The T. asperelloides LIBASP02 strain (Lopes et al., 2020) was cultured on Potato Dextrose Agar (PDA, HiMedia) in Petri dishes at 28°C for 7 days. The crude ethanolic extract of the mycelium and spores was obtained by washing the cultures with 95% ethanol (Merck) (Fukuzawa et al., 2008). The ethanolic suspension was incubated under agitation in an orbital shaker (Gerhardt) at 100 rpm for 24 h and then centrifuged at 10,000 × g for 20 min (BR4i-Jouan centrifuge). The final crude ethanolic extract (ExtTa) was lyophilized and solubilized in 100% DMSO (Sigma), resulting in a final concentration of 197 mg/mL.

Human cancer cell lines T98G (glioblastoma), MDA-MB-231 (breast adenocarcinoma), A549 (lung carcinoma), HT1080 (fibrosarcoma), LB373 (melanoma), Saos-2 (osteosarcoma), HCT116 (colorectal carcinoma) and WiDr (colorectal adenocarcinoma) were used. The human non-cancer cell lines GM637, HEK-293T, and the non-cancer mice cells NIH/3T3 and L929 were used as references. L929 and GM637 cells were kindly provided by Dr. Luiz Fernando Lima Reis (Hospital Sírio Libanês - São Paulo, Brazil), and all other cell lines are from ATCC. All cell lines were kept cryopreserved in liquid-phase nitrogen in the Laboratory of Inflammation and Cancer (LINC) at the Universidade Federal de Minas Gerais (UFMG—Minas Gerais, Brazil). All cell lines were kept cryopreserved in liquid-phase nitrogen in the Laboratory of Inflammation and Cancer (LINC) at the Universidade Federal de Minas Gerais (UFMG). Cell cultures were maintained in a humidified atmosphere regulated at 5% CO2 and 37°C in the specific medium for each cell line: Dulbeccos’s Modified Eagle’s Medium (DMEM - Gibco) for lines HEK-293T, NIH/3T3, L929, HCT116, A549, HT1080, DMEM supplemented with non-essential amino acids (Sigma) for lines GM637, Saos-2, T98G, WiDr; Iscove’s Modified Dulbecco’s Medium (Gibco) for LB373; and RPMI-1640 Medium (Gibco) for MDA-MB-231—supplemented with 10% FBS (Sigma), 2 mM L-glutamine (Sigma), 10 μg/mL penicillin-streptomycin (Sigma). Subculture was performed using trypsin 0.25%—EDTA 0.53 mM (Gibco) for cell detachment. All lines tested negative for mycoplasma contamination in a PCR assay using primers as described by Young et al (Young et al., 2010).

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma Aldrich, St. Louis, MO, United States) assay was used to assess cell viability in response to each treatment (van Meerloo et al., 2011; Stockert et al., 2018). Briefly, cells were cultured in wells of 96-well plates for 48 h in the presence of varying concentrations of ExtTa, DOX, or 5-FU. The number of cells seeded per well was determined in a pilot assay for standardization. Cells were seeded at concentrations ranging from 0.5 to 2.0 × 10⁵ cells/well, depending on the cell type and its growth rate. 0.5% DMSO was added to all wells, including those containing control cells and those with treated cells, to eliminate any potential bias caused by the vehicle concentration present in samples treated with higher concentrations of ExtTa and DOX. DMSO 0.5% was used as survival control and DMSO 20% as a 100% cell death control. Subsequently, MTT solution (5 mg/mL) was added (0.5 mg/mL final concentration) to each well, and cells were incubated for 3 h. Then, cell media was removed, and a 1:1 v/v N'N'Dimethylformamide solution (Synth) in 10% SDS (Affymetrix) was added for formazan solubilization. Absorbance was recorded at 595 nm using a Multiskan Spectrum reader (Thermo Scientific). Cell viability was quantified using the following equation: %   C e l l   v i a b i l i t y = A b s o r b a n c e   i n d i v i d u a l   t e s t   g r o u p − a b s o r b a n c e   b l a n k   g r o u p   A b s o r b a n c e   c o n t r o l   g r o u p − a b s o r b a n c e   b l a n k   g r o u p . The half-maximal inhibitory concentration (IC₅₀) was calculated based on the dose-response inhibition curve, using the GraphPad Prism software (v. 8.0.1, San Diego, California, United States). All assays were performed in technical quadruplicates with three independent biological replicates.

