene contents of the Bt isolates were ps1 (2.1), ps2 (40.4), and ps6 (5.4, 5.7, 5.10, and 7.1). The reference strain Bacillus thuringiensis subsp. dakota 4R2 for the ps2 gene was obtained from the Bacillus Genetic Stock Center (Ohio, USA).

Extraction, solubilization, and activation of parasporal inclusion proteins
Parasporal proteins become solubilized under alkaline conditions and are subsequently digested by proteolytic enzymes, resulting in the formation of active toxins [12]. In this present study, Bt dakota 4R2 carrying the ps2 gene was used as a reference strain for optimization of pH and proteinase K for effective solubilization of parasporal protein extraction. The extraction, solubilization, and activation of parasporal inclusion proteins were performed according to the methods of Brasseur et al. [13]. According to this methodology, Bt isolates were inoculated onto nutrient agar and incubated at 30 °C for 4 days. After observing the cell lysis with a phase contrast microscope, the cultures were harvested and washed with sterile dH2O twice. The presence of a spore crystal mixture in the pellet was confirmed under a phase contrast microscope and incubated with 500 µl solubilization buffer at different pH values of 10, 10.5, 11, and 11.5 containing 56 mM Na2CO3 and 11 mM dithiothreitol (DTT) at 37 °C for 1 h [13]. Then, the mixture was centrifuged at 13,200 rpm for 2 min to discard the insoluble protein. The supernatant was filtered through a 0.22 μm membrane filter, and the pH of the 250 µl filtrate was adjusted to 8 with 1 M Tris-HCl (pH 4.98). Parasporal proteins in each pH range were digested separately with 25, 50, 100, 150, and 200 µg/ml proteinase K at 37 °C for 1 h. Phenylmethylsulfonyl fluoride (PMSF) at a final concentration of 1 mM was added to stop proteolytic processing. After activation, the protein concentration was determined via the Bradford assay [14]. The protein patterns were determined by SDS‒PAGE with a 10% separating gel [15]. Additionally, the percent protein recovery was calculated via the formula [(protein concentration after solubilization/protein concentration before solubilization) × 100].

Cell lines and culture conditions
In this study, 5 different cancer cell lines, PC-3, A-549, MCF-7, HeLa, and Burkitt, and a healthy cell line, HEK-293, were used. The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 100 µg/ml penicillin/streptomycin in a humidified incubator at 37 °C with 5% CO2 and 95% air.

MTT assay
The cytotoxic effects of parasporal proteins on cancer lines were investigated via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. This method is based on the formation of water-insoluble purple formazan crystals by the mitochondrial enzymes of metabolically active cells, which reduce the tetrazolium dye MTT [16]. The cells (2 × 104 cells/well) were seeded in 96-well plates and incubated for 16–20 h at 37 °C in a 5% CO2 and 98% humidity incubator. Then, the activated parasporal crystal proteins (20 µg/ml) were added to each well and incubated at 37 °C for 48 h. After incubation, the cytopathic effect was examined under an inverted microscope. After 48 h of incubation, the medium in the 96-well plates was discarded, and 100 µl of a new medium was added. In the case of Burkitt cells, cell culture dishes with nonadherent cells were centrifuged at 25 °C and 800 rpm for 6 min. The old medium was discarded, and a new medium was added. Then, 5 mg/ml MTT was added to each well at a ratio of 1:10. The mixture was incubated for 4 hours at 37 °C, 5% CO2 and 98% humidity in an incubator. After incubation, 100 µl of DMSO was added, and the mixture was mixed for 5 min at 150 rpm. The absorbance at 540 nm was determined with a microplate reader (Thermo Fisher Scientific, USA). Compared with that of control cells, cell viability was calculated as a percentage via the following formula: viable cell percentage = [number of live cells/total number of live + dead cells]. Each treatment was performed in triplicate.

