Evaluation of the Anti-Histoplasma capsulatum Activity of Indole and Nitrofuran Derivatives and Their Pharmacological Safety in Three-Dimensional Cell Cultures
Abstract
:1. Introduction
2. Materials and Methods
2.1. Antifungal Drugs, Indole and Nitrofuran Derivatives
2.2. Microorganisms and Culture Conditions
2.3. Susceptibility of H. capsulatum to Nitrofuran and Indole Derivates and Antifungal Drugs and Determination of Minimum Fungicide Concentration (MFC)
2.3.1. Determination of Minimum Inhibitory Concentration (MIC90)
2.3.2. Determination of Minimum Fungicide Concentration (MFC)
2.4. Cell Lines Mantainance
2.4.1. Characterization of Three-Dimensional Cell Culture (3D)
Diameter Establishment
Cell Quantification and Viability Using Trypan Blue
Cell Viability—Qualitative
External Cell Morphology of Spheroids
Confocal Fluorescence Microscopy
2.4.2. Cytotoxicity Assay in Three-Dimensional (3D) and Monolayer (2D) Models by Resazurin Colorimetric Method
2.5. Determination of the Mechanisms of Action
2.5.1. Ergosterol Dosage
2.5.2. Evaluation of Morphology and Cell Wall Damage Using Calcofluor White Staining and Laser Scanning Confocal Microscopy
2.5.3. Quantification of Reactive Oxygen Species (ROS)
2.5.4. Apoptosis/Necrosis Assay
2.6. Statistical Analysis
3. Results
3.1. Determination of MIC90 and MFC
3.2. Three-Dimensional Cell Culture
3.2.1. Diameter
3.2.2. Quantification and Cell Viability Using Trypan Blue
3.2.3. Cell Viability Using the Resazurin Colorimetric Method
3.2.4. Scanning Electron Microscopy
3.2.5. Confocal Fluorescence Microscopy
3.2.6. Cytotoxicity Assay for Three-Dimensional and Monolayer Cell Culture
3.3. Determination of the Mechanisms of Action
3.3.1. Ergosterol Dosage
3.3.2. Evaluation of Morphology and Wall Damage
3.3.3. Reactive Oxygen Species (ROS)
3.3.4. Cell Death Assay (Necrosis–Apoptosis)
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Compound | Concentration (µg/mL) | Compound | Concentration (µg/mL) | Compound | Concentration (µg/mL) |
---|---|---|---|---|---|
1 | ≥125 | 20 | 7.8 | 39 | 7.8 |
2 | 15.6 | 21 | 7.8 | 40 | 62.5 |
3 | 1.95 | 22 | 31.25 | 41 | 7.8 |
4 | 0.98 | 23 | 31.25 | 42 | 31.25 |
5 | 0.25 | 24 | 62.5 | 43 | 62.5 |
6 | 62.5 | 25 | ≥125 | 44 | 125 |
7 | 0.122 | 26 | ≥125 | 45 | 62.5 |
8 | 31.25 | 27 | ≥125 | 46 | 31.25 |
9 | 0.98 | 28 | 31.25 | 47 | 125 |
10 | 7.8 | 29 | 31.25 | 48 | ≥125 |
11 | 0.25 | 30 | 15.6 | 49 | 125 |
12 | 7.8 | 31 | 3.9 | 50 | 3.9 |
13 | 62.5 | 32 | 3.9 | 51 | 31.25 |
14 | 3.9 | 33 | 1.95 | 52 | ≥125 |
15 | 7.8 | 34 | ≥125 | 53 | ≥125 |
16 | 31.25 | 35 | 125 | 54 | ≥125 |
