Two samples of lyophilized crude venom were extracted by electrical stimulation of numerous adult spiders of both sexes (Sisgen A23162A). The crude PnV, fractions, and subfractions were stored at −80°C and dissolved in a sterile culture medium immediately prior to use. The composition and reproducibility of the venom lots were verified by high-performance liquid chromatography (HPLC). The initial fractionation of the crude venom was performed using the Amicon ultra centrifugal filter (#UFC801008; Thermo Fisher Scientific, Suwannee, GA, United States). This procedure consisted of the separation of the crude venom by molecular mass using molecular filters, generating 3 main fractions that were denominated: F1 (LW = low weight, less than 3 kDa), F2 (IW = intermediate weight, between 3 kDa and 10 kDa), and F3 (HW = high weight, more than 10 kDa). From these, experiments were conducted to select the more significant fraction, considering the effects presented; then, F1 and F2 were chosen, and F3 was eliminated. A new purification of F1 and F2 together, using HPLC, was carried out, obtaining new components, which were denominated subfractions 1 to 11 (SF 1–SF 11). Reversed phase HPLC was performed using a Shimadzu VP-ODS column, with 0.1% trifluoroacetic acid (TFA) as the mobile phase and 90% acetonitrile 0.1% TFA as the eluent.

Human GB (NG97) cells were donated by a patient from the Hospital das Clínicas/Universidade Estadual de Campinas (HC/UNICAMP), and the cell line was established and characterized in a sequence of published studies (Machado et al., 2005; Schenka et al., 2005; Machado et al., 2008; Machado et al., 2009; Lin et al., 2020). NG97 cells were seeded at a density of 1 × 10⁴ per cm² in a 25 cm² culture bottle and were grown using Iscove’s modified Dulbecco’s medium (IMDM) containing 10% fetal bovine serum (FBS) and 100 UI/ml penicillin and streptomycin (pH 7.4) (Gibco), referred to as complete IMDM. The cell culture was maintained in a humidified atmosphere at 37°C and 5% CO₂ until semi-confluence (about 90% of the total surface area). For the assays, cells were transferred after careful scraping of 48- or 96-well plates (Corning, Inc.), at an initial density of 1 × 10⁴ cells per well, and incubated at 37°C for 72 h. The cells were then treated with 14 μg/ml and 280 μg/ml of PnV (Rapôso, 2017), F1, F2, and F3 (0.1, 1, and 10 μg/ml) (Barreto dos Santos et al., 2019), or SF 1–11 (0.1 and 1 μg/ml), for 1, 5, 24, and/or 72 h, depending on the method (Barreto dos Santos et al., 2019; Barreto et al., 2020), according to the following assays, while the control cells were maintained in the medium for the same times. For the mTOR inhibition assay, cells were incubated with 50 nM of rapamycin (#051357-13 Cayman Chemical) (Foster and Toschi, 2009). The inhibitor was first diluted in DMSO to a concentration of 10 mM, and then diluted with IMDM to the final concentration. GB NG97 cells were incubated for 1 h prior to the treatments. Subsequently, the inhibitor was replaced during the treatment periods (1, 5, 24, and/or 72 h) together with SF 1–11.

Thiazolyl blue tetrazolium bromide (MTT), whose reduction capacity indicates cellular activity, was used to determine the cytotoxicity of PnV, and its fractions and subfractions. MTT was added to each well and incubated at 37°C for 4 h, according to the manufacturer’s protocol. Acidified isopropanol was added to each well to solubilize the blue crystals of MTT. Absorbance at 540 nm, which indicates cell activity, was determined using a Multiskan GO microplate spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, United States). The MTT assay was performed with PnV 14 and 280 μg/ml and fractions (F1, F2, and F3; 0.1, 1, and 10 μg/ml, respectively), at 1, 2, and 5 h treatment times; after choosing and repurifying F1 and F2, the test was performed again using all SF (1–11), with or without mTOR inhibition, as mentioned before.