Non-cancer cell lines GM637, HEK-293T, NIH/3T3 and L929 were used as reference for selectivity index (SI) calculation following the equation (López-Lázaro, 2015): S I = I C 50   n o n − c a n c e r   c e l l   l i n e I C 50   c a n c e r   c e l l   l i n e

The CompuSyn software (CompuSyn, Inc., Paramus, NJ, United States) calculates a Combination Index (CI) that assigns numerical values to levels of synergism, additivity, or antagonism between compounds. The combinatorial effect of 8 different dose combinations of the ExtTa, DOX or 5-FU (dilutions 1:2, in a constant ratio) was calculated according to the Chou-Talalay Combination Index method (Chou, 2006; Chou, 2010) based on the cell viability assessed by MTT assay. The fraction of inhibited cells (Fa) in each dose of the treatment or the combination was calculated. Fa = 0 means non-inhibitory effect and Fa = 1 means a 100% inhibitory effect. The half-maximal inhibitory concentration (IC₅₀) of the drug is calculated by the dose-effect curve and median-effect. The x-axis intercept (logD) represents the medium-effect dose of IC₅₀. The CI was automatically determined by the CompuSyn software based on the median effect equation. CI < 1 points to synergism; CI = 1 means additive effect, and when CI > 1 the interaction is considered antagonistic. Additionally, the dose-reduction index (DRI) was calculated to assess if it is possible to reduce the dosage of the compounds to achieve the same inhibitory effect in a combination scenario. DRI >1 represents the possibility of a favorable dose reduction; DRI = no dose-reduction index; DRI <1 not favorable dose-reduction (Chou, 2010).

In the cell viability assays, non-linear regression was applied to find the IC₅₀. Results are presented as the mean ± SD of three independent experiments in triplicates. Statistical analyses and graphs were performed using the GraphPad Prism Version 8 program.

ExtTa exhibited cytotoxicity towards cells in a dose-dependent manner. The mean IC₅₀ values for non-cancer lines GM637, HEK-293T, NIH/3T3, and L929 after 48 h post-treatment with ExtTa were 70.62 ± 5.15, 53.82 ± 11.07, 49.23 ± 7.83, and 53.67 ± 6.54 μg/mL, respectively (Figure 1A). Most tumoral cell lines demonstrated greater sensitivity to ExtTa cytotoxic activity compared to non-cancer cells: T98G (mean IC₅₀ = 17.78 ± 3.20 μg/mL), MDA-MB-231 (IC₅₀ = 39.32 ± 9.87 μg/mL), A549 (IC₅₀ = 53.67 ± 0.08 μg/mL), HCT116 (IC₅₀ = 17.97 ± 4.34 μg/mL), LB373 (IC₅₀ = 37, 71 ± 2.06 μg/mL), HT1080 (IC₅₀ = 25.52 ± 6.66 μg/mL), WiDr (IC₅₀ = 26.05 ± 6.08 μg/mL) and Saos-2 (IC₅₀ = 25.59 ± 2.76 μg/mL) (Figure 1B). Treatment with ExtTa resulted in a dose-dependent decrease in cell proliferation across different levels. Notably, T98G (glioblastoma) and HCT116 (colorectal carcinoma) cells exhibited the highest sensitivity to the cytotoxic effects of ExtTa.

All non-tumoral cell lines tested in our study, GM637 (IC₅₀ = 0.04 ± 0.03 μg/mL), HEK-293T (IC₅₀ = 0.37 ± 0.07 μg/mL), NIH/3T3 (IC₅₀ = 0.01 ± 0.01 μg/mL), and L929 (IC₅₀ = 0.03 ± 0.08 μg/mL), exhibited sensitivity to DOX (Figure 1C). Among the tumor cell lines, varying profiles of cytotoxicity to DOX treatment were observed: T98G (IC₅₀ = 13.88 ± 2.91 μg/mL), MDA-MB-231 (IC₅₀ = 0.42 ± 0.09 μg/mL), A549 (IC₅₀ = 1.39 ± 0.08 μg/mL), HCT116 (IC₅₀ = 0.51 ± 0.05 μg/mL), LB373 (IC₅₀ = 3.45 ± 2.01 μg/mL), HT1080 (IC₅₀ = 0.04 ± 0.01 μg/mL), WiDr (IC₅₀ = 0.19 ± 0.14 μg/mL) and Saos-2 (IC₅₀ = 0.18 ± 0.14 μg/mL) (Figure 1D). Notably, non-cancer lines GM637, NIH/3T3, and L929 as well as the tumor cell line HT1080, exhibited higher sensitivity to DOX. Conversely, T98G and LB373 cell lines demonstrated less sensitivity to the cytotoxic effects of this drug.