Determination of apoptosis
The apoptotic effects of the parasporal protein of the Bt 5.4 isolate on A549 and PC-3 cells were investigated via an Annexin-V/PI assay via an Annexin V-FITC Apoptosis Detection Kit (BioVision, K101). During the apoptosis process, the asymmetric distribution of phospholipids in the plasma membrane changes, and phosphatidylserine located on the inner leaflet of the membrane translocates to the outer leaflet. This structure becomes available for binding with Annexin [17]. Briefly, A549 and PC-3 cells at a concentration of 2.5 × 105 cells/ml were treated with parasporal protein of Bt 5.4 isolate at 5, 10, and 20 µg/ml concentrations. After 24 h of incubation, the cells were collected and washed with phosphate-buffered saline (PBS). The cells were resuspended in 500 µl of 1x binding buffer. Five microliters of Annexin V-FITC and 5 µl of propidium iodide (PI) were added to the cells, which were then incubated at room temperature for 5 minutes in the dark and analyzed on a flow cytometer (BD FACSCanto) with BD FACSDiva software v6.13.

Cell cycle analysis
The DNA content of apoptotic cells is lower than that of living cells. This can be detected by staining cells with propidium iodide (PI), which has fluorescent properties and intercalates into DNA [18]. The amount of PI bound to double-stranded DNA was measured via flow cytometry, and the amount of fragmented DNA with cell cycle phases was determined. The method of Pozarowski and Darzynkiewicz [19] was followed with some modifications. A549 and PC-3 cells (5 × 105 cells/ml) were treated with different concentrations of parasporal proteins (5, 10, and 20 µg/ml) for 24 h. At the end of the treatment, the cells were harvested, washed with PBS, and centrifuged at 800 rpm for 5 min at 4 °C. The samples were subsequently fixed with 70% ethanol for 24 h at -20 °C. After incubation, the cells were centrifuged at 1200 rpm for 10 min at 4 °C. The pellet was resuspended in PBS and centrifuged again. The pellet was resuspended in PBS containing 0.1% Triton X-100. Then, 25 µl of RNAse from a 200 µg/ml stock mixture was added, and the mixture was incubated at 37 °C for 30 min. At the end of the incubation, 25 µl of PI (from a 1 mg/ml stock) was added, and the mixture was incubated for 15 min at room temperature in the dark. Flow cytometry analysis was performed, and the ModFit LT program was used to determine the amount of fragmented DNA in the cell cycle phases.

Caspase-3 activity
Caspase-3, a cysteine‒aspartic acid protease, has been recognized as a critical mediator of programmed cell death [20]. Caspase-3 is involved in both intrinsic and extrinsic pathways that stimulate apoptosis and cause DNA fragmentation via CAD (caspase-activated DNAse I) activation [21]. Caspase-3 activity was examined via a Caspase-3/CPP32 colorimetric assay kit (BioVision). A549 and PC-3 cells (106 cells/ml) were treated with 10 µg/ml and 20 µg/ml parasporal proteins, respectively. After 36–48 h of incubation, the cells were harvested and washed with PBS. The pellet was resuspended in 50 µl of lysis buffer and incubated on ice for 10 min. Then, the mixture was centrifuged at 13,500 rpm for 1 min. The protein concentration of the supernatant was adjusted to 50–200 µg/ml, and 50 µl of lysis buffer was added to each protein sample. Next, 2X reaction buffer containing 10 mM DTT and 5 µl of 4 mM DEVD-pNA was added to each sample (at a final concentration of 200 µM) and incubated at 37 °C for 1–2 h. After incubation, the absorbance was measured at 405 nm with a microplate reader (Thermo Fisher Scientific, USA).

Drug combination assay
Synergistic assays were conducted to determine the interaction of cytotoxic parasporal proteins with the anticancer drug cisplatin, which is currently used in the clinic. A549 and PC-3 cells (2 × 104 cells/well) were exposed to different concentrations (1.35, 3.75, 7.5, 15, or 30 µg/ml) of parasporal protein; cisplatin (12.5, 25, 50, 100, or 200 µg/ml); and cisplatin + parasporal protein at 37 °C in an incubator with 5% CO2 and 98% humidity. After incubation for 24 h, cell viability was determined via the MTT assay. IC50 values were determined via the ​​GraphPad Prism 7 program. The combination index (CI) was calculated according to the formula used by Chou [22] as follows: CI = (D1 ÷ DX1) + (D2 ÷ DX2). D1: IC50 value of cisplatin used in the cisplatin + parasporal protein combination causing 50% cell death; DX1: IC50 value of cisplatin causing 50% cell death; D2: IC50 value of the parasporal protein used in the cisplatin + parasporal protein combination causing 50% cell death; DX2: IC50 value of the parasporal protein causing 50% cell death.