After culturing, 2 × 10⁴ cells/mL were washed with sterile PBS and then resuspended in 1 ml PBS containing the CSFE probe (carboxyfluorescein succinimidyl ester, 5 μM) and maintained at room temperature for 5 minutes, protected from light. Then, complete IMDM was added to block the effect of CFSE, and the suspensions were washed twice by centrifugation at 300 g for 10 min, according to the manufactures’ protocol. Cells were then resuspended with complete IMDM, cultured, and treated with F1, F2, and F3 (0.1, 1, and 10 μg/ml). The culture was kept at 37°C, CO₂ 5%, for 24 and 72 h. The analysis of an aliquot of cells labeled with CSFE by flow cytometry was performed on the same day of labeling to define the maximum incorporation value of the probe. After treatment time, cells from each well were transferred to appropriate tubes and analyzed using the FACSVerse flow cytometer (BD Biosciences), and the results were computed using the BD FACSuite software.

To determine the extent of apoptosis and necrosis, after treatments with F1, F2, and F3 (0.1 μg/ml) for 1, 5, and 24 h, respectively, 2 × 10⁵ cells were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (Pi), using the Annexin V-FITC Apoptosis Detection Kit (Biolegend, San Diego, CA, United States #640914), following the manufacturer’s instructions. A total of 10,000 cells were analyzed, and apoptosis/necrosis was determined using the FACSVerse cytometer and FACSuite system (BD Biosciences). The data were analyzed using FlowJo software (v7.6.5). For cell cycle distribution, following the treatments, 2 × 10⁴ cells were fixed overnight at 4°C in 70% ethanol. The fixed cells were then washed with PBS and incubated with 1 ml solution containing 50 μg/ml Pi, and 0.5 µg RNase A, for 30 min, in the dark. Cell cycle distribution was assessed by flow cytometry using the FACSVerse cytometer and FACSuite system (BD Biosciences, Franklin Lakes, NJ, United States), and the data were analyzed using FlowJo software (v7.6.5).

Values were analyzed using the GraphPad Prism software package, v. 5.0 (San Diego, CA, United States). The level of significance was analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s and Tukey’s multiple comparisons tests. Unpaired Student’s t-test was used to compare each treatment with the control. Error bars show the standard error of the mean (SEM). A p-value < 0.05 indicates the statistical significance.

It was demonstrated there were no significant differences between the two extracted venom samples (Figure 1A). From the first PnV separation, three main fractions were obtained (Figure 1B). These fractions were still complex mixtures, and they were evaluated by the effects on viability, proliferation, death, and cell cycle to select those with the most significant effects on GB cells. Then, the purification of F1 and F2 (together) by HPLC generated eleven subfractions, named SF 1 to SF 11 (Figure 1C).

The cell viability assay (MTT) was performed using fractions F1, F2, and F3 (0.1, 1, and 10 μg/ml) (Figure 2). After 1 h of exposure, F1 at 0.1 μg/ml was the most effective treatment to reduce the viability of GB cells significantly compared to the untreated control (p < 0.01); F1 and F2, at 10 μg/ml, also induced a significant reduction in the viability of GB cells compared to the untreated control (p < 0.05), while no significant effect was observed in F3 fraction (2, A). After 5 h of exposure, the alterations were less significant than 1 h, and fractions F1 and F2 were still effective in reducing the viability of GB cells (p < 0.05) (2, B), while no significant effect was observed in F3 fraction. After 24 h of treatment, only F1 0.1 μg/ml showed a significant effect (2, C). Controls with PnV at 14 and 280 μg/ml induced a significant decrease of cell viability after 1 and 5 h, and after 24 h only the overconcentration (280 μg/ml) was significant.

The cell proliferation assay (CFSE) was performed using fractions F1, F2, and F3 (0.1, 1, and 10 μg/ml) (Figure 3). In the 24 h of treatment, F1 at a dose of 10 μg/ml was significantly effective in reducing proliferation, compared to control (p < 0.01), while F3, at 0.1 and 1 μg/ml, was significantly effective in inducing GB cell proliferation (p < 0.05 and p < 0.0001, respectively) (3.1, A). Results are represented in the histogram (3.1, B). After 72 h of treatment, F1 at a concentration of 1 μg/ml (p < 0.05) and F2 at 0.1 μg/ml (p < 0.001) were significantly effective in reducing the proliferation of GB cells (3.2, A). Results are represented in the histogram (3.2, B).