The selectivity indices for ExtTa and DOX are presented in Table 1. The cytotoxicity of ExtTa was approximately 2 times higher for Saos-2, MDA-MB-231, LB373, and HT1080 cell lines, and 3 to 4 times higher for T98G and HCT116 cells compared to non-cancer cell lines. The A549 cell line exhibited less sensitivity to ExtTa cytotoxicity, and no dose-dependent selectivity was observed for this tumor cell (Table 1).

In our study, DOX exhibited selectivity only for the HT1080 cell line compared to the non-cancer cell line HEK-293T, being 9.25 times more cytotoxic for this tumor cell line than for non-cancer cell (Table 1).

The results indicated that the selectivity index (SI) of ExtTa surpassed that of DOX for most of the evaluated cells. ExtTa demonstrated higher cytotoxicity for the cancer cell lines HCT116 and T98G than for the non-cancer cell line GM637, highlighting the selectivity of the ExtTa and its minor impact on healthy cells, a characteristic not observed in DOX-treated cells.

Addressing the challenge of making cancer treatment more personalized and effective remains one of the major hurdles in the healthcare system. Combinations of multidirectional synergistic drugs may offer effective and sustained clinical responses (Lawrence et al., 2014b; Zagidullin et al., 2019b). With this aim, the effects of combining ExtTa with DOX were evaluated in the tumor cell lines MDA-MB-231, Saos-2, and HT1080, which are sensitive to ExtTa and where DOX is commonly used for clinical treatment of these tumor types. Additionally, the combination of ExtTa with 5-Fluorouracil (5-FU) was assessed in the colorectal carcinoma cell line HCT116, considering that this chemotherapeutic agent is used as the first-line treatment for this type of tumor. The combinatorial effect of ExtTa and DOX on the Saos-2 osteosarcoma cell line was graphically represented in Figure 2 through dose-effect curves (Figure 2A), median-effect (Figure 2B), CI graphs (Figure 2C), and dose-reduction index (Figure 2D). The co-treatment of ExtTa with DOX was found to be more cytotoxic than each isolated treatment (Figure 2; Table 2). The most significant synergistic effects were observed at the lowest and highest concentrations of the compounds (Figure 2C; Table 2). Favorable ExtTa dose reduction was evident across all the inhibitory fractions (Fa) evaluated. Favorable DOX dose reduction was observed on the inhibition fractions from 0.45 to 0.97 (Figure 2D; Supplementary Table S1). The effect of ExtTa and 5-FU on the HCT116 colorectal carcinoma cell line was graphically represented in Figure 2 through dose-effect curves (Figure 2E), median-effect (Figure 2F), CI graphs (Figure 2G), and dose-reduction index (Figure 2H).

For colorectal carcinoma HCT116 cells, we also observed a synergistic action of the ExtTa and 5-FU (Figure 2G). The combination index indicated synergistic action for specific dose combinations (Table 2). For the synergistic combination of ExtTa with 5-FU, DRI values demonstrated dose reduction for both compounds for Fa from 0.3 to 0.90 (Figure 2H). As a result, the IC₅₀ values of each drug in combination decreased 2.53 and 1.44 times for ExtTa and 5-FU, respectively (Supplementary Table S2).

For the MDA-MB-231 breast cancer cell line, synergism between ExtTa and DOX was observed only at high doses, without an indication of a favorable dose reduction (Supplementary Figure S1).

An antagonistic effect was found when ExtTa and DOX were used simultaneously in the treatment of the HT1080 fibrosarcoma cell line (Supplementary Figure S2). In this case, the combination was less effective in inducing cytotoxicity than each compound isolated in all tested doses.