VEGF assay
Vascular endothelial growth factor is an important angiogenic factor. As a result of disruption of the natural angiogenesis mechanism, VEGF release increases in cells. Increased VEGF release causes uncontrolled proliferation of blood vessels in cancerous tissues and metastasis [23]. The effects of parasporal proteins on the suppression of VEGF production in A549 and PC-3 cells were determined via the Human VEGF ELISA Kit PicoKine (Boster Bio). A549 and PC-3 cells (2.5 × 103 cells/ml) treated with 10 µg/ml and 20 µg/ml parasporal proteins, respectively, were incubated for 12, 24, and 48 h. After incubation, the cells were collected and centrifuged at 14,000 rpm for 30 s. One hundred microliters of the supernatant was added to the microplate strips containing VEGF-specific monoclonal antibodies in the wells and incubated at 37 °C for 90 min. After incubation, the liquid in the plate was discarded, 100 µl of biotinylated anti-human VEGF antibody was added, and the mixture was incubated at 37 °C for 60 min. The plates were subsequently washed three times with PBS. After washing, 100 µl of 1X avidin-biotin peroxidase complex was added, and the mixture was incubated at 37 °C for 30 min. After incubation, the washes were repeated 5 times with PBS. Finally, 90 µl of color-developing reagent was added to each well and incubated at 37 °C for 25 min. One hundred microliters of TMB solution was added to stop color development. The color intensity in proportion to the amount of VEGF bound to the supernatants was read via a microplate reader at 450 nm. Lipopolysaccharide (LPS) at 10 µg/ml was used as a positive control. The experiments were performed in triplicate.

Statistical analyses
The differences between the results of the cytotoxicity assays were evaluated via one-way analysis of variance (ANOVA), Dunnett’s test, and Tukey’s test, where the variances were homogeneous. When the variances were not homogeneous, Kruskal‒Wallis (H test) analysis was applied. All analyses were performed via the GraphPad Prism 7 program.

Results

Optimization of solubilization and enzyme activation of parasporal inclusion proteins
To determine the optimum solubilization of parasporal proteins from Bt 4R2, solubilization buffers at four different pH values, pH 10, 10.5, 11, and 11.5, were tested. Solubilization buffer at pH 11.5 provided the highest protein concentration (Fig. 1A). The highest protein recovery (82%) was achieved when solubilization buffer at pH 11.5 was used, as shown in Fig. 1B.

Solubilized parasporal proteins at each pH value were treated separately with 25, 50, 100, 150, or 200 µg/ml proteinase K. The amount of protein resulting from digestion with all concentrations of proteinase K was greater at pH 11.5 than at other pH values (Fig. 2).

Therefore, the parasporal inclusion of the Bt 5.4 isolate was solubilized at pH 11.5 and digested with 150 µg/ml proteinase K (Fig. 3). Two major protein bands at approximately 135 kDa and 70 kDa were observed after solubilization. Proteinase K digestion resulted in a major band at 59 kD.

Cytotoxicity of parasporal proteins
Parasporal proteins were added to 96-well plates containing HEK293, HeLa, A549, MCF-7, PC-3, and Burkitt cells. According to Brasseur et al. [13], activated parasporin proteins from Bt 4R2 caused 50% cytotoxicity at 1–20 µg/ml on some of the cancer cell lines. Therefore, activated parasporin proteins from different Bt strains were used at 20 µg/ml in this present study. After 48 h of incubation, the MTT assay was performed. The parasporal proteins of the 5.7, 7.1, and 2.1 40.4 isolates significantly reduced the viability of healthy HEK293 cells, whereas the parasporal proteins of the 5.4 and 5.10 isolates did not have a significant cytotoxic effect on this cell line (Fig. 4A). Compared with those of the untreated cells, the parasporal proteins of the 5.4 and 2.1 isolates significantly reduced the viability of the A549 cells, with rates of 51 and 51.6%, respectively (Fig. 4B). In the case of the HeLa cells, parasporal proteins from the 5.10 and 2.1 isolates had the greatest effect (62% and 67%, respectively) on cell viability (Fig. 4C). All parasporal proteins caused a decrease in the viability of MCF-7 cells at different concentrations. Isolates 5.4 and 5.10 presented the most significant decrease in cell viability (Fig. 4D). In the case of PC-3 cells, parasporal proteins 5.7 and 2.1 were more effective at decreasing cell viability than other parasporal proteins were (Fig. 4E). In the Burkitt cell line, all parasporal proteins except 2.1 significantly reduced cell viability compared with the control at various levels. The most significant effect was shown for isolate 7.1 (p < 0.001) (Fig. 4F).

