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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

Effects of 1,3,4-thiadiazolium derivatives on hepatocytes morphology (the experimental conditions are described in the Materials and Methods section 2.6).Hepatocytes were incubated with the derivatives at 25 μM for 24 h. The images were obtained using inverted microscope. A: control (untreated cells); B-E: treatments by MI-D, MI-J, MI-4F and MI-2,4diF, respectively. The scale is indicated by black bars representing 50 μm. The photographs represent three different experiments in triplicate.
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pone.0130046.g008: Effects of 1,3,4-thiadiazolium derivatives on hepatocytes morphology (the experimental conditions are described in the Materials and Methods section 2.6).Hepatocytes were incubated with the derivatives at 25 μM for 24 h. The images were obtained using inverted microscope. A: control (untreated cells); B-E: treatments by MI-D, MI-J, MI-4F and MI-2,4diF, respectively. The scale is indicated by black bars representing 50 μm. The photographs represent three different experiments in triplicate.

Mentions: Considering the significant toxicity of the derivatives on HepG2 cells (Fig 2), we evaluated the induction of apoptosis in these cells by DNA fragmentation, a key event of cells undergoing apoptosis [47]. After 24 h of treatment with MI-J, MI-4F and MI-2,4diF (Fig 4C–4E), approximate increases of 12%, 9% and 8%, respectively, were observed, as evidenced by the higher number of cells in sub-G1 region. MI-D under the same conditions did not promote any significant alteration in DNA fragmentation, but increased the number of cells in G2/M phase (Fig 4B). The G1/G0 and G2/M phases were not significantly changed by the other derivatives. To further investigate the induction of apoptosis by mesoionic derivatives, HepG2 cells were simultaneously stained with FITC-conjugated annexin V and PI, and analyzed by flow cytometry (Fig 5). All compounds (at 25 μM for 24 h) increased the number of doubly-stained cells in comparison to control, reaching up to 76% for MI-J, 36% and 25% for MI-4F and MI-2,4diF, while a lower value of 11% was observed for MI-D. In addition, MI-J and MI-2,4diF promoted a slight increase (around 2.4%) in the number of PI-labeled cells. Since the differentiation between apoptosis and necrosis was not possible with such an assay, short incubation time (3 h) and reduced concentration (5 μM) were used for morphological analyzes [47]. Apoptotic bodies (blebs) were observed, and loss of cellular organization in monolayer was elicited for all compounds even at low concentration (Fig 6). Other characteristics of apoptosis induction, such as vacuolization, cellular shrinkage (with MI-D, MI-J and MI-4F) and nuclear pyknosis (with MI-4F and MI-2,4diF), were also observed. All together, these results suggest that apoptosis may be the death pathway induced by 1,3,4-thiadizolium derivatives on HepG2 cells. Cultured hepatocytes were also doubly stained with FITC-conjugated annexin V and PI, and analyzed by fluorescence microscopy (Fig 7), but no increase in annexin-FITC and PI labeling was observed for any compound (at 25 μM for 18–24 h) when compared to treatment with acetylsalicylic acid (at 20 mM for 18–24 h), used as a positive control [48]. The hepatocytes morphology was also verified: no alterations in normal characteristics of primary hepatocytes, as cubic form, monolayer organization and multinucleation was observed, supporting previous results suggesting that these derivatives slightly or not at all affected hepatocytes viability (Fig 8).


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)

Effects of 1,3,4-thiadiazolium derivatives on hepatocytes morphology (the experimental conditions are described in the Materials and Methods section 2.6).Hepatocytes were incubated with the derivatives at 25 μM for 24 h. The images were obtained using inverted microscope. A: control (untreated cells); B-E: treatments by MI-D, MI-J, MI-4F and MI-2,4diF, respectively. The scale is indicated by black bars representing 50 μm. The photographs represent three different experiments in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130046.g008: Effects of 1,3,4-thiadiazolium derivatives on hepatocytes morphology (the experimental conditions are described in the Materials and Methods section 2.6).Hepatocytes were incubated with the derivatives at 25 μM for 24 h. The images were obtained using inverted microscope. A: control (untreated cells); B-E: treatments by MI-D, MI-J, MI-4F and MI-2,4diF, respectively. The scale is indicated by black bars representing 50 μm. The photographs represent three different experiments in triplicate.
Mentions: Considering the significant toxicity of the derivatives on HepG2 cells (Fig 2), we evaluated the induction of apoptosis in these cells by DNA fragmentation, a key event of cells undergoing apoptosis [47]. After 24 h of treatment with MI-J, MI-4F and MI-2,4diF (Fig 4C–4E), approximate increases of 12%, 9% and 8%, respectively, were observed, as evidenced by the higher number of cells in sub-G1 region. MI-D under the same conditions did not promote any significant alteration in DNA fragmentation, but increased the number of cells in G2/M phase (Fig 4B). The G1/G0 and G2/M phases were not significantly changed by the other derivatives. To further investigate the induction of apoptosis by mesoionic derivatives, HepG2 cells were simultaneously stained with FITC-conjugated annexin V and PI, and analyzed by flow cytometry (Fig 5). All compounds (at 25 μM for 24 h) increased the number of doubly-stained cells in comparison to control, reaching up to 76% for MI-J, 36% and 25% for MI-4F and MI-2,4diF, while a lower value of 11% was observed for MI-D. In addition, MI-J and MI-2,4diF promoted a slight increase (around 2.4%) in the number of PI-labeled cells. Since the differentiation between apoptosis and necrosis was not possible with such an assay, short incubation time (3 h) and reduced concentration (5 μM) were used for morphological analyzes [47]. Apoptotic bodies (blebs) were observed, and loss of cellular organization in monolayer was elicited for all compounds even at low concentration (Fig 6). Other characteristics of apoptosis induction, such as vacuolization, cellular shrinkage (with MI-D, MI-J and MI-4F) and nuclear pyknosis (with MI-4F and MI-2,4diF), were also observed. All together, these results suggest that apoptosis may be the death pathway induced by 1,3,4-thiadizolium derivatives on HepG2 cells. Cultured hepatocytes were also doubly stained with FITC-conjugated annexin V and PI, and analyzed by fluorescence microscopy (Fig 7), but no increase in annexin-FITC and PI labeling was observed for any compound (at 25 μM for 18–24 h) when compared to treatment with acetylsalicylic acid (at 20 mM for 18–24 h), used as a positive control [48]. The hepatocytes morphology was also verified: no alterations in normal characteristics of primary hepatocytes, as cubic form, monolayer organization and multinucleation was observed, supporting previous results suggesting that these derivatives slightly or not at all affected hepatocytes viability (Fig 8).

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