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Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2).

Gozzi GJ, Pires Ado R, Valdameri G, Rocha ME, Martinez GR, Noleto GR, Acco A, Alves de Souza CE, Echevarria A, Moretto Dos Reis C, Di Pietro A, Suter Correia Cadena SM - PLoS ONE (2015)

Bottom Line: MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays.It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h).These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, Paraná, Brazil.

ABSTRACT
In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.

No MeSH data available.


Related in: MedlinePlus

Cytotoxic effects of 1,3,4-thiadiazolium derivatives on hepatocytes.A. MTT assay (the experimental conditions are described in the Materials and Methods section 2.5.1) The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 25 for 18–24 h. The results were expressed as % of viability in comparison to control. B. LDH release assay (the experimental conditions are described in the Materials and Methods section 2.5.2). Under the same treatment conditions described above, LDH activity was measured in the supernatants. Data represent means of four different experiments in quadruplicate. The results were expressed as % of viability in comparison to control. ** and *** denotes values significantly different from the control or between the different treatments at P< 0.01 and P< 0.0001, respectively.
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pone.0130046.g003: Cytotoxic effects of 1,3,4-thiadiazolium derivatives on hepatocytes.A. MTT assay (the experimental conditions are described in the Materials and Methods section 2.5.1) The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 25 for 18–24 h. The results were expressed as % of viability in comparison to control. B. LDH release assay (the experimental conditions are described in the Materials and Methods section 2.5.2). Under the same treatment conditions described above, LDH activity was measured in the supernatants. Data represent means of four different experiments in quadruplicate. The results were expressed as % of viability in comparison to control. ** and *** denotes values significantly different from the control or between the different treatments at P< 0.01 and P< 0.0001, respectively.

Mentions: The viability of HepG2 cells was determined after 24 h of treatment with derivatives at 5, 25 and 50 μM, by both MTT and LDH-release assays. As observed in Fig 2, upper panel, MI-J, MI-4F and MI-2,4diF reduced HepG2 cells viability by about 50% at 25 μM when analyzed by MTT. MI-D only reduced by 28% the cell viability, requiring 50 μM to reach 50%. The results of the LDH-release assay (Fig 2, lower panel) also demonstrated the reduction of cell viability by MI-J, MI-4F and MI-2,4diF treatments. The enzymatic activity of the culture medium was increased by 55, 24 and 16%, respectively, for MI-J, MI-4F and MI-2,4diF at 25 μM, in comparison to controls without mesoionic derivative. MI-D, on the contrary, did not significantly affect the LDH activity. The viability of primary hepatocytes was also determined in order to verify the selectivity of derivatives for tumor cells. As observed in Fig 3, no cytotoxicity was observed in MTT assays (upper panel), except for MI-2,4diF producing a 36% effect (at 25 μM for 18–24 h). However, no increase in LDH activity was observed with any derivative (lower panel). Interestingly, MI-D induced a reduction of LDH activity (~ 17% at 25 μM).


Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2).

Gozzi GJ, Pires Ado R, Valdameri G, Rocha ME, Martinez GR, Noleto GR, Acco A, Alves de Souza CE, Echevarria A, Moretto Dos Reis C, Di Pietro A, Suter Correia Cadena SM - PLoS ONE (2015)

Cytotoxic effects of 1,3,4-thiadiazolium derivatives on hepatocytes.A. MTT assay (the experimental conditions are described in the Materials and Methods section 2.5.1) The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 25 for 18–24 h. The results were expressed as % of viability in comparison to control. B. LDH release assay (the experimental conditions are described in the Materials and Methods section 2.5.2). Under the same treatment conditions described above, LDH activity was measured in the supernatants. Data represent means of four different experiments in quadruplicate. The results were expressed as % of viability in comparison to control. ** and *** denotes values significantly different from the control or between the different treatments at P< 0.01 and P< 0.0001, respectively.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4470815&req=5

pone.0130046.g003: Cytotoxic effects of 1,3,4-thiadiazolium derivatives on hepatocytes.A. MTT assay (the experimental conditions are described in the Materials and Methods section 2.5.1) The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 25 for 18–24 h. The results were expressed as % of viability in comparison to control. B. LDH release assay (the experimental conditions are described in the Materials and Methods section 2.5.2). Under the same treatment conditions described above, LDH activity was measured in the supernatants. Data represent means of four different experiments in quadruplicate. The results were expressed as % of viability in comparison to control. ** and *** denotes values significantly different from the control or between the different treatments at P< 0.01 and P< 0.0001, respectively.
Mentions: The viability of HepG2 cells was determined after 24 h of treatment with derivatives at 5, 25 and 50 μM, by both MTT and LDH-release assays. As observed in Fig 2, upper panel, MI-J, MI-4F and MI-2,4diF reduced HepG2 cells viability by about 50% at 25 μM when analyzed by MTT. MI-D only reduced by 28% the cell viability, requiring 50 μM to reach 50%. The results of the LDH-release assay (Fig 2, lower panel) also demonstrated the reduction of cell viability by MI-J, MI-4F and MI-2,4diF treatments. The enzymatic activity of the culture medium was increased by 55, 24 and 16%, respectively, for MI-J, MI-4F and MI-2,4diF at 25 μM, in comparison to controls without mesoionic derivative. MI-D, on the contrary, did not significantly affect the LDH activity. The viability of primary hepatocytes was also determined in order to verify the selectivity of derivatives for tumor cells. As observed in Fig 3, no cytotoxicity was observed in MTT assays (upper panel), except for MI-2,4diF producing a 36% effect (at 25 μM for 18–24 h). However, no increase in LDH activity was observed with any derivative (lower panel). Interestingly, MI-D induced a reduction of LDH activity (~ 17% at 25 μM).

Bottom Line: MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays.It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h).These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, Paraná, Brazil.

ABSTRACT
In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.

No MeSH data available.


Related in: MedlinePlus