Inverted microscope analysis
The effects of the parasporal protein of the Bt 5.4 isolate were examined to determine the morphological changes in the A549 and PC-3 cancer cell lines. Observation was performed via an inverted microscope after 4, 10, and 24 h of incubation of cancer cells with parasporal inclusions. The microscopy results showed that the parasporal protein of Bt 5.4 caused cell shrinkage, rounding, and membrane blebbing, which occurred as early as after 4 h of treatment with parasporin. These morphological changes are characteristics of apoptotic cell death. It was obvious that cells did not look healthy, and cell death seemed to occur after treatment with parasporin in both cell lines (Fig. 5).

Flow cytometric analysis
The apoptotic effects of the parasporal protein of the Bt 5.4 isolate on A549 and PC-3 cells were investigated. Apoptosis was determined at concentrations of 5, 10, and 20 µg/ml parasporal protein at 12 and 24 h. After 12 h of incubation with A549 cells, 7.1%, 8.6%, and 12.1% apoptosis and 1.5%, 2.2%, and 1.7% necrosis were observed at increasing parasporal protein concentrations, respectively. After 24 h of incubation, the percentage of apoptotic cells was 10.5, 71.6, and 62.2%, and the percentage of necrotic cells was 0.6, 0.7, and 1.2%, respectively (Table 1; Fig. 6). After PC-3 cells were incubated for 12 h, the percentages of apoptotic cells were 15.9%, 32.1%, and 32.9%, while the percentages of necrotic cells were 0.8%, 0.7%, and 0.9%, respectively. After 24 h of incubation, the percentages of apoptotic cells were 15.4%, 17.2%, and 31.1%, whereas the percentages of necrotic cells were 1%, 3.3%, and 5.2%, respectively (Table 2; Fig. 7).

Caspase-3 activity
The induction of apoptosis in cancer cells was also determined via a Caspase-3 assay. The effects of the parasporal proteins Bt 5.4 and 4R2 on A549 and PC-3 cells were examined. When the parasporal protein of the Bt 5.4 isolate or 4R2 was applied to A549 cells, caspase-3 activity was determined to increase 4.5-fold (p < 0.01) and 2.2-fold (p < 0.05) compared with that of the control, respectively (Fig. 8A). In PC-3 cells, the Bt 5.4 isolate increased the amount of caspase-3 by 3.6-fold and that of 4R2 by 4.5-fold (Fig. 8B). These data showed that parasporal proteins caused apoptotic death in these cancer cells.

Effects of parasporal proteins on the cell cycle
The data obtained from flow cytometry analysis were edited via the ModFit LT program. The number of A549 and PC-3 cells in the G1 phase increased with increasing doses of the Bt 5.4 parasporal protein, whereas the number of cells in the G2 and S phases decreased. In other words, the parasporal protein Bt 5.4 caused A549 and PC-3 cancer cells to pause in the G1 phase of the cell cycle (Figs. 9 and 10).

Synergistic effects of cisplatin and parasporal proteins
The synergistic interactions of cisplatin with the Bt 5.4 and 4R2 parasporal proteins were investigated in A549 and PC-3 cells. The results were interpreted according to the combination index value (CI) [22]. The CI value is interpreted according to the following ranges: CI >1.3 means antagonism; CI = 1.1–1.3 means moderate antagonism; CI = 0.9–1.1 means additive effect; CI = 0.8–0.9 means slight synergism; 0.6–0.8 means moderate synergism; CI = 0.4–0.6 means synergism; and CI = 0.2–0.4 means strong synergism. The results of A549 cells indicated that while cisplatin had a strong synergistic effect when combined with the parasporal protein 4R2, a moderate synergistic effect was observed with the parasporal protein Bt 5.4 (Fig. 11; Table 3). No synergistic interaction, but an additive effect was observed between cisplatin and the Bt 5.4 parasporal protein in the PC-3 cell line (Fig. 12; Table 3).

