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THE CYTOTOXIC EFFECT OF BENZOIC ACID ON TEN DIFFERENT CANCER CELL LINES

Year 2024, Volume: 25 Issue: 1, 66 - 77, 28.03.2024
https://doi.org/10.18038/estubtda.1327658

Abstract

Cancer, having numerous types, is among the most dangerous and complex chronic diseases in the world affecting the wellbeing of humans, society and economy. The exploration and reassessment of effective chemicals, compounds, and natural products as potential agents for alleviating the adverse effects of cancer and its related symptoms continue on a global scale. This process involves an initial evaluation of the cytotoxic activities of potential drug candidates or treatment regimens on diverse cancer cell types in an ex vivo context. Benzoic acid (BA), an aromatic carboxylic acid that is widely available and used in the food industry, is one of the phenolic acids that may bear considerable anti-cancer potential. It is useful to find out the comparable effect of BA on various cancer types. Therefore, in this study, we tested the cytotoxicity of BA using MTT assay, on a number of ten different cancer cell lines and one normal cell type, namely prostate cancer (PC3), cervical cancer (HeLA), liver cancer (HUH7), colon cancer (CaCO2, HT29, SW48), bone cancer (MG63 and A673), pharyngeal cancer (2A3), lung cancer (CRM612) and kidney epithelial control cell line (Phoenix), respectively. IC50 (µg/ml) values after 48 and 72-hour exposure to BA were found to differ between 85.54±3.17 to 670.6±43.26, while the IC values for the control cell line Phoenix were 410.54±32.29 and 231.16±25.25, respectively. Taking into account of statistical evaluation of the IC50 values for BA on 11 cell types, we suggest that the molecular and omics approaches can be implemented in more details in order to find cellular and biochemical targets of BA as well as elucidating molecular mode of action, especially starting with the cancer cell lines of MG63, CRM612 and A673, in which the IC50 levels are relatively the lowest compared to those of the control cell line.

Thanks

This study, Biotechnology Research Center and Proteogenome Technologies research laboratory at Mugla Sitki Kocman University Research Laboratories Center provided support in Turkey. Their support was gratefully acknowledged.

