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DNA damage and inhibition of akt pathway in mcf-7 cells and ehrlich tumor in mice treated with 1,4-naphthoquinones in combination with ascorbate.

Ourique F, Kviecinski MR, Felipe KB, Correia JF, Farias MS, Castro LS, Grinevicius VM, Valderrama J, Rios D, Benites J, Calderon PB, Pedrosa RC - Oxid Med Cell Longev (2015)

Bottom Line: Juglone, phenylaminonaphthoquinone-7, and 9 (Q7/Q9) were evaluated for effects on CT-DNA and DNA of cancer cells.Treatments caused DNA damage and reduced viability and proliferation of MCF-7 cells.Juglone and Q7 plus ascorbate caused enhanced ROS and DNA damage and inhibited pAkt also in Ehrlich carcinoma cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Bioquímica Experimental, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis, Brazil.

ABSTRACT
The aim of this study was to enhance the understanding of the antitumor mechanism of 1,4-naphthoquinones and ascorbate. Juglone, phenylaminonaphthoquinone-7, and 9 (Q7/Q9) were evaluated for effects on CT-DNA and DNA of cancer cells. Evaluations in MCF-7 cells are DNA damage, ROS levels, viability, and proliferation. Proteins from MCF-7 lysates were immunoblotted for verifying PARP integrity, γH2AX, and pAkt. Antitumor activity was measured in Ehrlich ascites carcinoma-bearing mice. The same markers of molecular toxicity were assessed in vivo. The naphthoquinones intercalate into CT-DNA and caused oxidative cleavage, which is increased in the presence of ascorbate. Treatments caused DNA damage and reduced viability and proliferation of MCF-7 cells. Effects were potentiated by ascorbate. No PARP cleavage was observed. Naphthoquinones, combined with ascorbate, caused phosphorylation of H2AX and inhibited pAkt. ROS were enhanced in MCF-7 cells, particularly by the juglone and Q7 plus ascorbate. Ehrlich carcinoma was inhibited by juglone, Q7, or Q9, but the potentiating effect of ascorbate was reproduced in vivo only in the cases of juglone and Q7, which caused up to 60% inhibition of tumor and the largest extension of survival. Juglone and Q7 plus ascorbate caused enhanced ROS and DNA damage and inhibited pAkt also in Ehrlich carcinoma cells.

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The fluorescence of CT-DNA stained with ethidium bromide is reduced by doxorubicin and juglone, Q7, or Q9, mainly in presence of ascorbate (Asc) (a). Absorbance of thiobarbituric reactive species (TBARS) in CT-DNA treated with juglone, Q7, or Q9 alone and combined with ascorbate. Negative control (NC): phosphate buffer. Positive control (PC): [Fe(EDTA)]2−/H2O2 (b). DNA damage index was determined by the comet assay in MCF-7 cells treated for 24 h with juglone 75 μM, Q7 50 μM, or Q9 50 μM with or without ascorbate 1 mM (c). γH2AX was assessed by immunoelectrophoresis in MCF-7 cells treated for 2 h with juglone (Jug), Q7, or Q9 at 20 μM with or without ascorbate (Asc) at 1 mM (d). (∗∗) and (∗∗∗) denote statistical differences at P < 0.01 and P < 0.001 compared to indicated treatments.
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fig1: The fluorescence of CT-DNA stained with ethidium bromide is reduced by doxorubicin and juglone, Q7, or Q9, mainly in presence of ascorbate (Asc) (a). Absorbance of thiobarbituric reactive species (TBARS) in CT-DNA treated with juglone, Q7, or Q9 alone and combined with ascorbate. Negative control (NC): phosphate buffer. Positive control (PC): [Fe(EDTA)]2−/H2O2 (b). DNA damage index was determined by the comet assay in MCF-7 cells treated for 24 h with juglone 75 μM, Q7 50 μM, or Q9 50 μM with or without ascorbate 1 mM (c). γH2AX was assessed by immunoelectrophoresis in MCF-7 cells treated for 2 h with juglone (Jug), Q7, or Q9 at 20 μM with or without ascorbate (Asc) at 1 mM (d). (∗∗) and (∗∗∗) denote statistical differences at P < 0.01 and P < 0.001 compared to indicated treatments.

Mentions: Intercalation of juglone, Q7, and Q9 into CT-DNA was examined by using the fluorescent intercalating agent ethidium bromide. Compounds that are able to intercalate into DNA compete with ethidium bromide and reduce its fluorescence when read in a fluorimeter. As show in Figure 1(a), when DNA and ethidium bromide were incubated with juglone, Q7, or Q9, the fluorescence was reduced, indicating that the compounds can intercalate into CT-DNA. The intercalating capacity of juglone, Q7, and Q9 was always higher when the incubations were performed in the presence of ascorbate. As expected, doxorubicin, which is a known intercalating agent, also reduced the fluorescence signal [27].