Effects of parasporal protein on vascular endothelial growth factor
The effects of the Bt 5.4 parasporal protein on VEGF secretion were investigated in A549 and PC-3 cells. After 12 h, LPS significantly increased VEGF secretion in A549 cells, but treatment with Bt 5.4 alone or with 5.4 and LPS together resulted in no significant difference compared with the control. After 24 h, LPS significantly increased the amount of VEGF (78 pg/ml), whereas the 5.4 isolate decreased it to 20 pg/ml. After 48 h, no difference was observed among the treatments (Fig. 13A). In PC-3 cells, parasporal protein from the Bt 5.4 isolate decreased VEGF secretion to 25 pg/ml after 12 h. After 24 h, LPS still significantly increased the effect (160 pg/ml), whereas the combination of Bt 5.4 and LPS decreased VEGF secretion to 70 pg/ml and 85 pg/ml, respectively. After 48 h, all the treatments significantly decreased the amount of VEGF secreted (Fig. 13B).

Discussion
Cancer is one of the most serious health problems that threatens public health and has a high mortality rate. Owing to some side effects and inadequacies of traditional methods in the fight against cancer, alternative treatment methods are being sought, and parasporin toxins obtained from Bt are attracting attention as biological preparations. Studies on parasporin proteins in the literature have shown that each one (PS1-PS6) could be effective against different target cell lines at different IC50 doses [10, 24]. Therefore, screening new Bt isolates will increase the possibility of finding isolates with cytotoxic activity and revealing new parasporins. Therefore, the cytotoxic, apoptotic, angiogenic, and synergistic activities of native Bt isolates that carry parasporin genes [11] were examined in the present study. Since these Bt strains were isolated from different locations and geographic locations than those reported in the literature, they have diverse abilities in screening for cytotoxicity.
It has been reported in the literature that the solubility of toxins under different alkaline conditions and the type of enzyme used in proteolytic cleavage affect their cytocidal activity. Therefore, parasporal inclusions were obtained from native Bt strains and activated according to the optimized solubilization and digestion conditions in the present study. Although the pH of alkaline buffers generally used is 10.5 [25–27], some studies have used pH values of 10.2 and 11.4 [13, 28]. In this work, four different pH values (10, 10.5, 11, and 11.5) were tested for solubilization of the toxins, and the pH with the highest protein recovery was 11.5 (Fig. 1). Studies have shown that parasporal proteins treated with proteinase K have greater cytocidal activity than other enzymes do [29, 30]. Therefore, we preferred this enzyme in our experiments and determined the appropriate proteolytic enzyme concentration. When parasporal proteins were treated with proteinase K at five different concentrations (25, 50, 100, 150, and 200 µg/ml), the most appropriate concentration was 150 µg/ml (Fig. 2). SDS‒PAGE analysis of the parasporal protein profile of Bt 5.4 revealed 2 bands at approximately 70 kDa and 135 kDa. Proteolytic digestion with proteinase K (150 µg/ml) gave rise to 1 band at approximately 59 kDa (Fig. 3). According to Nagamatsu et al. [28], parasporin 6Aa1 is approximately 73 kDa, and trypsin digestion results in 2 bands at 14 and 59 kDa. The differences observed in our Bt 5.4 strains could be due to the type of enzyme used in proteolytic digestion and the different geographical locations of the Bt isolates.
To test the cytotoxic activity of our parasporal proteins, we chose five types of cancer cells, namely, human lung, breast, prostate, uterine cervix, and leukemia cells, which have high incidence and mortality rates [1]. In addition, healthy embryonic kidney cells were tested to determine the selectivity of the cytotoxic activity of parasporal proteins. After the MTT assays were performed, parasporal proteins of isolates other than Bt 5.4 and 5.10 were excluded because they also had cytotoxic effects on healthy cell lines. In addition, the isolates Bt 5.4 and Bt 5.10 were found to be significantly cytotoxic toward all the tested cancer cells (Fig. 4). Kesik Oktay [11] reported that Bt 5.4 and 5.10 isolates harbored the parasporin 6 gene. There is very limited research in the literature on PS6. Previous studies have shown that the PS6 protein is selectively toxic to HeLa and HepG2 cells [28].