References

  • [1] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100(1), 57-70. https://doi.org/10.1016/S0092-8674(00)81683-9
  • [2] Lund AH, van Lohuizen M. Epigenetics and cancer. Genes Dev 2004; 18(19), 2315-2335
  • [3] Sung H, Ferlay J, Siege RL, Laversanne M, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA-Cancer J Clin 2021; 71(3), 209-249.
  • [4] Oto Murat. Osteosarkom ve varyantlarına tanısal yaklaşım modaliteleri. Pamukkale Tıp Dergisi 2008; (1), 59-64.
  • [5] Cho WH, Lee HJ, Choi YJ, Oh JH, Kim HS, Cho HS. Capsaicin induces apoptosis in MG63 human osteosarcoma cells via the caspase cascade and the antioxidant enzyme system. Mol Med Rep 2013; 8(6), 1655-1662.
  • [6] Wang M, Yin B, Wang HY, Wang RF. Current advances in T-cell-based cancer immunotherapy. Immunotherapy 2014; 6(12), 1265-1278.
  • [7] Anantharaju PG, Gowda PC, Vimalambike MG, Madhunapantula SV. An overview on the role of dietary phenolics for the treatment of cancers. Nutr J 2016; 15(1), 1-16.
  • [8] Devi KP, Rajavel T, Habtemariam S, Nabavi SF, Nabavi SM. Molecular mechanisms underlying anticancer effects of myricetin. Life Sci 2015; 142, 19-25.
  • [9] Kennedy DO, Wightman EL. Herbal extracts and phytochemicals: plant secondary metabolites and the enhancement of human brain function. Adv Nutr 2011; 2(1), 32-50.
  • [10] Soto-Vaca A, Gutierrez A, Losso JN, Xu Z, Finley JW. Evolution of phenolic compounds from color and flavor problems to health benefits. J Agr Food Chem 2012; 60(27), 6658-6677.
  • [11] Onat KA, Kürkçü-Sezer M, Çöl B. Some biological activities of phenolic compounds cinnamic acid, caffeic acid and p-coumaric acid. J Ins Sci Tech 2021; 11 (4), 2587-2598.
  • [12] Onat-Taşdelen, KA, Öztürkel-Kabakaş H, Yüksektepe E, et al. Functional groups matter: metabolomics analysis of Escherichia coli exposed to trans-cinnamic acid, and its derivatives unveils common and unique targets. World J Microbiol Biotechnol 2024; 40(2), 47.
  • [13] Öztürkel-Kabakaş H, Taşdelen-Onat KA, Kürkçü-Sezer M, Çöl B. Investigation of the food protective activities of benzoic acid and derivatives occuring naturally ın foods. Ege 7th International Conference on Applied Sciences 2022; 803-810.
  • [14] Maki T, Takeda K. Benzoic acid, and derivatives. Ullmann’s encyclopedia of industrial chemistry 2000; 3. https://doi.org/10.1002/14356007.a03_555
  • [15] Wildermuth MC. Variations on a theme: synthesis and modification of plant benzoic acids. Curr Opin Plant Biol 2006; 9(3), 288-296.
  • [16] Block A, Widhalm JR, Fatihi A, et al. The origin and biosynthesis of the benzenoid moiety of ubiquinone (coenzyme Q) in Arabidopsis. Plant Cell 2014; 26(5), 1938-1948.
  • [17] Widhalm JR, Dudareva N. A familiar ring to it: biosynthesis of plant benzoic acids. Mol Plant 2015; 8(1), 83-97.
  • [18] Davidson PM, Sofos, JN, Branen AL. Antimicrobials in food. CRC press 2005.
  • [19] Xiang L, Moore BS. Biochemical characterization of a prokaryotic phenylalanine ammonia lyase. J Bacteriol 2005; 187(12), 4286-4289.
  • [20] Torrallardona D, Badiola I, Broz J. Effects of benzoic acid on performance and ecology of gastrointestinal microbiota in weanling piglets. Livest Sci 2007; 108(1-3), 210-213.
  • [21] Benjamin MM, Datta AR. Acid tolerance of enterohemorrhagic Escherichia coli. Appl Environ Microbiol 1995; 61(4), 1669-1672.
  • [22] Leth T, Christensen T, Larsen IK. Estimated intake of benzoic and sorbic acids in Denmark. Food Addit Contam A 2010; 27(6), 783-792.
  • [23] Sadeghi-Ekbatan S, Li XQ, Ghorbani M, Azadi B, Kubow S. Chlorogenic acid and its microbial metabolites exert anti-proliferative effects, S-phase cell-cycle arrest and apoptosis in human colon cancer Caco-2 cells. Int J Mol Sci 2018; 19(3), 723.
  • [24] Koochekpour S, Marlowe T, Singh KK, Attwood K, Chandra D. Reduced mitochondrial DNA content associates with poor prognosis of prostate cancer in African American men. PloS one 2013; 8(9), e74688.