The apoptosis/necrosis assay (Annexin V/PI) was performed using fractions F1, F2, and F3 at 0.1 μg/ml dose since this concentration induced distinctive effects in viability and proliferation tests (Figure 4). After 1 h of treatment, F3 induced a significant increase of GB cells necrosis compared to the untreated control (p < 0.01), whereas all fractions, F1, F2, and F3, were significantly effective in inducing apoptosis (p < 0.05) (4.1, A). After 5 h of treatment, F1 induced a significant increase of necrosis, compared to control; F2 apparently increased both necrosis and apoptosis, but it was not significant (4.2, A). F3 significantly decreased necrosis at this time-point. At 24 h of treatment, no significant differences were observed between the untreated control and the treated GB cells (4.3, A). Results are also represented in the histograms (4.1, B; 4.2, B; and 4.3, B).

The cell cycle assay (Pi) was performed using fractions F1, F2, and F3 at 0.1 μg/ml (Figure 5). After 1 h of treatment, F2 and F3 significantly increased the percentage of GB cells in the G1 phase of the cell cycle compared to the untreated control, while F3 significantly increased cells in the S phase; F1, F2, and F3 significantly decreased the percentage of cells in the G2 phase (6.1, A). After 5 h of exposure, F1 and F2 significantly increased the number of GB cells in the S phase (5.2, A). At 24 h of treatment, only F2 significantly increased the number of GB cells in the S phase (5.3, A). The results are represented in the histogram (5.1, B; 5.2, B; and 5.3, B).

The cell viability assay (MTT) was performed using subfractions SF 1 to SF 11 (0.1 and 1 μg/ml) from F1 and F2 separation by HPLC. The results showed, in general, that SF 3 and SF 4 were the most effective in decreasing viability of GB cells, and 0.1 μg/ml concentration was more effective than 1 μg/ml; when the cells were pretreated with rapamycin, the effect of SFs was exacerbated, except in 5 h of 0.1 ug/mL treatment (interestingly, the most effective time and concentration) (Figure 6).

This screening revealed that SF 3, SF 4, SF 7, and SF 8 subfractions at 0.1 μg/ml and 1 h of treatment (Figure 6A) were more effective in reducing the viability of GB cells than the control group. When cells were pretreated with rapamycin, SF 1 to SF 10 (Figure 6B) showed a reduction in cell viability compared to the control group, and the reduction was more significant than cells without the inhibitor. After 5 h of treatment (Figure 6C), all SFs showed a reduction in cell viability compared to the control group. In cells pretreated with rapamycin (Figure 6D), all SFs also showed a reduction of viability compared to the control group; however, the data were not as significant as those without mTOR inhibition. At the 24 h treatment (Figure 6E), SF 2, SF 3, SF 4, SF 5, SF 9, and SF 11 induced a significant reduction in cell viability compared to the control group. In the cells pretreated with rapamycin (Figure 6F) all SFs induced a decrease of viability compared to the control group, which was more statistically significant than that without the inhibitor.

After 1 h of treatment, at 1 μg/ml (Figure 7A), the SFs 7, 10, and 11 showed a significant reduction of cell viability compared to the control group. In the cells pretreated with rapamycin (Figure 7B), all SFs, excepting SF 2 and 5, induced a significant reduction of viability compared to the control group. After 5 h of exposure (Figure 7C), SF 1, SF 3, SF 7, and SF 8 showed a reduction in cell viability compared to the control group. While cells were pretreated with rapamycin (Figure 7D), all SFs, except SF9, induced a significant reduction in cell viability compared to the untreated cells. At 24 h treatment (Figure 7E), no subfractions showed effective reduction in cell viability, and when the cells were pretreated with rapamycin (Figure 7F), SF 3, SF 4, and SF 9 showed a significant reduction in cell viability compared to the control group.