17 | ≥125 | 36 | ≥125 | ||
18 | 31.25 | 37 | 15.6 | ||
19 | 15.6 | 38 | ≥125 |
EH-315 (µg/mL) | ATCC G217-B (µg/mL) | ||||
---|---|---|---|---|---|
Compounds | Derivate | MIC90 | MFC | MIC90 | MFC |
3 | Nitrofuran | 1.95 | 1.95 | 3.9 | 3.9 |
4 | Nitrofuran | 0.98 | 0.98 | 3.9 | 3.9 |
5 | Nitrofuran | 0.24 | 0.24 | 1.95 | 1.95 |
7 | Nitrofuran/Indole | 0.122 | 0.122 | 0.98 | 0.98 |
9 | Nitrofuran | 0.98 | 0.98 | 3.9 | 3.9 |
10 | Nitrofuran | 7.81 | 7.81 | 7.81 | 15.62 |
11 | Nitrofuran | 0.24 | 0.24 | 0.48 | 0.48 |
12 | Nitrofuran | 7.81 | 7.81 | 7.81 | 15.62 |
14 | Nitrofuran | 3.90 | 7.81 | 7.81 | 7.81 |
15 | Nitrofuran | 7.81 | 15.62 | >250 | >250 |
20 | Indole | 7.81 | 7.81 | 7.81 | 7.81 |
21 | Indole | 7.81 | 7.81 | 7.81 | 7.81 |
31 | Nitrofuran/indole | 3.90 | 3.90 | 31.25 | 31.25 |
32 | Indole | 3.90 | 3.90 | 3.90 | 3.90 |
33 | Indole | 1.95 | 1.95 | 3.90 | 3.90 |
39 | Indole | 7.81 | 7.81 | 15.62 | 15.62 |
41 | Indole | 7.81 | 7.81 | 61.25 | 61.25 |
50 | Indole | 3.90 | 3.90 | 31.25 | 31.25 |
AmB | Polyene | 0.06 | - | 0.03 | - |
ITZ | Azole | 0.125 | - | 0.007 | - |
Cell Line | Model | Mean—Fourth Day | Mean—Seventh Day | Mean—Tenth Day |
---|---|---|---|---|
A549 | Monolayer | 5.23 × 104 ± 7.56 × 103 | 8.50 × 104 ± 2.73 × 104 | 1.03 × 105 ± 2.08 × 103 |
Spheroid | 8.33 × 103 ± 2.36 × 103 | 1.23 × 104 ± 1.53 × 103 | 1.25 × 104 ± 2.08 × 103 | |
MRC-5 | Monolayer | 1.91 × 105 ± 1.53 × 104 | 3.56 × 105 ± 3.96 × 104 (*) | 3.06 × 105 ± 3.58 × 104 |
Spheroid | 2.69 × 103 ± 6.67 × 102 | 4.25 × 104 ± 3.75 × 103 (***) | 3.13 × 104 ± 7.60 × 103 (**) |
A549 | ||||||
---|---|---|---|---|---|---|
Monolayer | Spheroid | |||||
EH-315 | ATCC G217-B | EH-315 | ATCC G217-B | |||
Compounds | CC50 (µg/mL) | SI | SI | CC50 (µg/mL) | SI | SI |
3 | 231.2 | 118.56 | 59.28 | >250 | >128.20 | >64.10 |
4 | 29.50 | 30.10 | 7.56 | 62.96 | 64.24 | 16.14 |
5 | 30.11 | 125.45 | 15.44 | 43.01 | 179.20 | 22.05 |
7 | 8.58 | 70.32 | 8.75 | >250 | >2049.18 | >255.1 |
9 | 23.41 | 23.88 | 6.00 | 17.25 | 17.60 | 4.42 |
10 | 38.67 | 4.95 | 4.95 | 32.97 | 4.22 | 4.22 |
11 | 12.24 | 50.16 | 25.50 | 56.06 | 229.75 | 116.79 |
12 | >250 | >32.01 | >32.01 | 104.8 | 13.41 | 13.41 |
14 | 33.44 | 8.83 | 4.4 | 27.61 | 7.07 | 3.53 |
20 | 81.41 | 10.42 | 10.42 | 81.41 | 10.42 | 10.42 |
21 | 40.00 | 5.12 | 5.12 | 40.00 | 5.12 | 5.12 |
32 | 81.41 | 20.87 | 20.87 | 81.41 | 20.76 | 20.76 |
33 | 12.26 | 6.28 | 3.14 | 13.22 | 6.77 | 1.69 |
39 | 42.79 | 5.47 | 2.73 | 60.5 | 7.74 | 3.87 |
MRC-5 | ||||||
---|---|---|---|---|---|---|
Monolayer | Spheroid | |||||
EH-315 | ATCC G217-B | EH-315 | ATCC G217-B | |||