  • [25] Roth SY, Denu JM, Allis CD. Histone acetyltransferases. Annu Rev Biochem 2001; 70(1), 81-120.
  • [26] Marks PA, Richon VM, Rifkind RA. Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells. J Natl Cancer Ins 2000; 92(15), 1210-1216.
  • [27] Anantharaju PG, Reddy BD, Padukudru MA, Kumari-Chitturi CM, Vimalambike MG, Madhunapantula SV. Naturally occurring benzoic acid derivatives retard cancer cell growth by inhibiting histone deacetylases (HDAC). Cancer Biol Ther 2017; 18(7), 492-504.
  • [28] Seidel C, Schnekenburger M, Dicato M, Diederich M. Antiproliferative and proapoptotic activities of 4-hydroxybenzoic acid-based inhibitors of histone deacetylases. Cancer Lett 2014; 343(1), 134-146.
  • [29] Çöl B, Kürkçü-Sezer M, Sorucu A. Okaliptüs balının PC3, CaCO2, HeLa ve HuH7 kanser hücre hatları üzerinde sitotoksisitesinin değerlendirilmesi. International Journal of Innovative Approaches in Science Research 2021; 5 (4), 195-206.
  • [30] Güner A, Nalbantsoy A, Sukatar A, Karabay-Yavaşoğlu NÜ. Apoptosis-inducing activities of Halopteris scoparia L. Sauvageau (Brown algae) on cancer cells and its biosafety and antioxidant properties. Cytotechnology 2019; 71(3), 687-704.
  • [31] Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: How are they linked. Free Radic Biol Med 2010; 49(11), 1603-1616.
  • [32] Chiva-Blanch G, Badimon L. Effects of polyphenol intake on metabolic syndrome: current evidence from human trials. Oxid Med Cell Longev 2017; 1-17.
  • [33] Zakłos-Szyda M, Nowak A, Pietrzyk N, Podsędek A. Viburnum opulus L. juice phenolic compounds influence osteogenic differentiation in human osteosarcoma Saos-2 cells. Int J Mol Sci 2020; 21(14), 4909.
  • [34] Kandaswami C, Middleton E, Free radical scavenging, and antioxidant activity of plant flavonoids. Adv Exp Med Biol 1994; 351-376.
  • [35] Zhang B, Wu D, Hu L, et al. Ligustilide inhibits the proliferation of human osteoblastoma MG63 cells through the TLR4-ERK pathway. Life Sci 2022; 288, 118993.
  • [36] Dorfman HD. Czerniak B. Bone cancers. Cancer 1995; 75(S1), 203-210.
  • [37] Hosseini F, Shanehbandi D, Soleimanpour J, Yousefi B, Alemi F. Melatonin ıncreases the sensitivity of osteosarcoma cells to chemotherapy drug Cisplatin. Drug Res 2022; 312-318.
  • [38] Reinecke JB, Roberts RD. Targetable intercellular signaling pathways facilitate lung colonization in osteosarcoma. Adv Exp Med Biol 2020; 111-123.
  • [39] Meazza C, Scanagatta P, Metastatic osteosarcoma: a challenging multidisciplinary treatment. Expert Rev Anticancer Ther 2016; 16(5), 543-556.
  • [40] Bade BC, Cruz CSD. Lung cancer 2020: epidemiology, etiology, and prevention. Clin Chest Med 2020; 41(1), 1-24.
  • [41] Illian DN, Hafiz I, Meila O, et al. Distribution, and future directions of natural products against colorectal cancer in Indonesia: A systematic review. Molecules 2021; 26(16), 4984.
  • [42] O’Keefe SJ, Li JV, Lahti L, et al. Fat, fibre and cancer risk in African Americans and rural Africans. Nat Commun 2015; 6(1), 1-14.
  • [43] O'Keefe SJ. Diet, microorganisms and their metabolites, and colon cancer. Nat Rev Gastroenterol Hepatol 2016; 13(12), 691-706.
  • [44] Reynolds NA, Wagstaff AJ. Cetuximab. Drugs 2004; 64 (1), 109-118.
  • [45] Kuş S, Uygur T, Candan İ. Kolorektal kanser tedavisinde kullanılan Setuksimab’a bağlı akneiform erüpsiyon. TÜRKDERM-Deri Hastalıkları ve Frengi Arşivi 2006; 40(Supp: B), 85-87.
  • [46] Graziani V, Potenza N, D’Abrosca B, Troiani T. NMR Profiling of Ononis diffusa Identifies Cytotoxic Compounds against Cetuximab-Resistant Colon Cancer Cell Lines. Molecules 2021; 26(11), 3266.
  • [47] Elgin ES, Çatav ŞS., Babayeva A, et al. NMR metabolomics analysis of Escherichia coli cells treated with Turkish propolis water extract reveals nucleic acid metabolism as the major target. J. Appl Microbiol 2023; 134(1), lxac031.
  • [48] Canbek U, Dibek E, Akgün U, et al. Analysis of the fluid Biochemistry in patients with prolonged wound drainage after hip hemiarthroplasty. Injury 2021; 5 (2), 918-925.