DNA damage and inhibition of akt pathway in mcf-7 cells and ehrlich tumor in mice treated with 1,4-naphthoquinones in combination with ascorbate.

Ourique F, Kviecinski MR, Felipe KB, Correia JF, Farias MS, Castro LS, Grinevicius VM, Valderrama J, Rios D, Benites J, Calderon PB, Pedrosa RC - Oxid Med Cell Longev (2015)

The fluorescence of CT-DNA stained with ethidium bromide is reduced by doxorubicin and juglone, Q7, or Q9, mainly in presence of ascorbate (Asc) (a). Absorbance of thiobarbituric reactive species (TBARS) in CT-DNA treated with juglone, Q7, or Q9 alone and combined with ascorbate. Negative control (NC): phosphate buffer. Positive control (PC): [Fe(EDTA)]2−/H2O2 (b). DNA damage index was determined by the comet assay in MCF-7 cells treated for 24 h with juglone 75 μM, Q7 50 μM, or Q9 50 μM with or without ascorbate 1 mM (c). γH2AX was assessed by immunoelectrophoresis in MCF-7 cells treated for 2 h with juglone (Jug), Q7, or Q9 at 20 μM with or without ascorbate (Asc) at 1 mM (d). (∗∗) and (∗∗∗) denote statistical differences at P < 0.01 and P < 0.001 compared to indicated treatments.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4352476&req=5

fig1: The fluorescence of CT-DNA stained with ethidium bromide is reduced by doxorubicin and juglone, Q7, or Q9, mainly in presence of ascorbate (Asc) (a). Absorbance of thiobarbituric reactive species (TBARS) in CT-DNA treated with juglone, Q7, or Q9 alone and combined with ascorbate. Negative control (NC): phosphate buffer. Positive control (PC): [Fe(EDTA)]2−/H2O2 (b). DNA damage index was determined by the comet assay in MCF-7 cells treated for 24 h with juglone 75 μM, Q7 50 μM, or Q9 50 μM with or without ascorbate 1 mM (c). γH2AX was assessed by immunoelectrophoresis in MCF-7 cells treated for 2 h with juglone (Jug), Q7, or Q9 at 20 μM with or without ascorbate (Asc) at 1 mM (d). (∗∗) and (∗∗∗) denote statistical differences at P < 0.01 and P < 0.001 compared to indicated treatments.
Mentions: Intercalation of juglone, Q7, and Q9 into CT-DNA was examined by using the fluorescent intercalating agent ethidium bromide. Compounds that are able to intercalate into DNA compete with ethidium bromide and reduce its fluorescence when read in a fluorimeter. As show in Figure 1(a), when DNA and ethidium bromide were incubated with juglone, Q7, or Q9, the fluorescence was reduced, indicating that the compounds can intercalate into CT-DNA. The intercalating capacity of juglone, Q7, and Q9 was always higher when the incubations were performed in the presence of ascorbate. As expected, doxorubicin, which is a known intercalating agent, also reduced the fluorescence signal [27].

Bottom Line: Juglone, phenylaminonaphthoquinone-7, and 9 (Q7/Q9) were evaluated for effects on CT-DNA and DNA of cancer cells.Treatments caused DNA damage and reduced viability and proliferation of MCF-7 cells.Juglone and Q7 plus ascorbate caused enhanced ROS and DNA damage and inhibited pAkt also in Ehrlich carcinoma cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Bioquímica Experimental, Departamento de Bioquímica, Universidade Federal de Santa Catarina, Florianópolis, Brazil.

ABSTRACT
The aim of this study was to enhance the understanding of the antitumor mechanism of 1,4-naphthoquinones and ascorbate. Juglone, phenylaminonaphthoquinone-7, and 9 (Q7/Q9) were evaluated for effects on CT-DNA and DNA of cancer cells. Evaluations in MCF-7 cells are DNA damage, ROS levels, viability, and proliferation. Proteins from MCF-7 lysates were immunoblotted for verifying PARP integrity, γH2AX, and pAkt. Antitumor activity was measured in Ehrlich ascites carcinoma-bearing mice. The same markers of molecular toxicity were assessed in vivo. The naphthoquinones intercalate into CT-DNA and caused oxidative cleavage, which is increased in the presence of ascorbate. Treatments caused DNA damage and reduced viability and proliferation of MCF-7 cells. Effects were potentiated by ascorbate. No PARP cleavage was observed. Naphthoquinones, combined with ascorbate, caused phosphorylation of H2AX and inhibited pAkt. ROS were enhanced in MCF-7 cells, particularly by the juglone and Q7 plus ascorbate. Ehrlich carcinoma was inhibited by juglone, Q7, or Q9, but the potentiating effect of ascorbate was reproduced in vivo only in the cases of juglone and Q7, which caused up to 60% inhibition of tumor and the largest extension of survival. Juglone and Q7 plus ascorbate caused enhanced ROS and DNA damage and inhibited pAkt also in Ehrlich carcinoma cells.

Show MeSH
Related in: MedlinePlus