Compounds | CC50 (µg/mL) | SI | SI | CC50 (µg/mL) | SI | SI |
3 | >250 | >128.20 | >64.10 | >250 | >128.20 | >64.10 |
64 | 135.2 | 137.95 | 34.66 | >250 | > 255.10 | >64.10 |
5 | 39.76 | 165.66 | 20.38 | 81.41 | 339.20 | 41.74 |
7 | 71.24 | 583.93 | 72.69 | 73.50 | 602.45 | 75.00 |
9 | 47.23 | 48.19 | 12.11 | 169.6 | 173.06 | 43.48 |
10 | 215.4 | 27.58 | 27.58 | 57.92 | 7.41 | 7.41 |
11 | 62.56 | 256.39 | 130.33 | 67.79 | 277.82 | 141.22 |
12 | 228.0 | 29.19 | 29.19 | 76.12 | 9.74 | 9.74 |
14 | 26.8 | 6.87 | 3.43 | 7.68 | 1.96 | 0.98 |
20 | 20.39 | 2.61 | 2.61 | 25.35 | 3.57 | 3.57 |
21 | 33.77 | 4.32 | 4.32 | 52.34 | 6.70 | 6.70 |
32 | 24.7 | 6.33 | 6.33 | 26.14 | 6.70 | 6.70 |
33 | 18.74 | 9.56 | 4.78 | 14.03 | 7.19 | 3.59 |
39 | 40.00 | 5.12 | 2.56 | 40.00 | 5.12 | 2.56 |
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Vaso, C.O.; Bila, N.M.; Pandolfi, F.; De Vita, D.; Bortolami, M.; Bonatti, J.L.C.; De Moraes Silva, R.A.; Gonçalves, L.N.C.; Tudino, V.; Costi, R.; et al. Evaluation of the Anti-Histoplasma capsulatum Activity of Indole and Nitrofuran Derivatives and Their Pharmacological Safety in Three-Dimensional Cell Cultures. Pharmaceutics 2022, 14, 1043. https://doi.org/10.3390/pharmaceutics14051043
Vaso CO, Bila NM, Pandolfi F, De Vita D, Bortolami M, Bonatti JLC, De Moraes Silva RA, Gonçalves LNC, Tudino V, Costi R, et al. Evaluation of the Anti-Histoplasma capsulatum Activity of Indole and Nitrofuran Derivatives and Their Pharmacological Safety in Three-Dimensional Cell Cultures. Pharmaceutics. 2022; 14(5):1043. https://doi.org/10.3390/pharmaceutics14051043
Chicago/Turabian StyleVaso, Carolina Orlando, Níura Madalena Bila, Fabiana Pandolfi, Daniela De Vita, Martina Bortolami, Jean Lucas Carvalho Bonatti, Rosângela Aparecida De Moraes Silva, Larissa Naiara Carvalho Gonçalves, Valeria Tudino, Roberta Costi, and et al. 2022. "Evaluation of the Anti-Histoplasma capsulatum Activity of Indole and Nitrofuran Derivatives and Their Pharmacological Safety in Three-Dimensional Cell Cultures" Pharmaceutics 14, no. 5: 1043. https://doi.org/10.3390/pharmaceutics14051043
APA StyleVaso, C. O., Bila, N. M., Pandolfi, F., De Vita, D., Bortolami, M., Bonatti, J. L. C., De Moraes Silva, R. A., Gonçalves, L. N. C., Tudino, V., Costi, R., Di Santo, R., Mendes-Giannini, M. J. S., Costa-Orlandi, C. B., Scipione, L., & Fusco-Almeida, A. M. (2022). Evaluation of the Anti-Histoplasma capsulatum Activity of Indole and Nitrofuran Derivatives and Their Pharmacological Safety in Three-Dimensional Cell Cultures. Pharmaceutics, 14(5), 1043. https://doi.org/10.3390/pharmaceutics14051043