THE CYTOTOXIC EFFECT OF BENZOIC ACID ON TEN DIFFERENT CANCER CELL LINES

Year 2024, Volume: 25 Issue: 1, 66 - 77, 28.03.2024
https://doi.org/10.18038/estubtda.1327658

Abstract

Cancer, having numerous types, is among the most dangerous and complex chronic diseases in the world affecting the wellbeing of humans, society and economy. The exploration and reassessment of effective chemicals, compounds, and natural products as potential agents for alleviating the adverse effects of cancer and its related symptoms continue on a global scale. This process involves an initial evaluation of the cytotoxic activities of potential drug candidates or treatment regimens on diverse cancer cell types in an ex vivo context. Benzoic acid (BA), an aromatic carboxylic acid that is widely available and used in the food industry, is one of the phenolic acids that may bear considerable anti-cancer potential. It is useful to find out the comparable effect of BA on various cancer types. Therefore, in this study, we tested the cytotoxicity of BA using MTT assay, on a number of ten different cancer cell lines and one normal cell type, namely prostate cancer (PC3), cervical cancer (HeLA), liver cancer (HUH7), colon cancer (CaCO2, HT29, SW48), bone cancer (MG63 and A673), pharyngeal cancer (2A3), lung cancer (CRM612) and kidney epithelial control cell line (Phoenix), respectively. IC50 (µg/ml) values after 48 and 72-hour exposure to BA were found to differ between 85.54±3.17 to 670.6±43.26, while the IC values for the control cell line Phoenix were 410.54±32.29 and 231.16±25.25, respectively. Taking into account of statistical evaluation of the IC50 values for BA on 11 cell types, we suggest that the molecular and omics approaches can be implemented in more details in order to find cellular and biochemical targets of BA as well as elucidating molecular mode of action, especially starting with the cancer cell lines of MG63, CRM612 and A673, in which the IC50 levels are relatively the lowest compared to those of the control cell line.