Bt 5.4 was chosen for further analysis of A549 and PC-3 cells. First, the morphological appearance of parasporin-treated cells was examined with an inverted microscope because before studying cell death mechanisms, it is necessary to ensure that cell death has happened. The results revealed that Bt toxin treatment induced significant alterations in cell morphology, including cell shrinkage, rounding, membrane blebbing, and cell breakage into small apoptotic bodies indicative of cytotoxic effects and possible apoptotic cell death [13]. In fact, similar morphological changes were observed in parasporin-treated cells in other studies as well [30–32]. Afterwards, an Annexin-V/PI assay along with flow cytometry was performed to determine whether observed morphological changes were related to apoptosis. Indeed, apoptotic cell death was detected in flow cytometry analysis. The apoptosis rate increased in A549 cells in a time- and dose-dependent manner. The percentage of apoptotic cells was 12.1% at the highest dose after 12 h of incubation, whereas it was 71.6% at 24 h. In PC-3 cells, the apoptosis rate increased in a dose-dependent manner, but no change was observed over time. As a result, the parasporal protein Bt 5.4 induced apoptosis in A549 and PC-3 cells, supporting the observed morphological changes. In addition, the DNA content profile was analyzed via PI staining and the ModFit mathematical model to determine the percentage of cells in different stages of the cell cycle. Analysis revealed that the cells were arrested in the G1 stage and could not pass to other stages.
The most important feature of cancer drugs is that they cause apoptotic death of cancer cells. A single method may not be sufficient to determine apoptosis in the cell. For this reason, in addition to morphological and Annexin-V/PI assays, apoptosis was determined by an increase in caspase-3 activity. The results of this assay revealed that caspase-3 activity significantly increased with the parasporal proteins of Bt toxins in both cell lines (Fig. 8). Other studies on the effects of parasporal proteins on caspase-3 activity support our findings that different parasporal proteins have different effects on caspase-3 activity depending on the dose [13, 33, 34].
Drug combinations have been used to treat many diseases. The aim of synergistic therapeutic interactions is to reduce the dose and toxicity of the drug and prevent the development of drug resistance. Cisplatin is a frequently used chemotherapeutic drug. It is used to treat many types of cancer, such as bladder, lung, ovarian, and testicular cancer [22]. Some studies have shown that cisplatin has synergistic interactions with many molecules [35–37], but the synergism between Bt parasporal proteins and cancer drugs has not yet been explored and needs to be elucidated. Our synergistic experiments combined with cisplatin revealed a strong synergistic effect with the parasporal protein of the 4R2 strain and a moderate synergistic effect with the parasporal protein of Bt 5.4 on A549 cells. Surprisingly, the combination of the parasporal protein of the 4R2 strain and Bt 5.4 had antagonistic and additive effects on PC-3 cells. Wong et al. [38], with a similar approach, performed heterologous competitive binding assays and reported a decrease in the percentage of bound biotinylated purified Bt 18 toxin in CEM-SS (human T4-lymphoblastoid cell line) cells treated with various anticancer drugs at 59.26 nM.
VEGF, a key mediator of angiogenesis, is an attractive target in cancer therapy [39]. In this study, the effects of the parasporal protein Bt 5.4 on VEGF release were investigated. In PC-3 cells, parasporal protein significantly decreased VEGF release in a time-independent manner, whereas in A549 cells, it was reduced only after 24 h (Fig. 13). The parasporal proteins of the Bt 5.4 isolate are cytotoxic toward A549 and PC-3 cells and induce apoptosis. Therefore, these parasporal proteins have the potential to prevent metastasis by preventing blood vessel formation while causing the death of cancer cells.

Conclusion
In conclusion, the cytotoxic effects of parasporin proteins obtained from parasporin gene-positive native Bt strains were investigated in various cancer cell lines. Among all the native Bt strains tested, Bt 5.4 was found to be the most effective strain on cytotoxicity against different cancer cell lines but not against normal cell lines. Selective cytotoxicity of Bt 5.4 has made it be further analyzed for its apoptotic, synergistic, and antiangiogenic activities. Compared with that in untreated cells, the expression of the parasporal protein Bt 5.4 had significant apoptotic and antiangiogenic effects on A549 and PC-3 cells. In addition, a moderate synergistic effect of the parasporal protein of this isolate with the cancer drug cisplatin was shown. These data indicate that the Bt 5.4 isolate has important potential in drug development against cancer cells. Future experiments related to the recombinant production of Bt 5.4 parasporin protein and demonstrating its anticancer effects in vitro will allow us to clarify its in vivo anticancer activities as well.

Supplementary Information
Below is the link to the electronic supplementary material.