References

  • [1] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100(1), 57-70. https://doi.org/10.1016/S0092-8674(00)81683-9
  • [2] Lund AH, van Lohuizen M. Epigenetics and cancer. Genes Dev 2004; 18(19), 2315-2335
  • [3] Sung H, Ferlay J, Siege RL, Laversanne M, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA-Cancer J Clin 2021; 71(3), 209-249.
  • [4] Oto Murat. Osteosarkom ve varyantlarına tanısal yaklaşım modaliteleri. Pamukkale Tıp Dergisi 2008; (1), 59-64.
  • [5] Cho WH, Lee HJ, Choi YJ, Oh JH, Kim HS, Cho HS. Capsaicin induces apoptosis in MG63 human osteosarcoma cells via the caspase cascade and the antioxidant enzyme system. Mol Med Rep 2013; 8(6), 1655-1662.
  • [6] Wang M, Yin B, Wang HY, Wang RF. Current advances in T-cell-based cancer immunotherapy. Immunotherapy 2014; 6(12), 1265-1278.
  • [7] Anantharaju PG, Gowda PC, Vimalambike MG, Madhunapantula SV. An overview on the role of dietary phenolics for the treatment of cancers. Nutr J 2016; 15(1), 1-16.
  • [8] Devi KP, Rajavel T, Habtemariam S, Nabavi SF, Nabavi SM. Molecular mechanisms underlying anticancer effects of myricetin. Life Sci 2015; 142, 19-25.
  • [9] Kennedy DO, Wightman EL. Herbal extracts and phytochemicals: plant secondary metabolites and the enhancement of human brain function. Adv Nutr 2011; 2(1), 32-50.
  • [10] Soto-Vaca A, Gutierrez A, Losso JN, Xu Z, Finley JW. Evolution of phenolic compounds from color and flavor problems to health benefits. J Agr Food Chem 2012; 60(27), 6658-6677.
  • [11] Onat KA, Kürkçü-Sezer M, Çöl B. Some biological activities of phenolic compounds cinnamic acid, caffeic acid and p-coumaric acid. J Ins Sci Tech 2021; 11 (4), 2587-2598.
  • [12] Onat-Taşdelen, KA, Öztürkel-Kabakaş H, Yüksektepe E, et al. Functional groups matter: metabolomics analysis of Escherichia coli exposed to trans-cinnamic acid, and its derivatives unveils common and unique targets. World J Microbiol Biotechnol 2024; 40(2), 47.
  • [13] Öztürkel-Kabakaş H, Taşdelen-Onat KA, Kürkçü-Sezer M, Çöl B. Investigation of the food protective activities of benzoic acid and derivatives occuring naturally ın foods. Ege 7th International Conference on Applied Sciences 2022; 803-810.
  • [14] Maki T, Takeda K. Benzoic acid, and derivatives. Ullmann’s encyclopedia of industrial chemistry 2000; 3. https://doi.org/10.1002/14356007.a03_555
  • [15] Wildermuth MC. Variations on a theme: synthesis and modification of plant benzoic acids. Curr Opin Plant Biol 2006; 9(3), 288-296.
  • [16] Block A, Widhalm JR, Fatihi A, et al. The origin and biosynthesis of the benzenoid moiety of ubiquinone (coenzyme Q) in Arabidopsis. Plant Cell 2014; 26(5), 1938-1948.
  • [17] Widhalm JR, Dudareva N. A familiar ring to it: biosynthesis of plant benzoic acids. Mol Plant 2015; 8(1), 83-97.
  • [18] Davidson PM, Sofos, JN, Branen AL. Antimicrobials in food. CRC press 2005.
  • [19] Xiang L, Moore BS. Biochemical characterization of a prokaryotic phenylalanine ammonia lyase. J Bacteriol 2005; 187(12), 4286-4289.
  • [20] Torrallardona D, Badiola I, Broz J. Effects of benzoic acid on performance and ecology of gastrointestinal microbiota in weanling piglets. Livest Sci 2007; 108(1-3), 210-213.
  • [21] Benjamin MM, Datta AR. Acid tolerance of enterohemorrhagic Escherichia coli. Appl Environ Microbiol 1995; 61(4), 1669-1672.
  • [22] Leth T, Christensen T, Larsen IK. Estimated intake of benzoic and sorbic acids in Denmark. Food Addit Contam A 2010; 27(6), 783-792.
  • [23] Sadeghi-Ekbatan S, Li XQ, Ghorbani M, Azadi B, Kubow S. Chlorogenic acid and its microbial metabolites exert anti-proliferative effects, S-phase cell-cycle arrest and apoptosis in human colon cancer Caco-2 cells. Int J Mol Sci 2018; 19(3), 723.
  • [24] Koochekpour S, Marlowe T, Singh KK, Attwood K, Chandra D. Reduced mitochondrial DNA content associates with poor prognosis of prostate cancer in African American men. PloS one 2013; 8(9), e74688.
  • [25] Roth SY, Denu JM, Allis CD. Histone acetyltransferases. Annu Rev Biochem 2001; 70(1), 81-120.
  • [26] Marks PA, Richon VM, Rifkind RA. Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells. J Natl Cancer Ins 2000; 92(15), 1210-1216.
  • [27] Anantharaju PG, Reddy BD, Padukudru MA, Kumari-Chitturi CM, Vimalambike MG, Madhunapantula SV. Naturally occurring benzoic acid derivatives retard cancer cell growth by inhibiting histone deacetylases (HDAC). Cancer Biol Ther 2017; 18(7), 492-504.
  • [28] Seidel C, Schnekenburger M, Dicato M, Diederich M. Antiproliferative and proapoptotic activities of 4-hydroxybenzoic acid-based inhibitors of histone deacetylases. Cancer Lett 2014; 343(1), 134-146.
  • [29] Çöl B, Kürkçü-Sezer M, Sorucu A. Okaliptüs balının PC3, CaCO2, HeLa ve HuH7 kanser hücre hatları üzerinde sitotoksisitesinin değerlendirilmesi. International Journal of Innovative Approaches in Science Research 2021; 5 (4), 195-206.
  • [30] Güner A, Nalbantsoy A, Sukatar A, Karabay-Yavaşoğlu NÜ. Apoptosis-inducing activities of Halopteris scoparia L. Sauvageau (Brown algae) on cancer cells and its biosafety and antioxidant properties. Cytotechnology 2019; 71(3), 687-704.
  • [31] Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: How are they linked. Free Radic Biol Med 2010; 49(11), 1603-1616.
  • [32] Chiva-Blanch G, Badimon L. Effects of polyphenol intake on metabolic syndrome: current evidence from human trials. Oxid Med Cell Longev 2017; 1-17.
  • [33] Zakłos-Szyda M, Nowak A, Pietrzyk N, Podsędek A. Viburnum opulus L. juice phenolic compounds influence osteogenic differentiation in human osteosarcoma Saos-2 cells. Int J Mol Sci 2020; 21(14), 4909.
  • [34] Kandaswami C, Middleton E, Free radical scavenging, and antioxidant activity of plant flavonoids. Adv Exp Med Biol 1994; 351-376.
  • [35] Zhang B, Wu D, Hu L, et al. Ligustilide inhibits the proliferation of human osteoblastoma MG63 cells through the TLR4-ERK pathway. Life Sci 2022; 288, 118993.
  • [36] Dorfman HD. Czerniak B. Bone cancers. Cancer 1995; 75(S1), 203-210.
  • [37] Hosseini F, Shanehbandi D, Soleimanpour J, Yousefi B, Alemi F. Melatonin ıncreases the sensitivity of osteosarcoma cells to chemotherapy drug Cisplatin. Drug Res 2022; 312-318.
  • [38] Reinecke JB, Roberts RD. Targetable intercellular signaling pathways facilitate lung colonization in osteosarcoma. Adv Exp Med Biol 2020; 111-123.
  • [39] Meazza C, Scanagatta P, Metastatic osteosarcoma: a challenging multidisciplinary treatment. Expert Rev Anticancer Ther 2016; 16(5), 543-556.
  • [40] Bade BC, Cruz CSD. Lung cancer 2020: epidemiology, etiology, and prevention. Clin Chest Med 2020; 41(1), 1-24.
  • [41] Illian DN, Hafiz I, Meila O, et al. Distribution, and future directions of natural products against colorectal cancer in Indonesia: A systematic review. Molecules 2021; 26(16), 4984.
  • [42] O’Keefe SJ, Li JV, Lahti L, et al. Fat, fibre and cancer risk in African Americans and rural Africans. Nat Commun 2015; 6(1), 1-14.
  • [43] O'Keefe SJ. Diet, microorganisms and their metabolites, and colon cancer. Nat Rev Gastroenterol Hepatol 2016; 13(12), 691-706.
  • [44] Reynolds NA, Wagstaff AJ. Cetuximab. Drugs 2004; 64 (1), 109-118.
  • [45] Kuş S, Uygur T, Candan İ. Kolorektal kanser tedavisinde kullanılan Setuksimab’a bağlı akneiform erüpsiyon. TÜRKDERM-Deri Hastalıkları ve Frengi Arşivi 2006; 40(Supp: B), 85-87.
  • [46] Graziani V, Potenza N, D’Abrosca B, Troiani T. NMR Profiling of Ononis diffusa Identifies Cytotoxic Compounds against Cetuximab-Resistant Colon Cancer Cell Lines. Molecules 2021; 26(11), 3266.
  • [47] Elgin ES, Çatav ŞS., Babayeva A, et al. NMR metabolomics analysis of Escherichia coli cells treated with Turkish propolis water extract reveals nucleic acid metabolism as the major target. J. Appl Microbiol 2023; 134(1), lxac031.
  • [48] Canbek U, Dibek E, Akgün U, et al. Analysis of the fluid Biochemistry in patients with prolonged wound drainage after hip hemiarthroplasty. Injury 2021; 5 (2), 918-925.
There are 48 citations in total.

Details

Primary Language English
Subjects Biologically Active Molecules
Journal Section Articles
Authors

Hatice Öztürkel Kabakaş 0000-0002-2882-0401

Merve Sezer Kürkçü 0000-0003-0947-2912

Kadriye Aslıhan Onat Taşdelen 0000-0002-5893-5728

Bekir Çöl 0000-0001-8997-4116

Publication Date March 28, 2024
Published in Issue Year 2024 Volume: 25 Issue: 1

Cite

AMA Öztürkel Kabakaş H, Sezer Kürkçü M, Onat Taşdelen KA, Çöl B. THE CYTOTOXIC EFFECT OF BENZOIC ACID ON TEN DIFFERENT CANCER CELL LINES. Estuscience - Se. March 2024;25(1):66-77. doi:10.18038/estubtda.1327658