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Improving anticancer efficacy of (-)-epigallocatechin-3-gallate gold nanoparticles in murine B16F10 melanoma cells.

Chen CC, Hsieh DS, Huang KJ, Chan YL, Hong PD, Yeh MK, Wu CJ - Drug Des Devel Ther (2014)

Bottom Line: (-)-Epigallocatechin-3-gallate (EGCG), the major bioactive constituent in green tea, has been reported to effectively inhibit the formation and development of tumors.To maximize the effectiveness of EGCG, we attached it to nanogold particles (EGCG-pNG) in various ratios to examine in vitro cytotoxicity and in vivo anti-cancer activity.EGCG-pNG showed improved anti-cancer efficacy in B16F10 murine melanoma cells; the cytotoxic effect in the melanoma cells treated with EGCG-pNG was 4.91 times higher than those treated with EGCG.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan, Republic of China ; Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China.

ABSTRACT
(-)-Epigallocatechin-3-gallate (EGCG), the major bioactive constituent in green tea, has been reported to effectively inhibit the formation and development of tumors. To maximize the effectiveness of EGCG, we attached it to nanogold particles (EGCG-pNG) in various ratios to examine in vitro cytotoxicity and in vivo anti-cancer activity. EGCG-pNG showed improved anti-cancer efficacy in B16F10 murine melanoma cells; the cytotoxic effect in the melanoma cells treated with EGCG-pNG was 4.91 times higher than those treated with EGCG. The enhancement is achieved through mitochondrial pathway-mediated apoptosis as determined by annexin V assay, JC-10 staining, and caspase-3, -8, -9 activity assay. Moreover, EGCG-pNG was 1.66 times more potent than EGCG for inhibition of tumor growth in a murine melanoma model. In the hemolysis assay, the pNG surface conjugated with EGCG is most likely the key factor that contributes to the decreased release of hemoglobin from human red blood cells.

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Enhanced apoptosis induction of EGCG by pNG in B16F10 cells.Notes: (A) The fractions of annexin V-positive B16F10 cells were 3.59%±0.30%, 12.63%±0.61%, 20.93%±0.55%, and 25.7%±0.54%, after treatment with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® (Sigma-Aldrich; St Louis, MO, USA) 0.5 μM, respectively, at 24 hours; (B) the fractions of annexin V-positive and PI-negative B16F10 cells were 1.0% ± 0.17%, 3.2% ± 0.23%, 6.5% ± 0.23%, and 11.7% ± 0.41%, after treatment with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® 0.5 μM, respectively, at 24 hours; (C) cells treated with EGCG-pNG for 24 hours expressed more green fluorescence than those treated with EGCG (magnification ×200). Depolarized mitochondria are indicated by green fluorescence (JC-10 monomer), and polarized mitochondria are indicated by orange fluorescence (aggregated JC-10). Cell nuclei are indicated by blue fluorescence coupled with Hoechst 33342 staining. Scale bar, 10 μm; (D) cells treated with EGCG-pNG for 24 hours showed a significant increase of caspase-3, -8, and -9 activity compared with those treated with EGCG; (E) initiation of time-dependent apoptotic activation by EGCG-pNG treatment via the caspase pathway in B16F10 melanoma cells. Data shown are mean ± standard deviation for three samples. Data containing asterisks are significantly different from the control values at *P<0.05; **P<0.01; ***P<0.001.Abbreviations: PI, propidium iodide; EGCG, (–)-epigallocatechin-3-gallate; pNG, physical nanogold; P2.5, pNG 2.5 ppm; E50, EGCG 50 μM; E50-P2.5, EGCG-pNG 50 μM:2.5 ppm; ZVAD, Z-VAD-FMK, N-Benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone.
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f3-dddt-8-459: Enhanced apoptosis induction of EGCG by pNG in B16F10 cells.Notes: (A) The fractions of annexin V-positive B16F10 cells were 3.59%±0.30%, 12.63%±0.61%, 20.93%±0.55%, and 25.7%±0.54%, after treatment with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® (Sigma-Aldrich; St Louis, MO, USA) 0.5 μM, respectively, at 24 hours; (B) the fractions of annexin V-positive and PI-negative B16F10 cells were 1.0% ± 0.17%, 3.2% ± 0.23%, 6.5% ± 0.23%, and 11.7% ± 0.41%, after treatment with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® 0.5 μM, respectively, at 24 hours; (C) cells treated with EGCG-pNG for 24 hours expressed more green fluorescence than those treated with EGCG (magnification ×200). Depolarized mitochondria are indicated by green fluorescence (JC-10 monomer), and polarized mitochondria are indicated by orange fluorescence (aggregated JC-10). Cell nuclei are indicated by blue fluorescence coupled with Hoechst 33342 staining. Scale bar, 10 μm; (D) cells treated with EGCG-pNG for 24 hours showed a significant increase of caspase-3, -8, and -9 activity compared with those treated with EGCG; (E) initiation of time-dependent apoptotic activation by EGCG-pNG treatment via the caspase pathway in B16F10 melanoma cells. Data shown are mean ± standard deviation for three samples. Data containing asterisks are significantly different from the control values at *P<0.05; **P<0.01; ***P<0.001.Abbreviations: PI, propidium iodide; EGCG, (–)-epigallocatechin-3-gallate; pNG, physical nanogold; P2.5, pNG 2.5 ppm; E50, EGCG 50 μM; E50-P2.5, EGCG-pNG 50 μM:2.5 ppm; ZVAD, Z-VAD-FMK, N-Benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone.

Mentions: EGCG has been widely reported to induce apoptosis in various cancer cells.32–34 To further determine whether the enhanced cytotoxicity of EGCG-pNG in B16F10 cells also occurred via the same mechanism, B16F10 cells were first treated with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® 0.5 μM, respectively, for 24 hours. Annexin V and PI staining for apoptotic bodies were evaluated by flow cytometry analysis with 10,000 cells. The fractions of annexin V-positive B16F10 cells were 3.59% ± 0.30%, 12.63% ± 0.61%, 20.93% ± 0.55%, and 25.7% ± 0.54% after treatment with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® 0.5 μM, respectively, at 24 hours (Figure 3A). The fractions of annexin V-positive and PI-negative B16F10 cells were 1.0% ± 0.17%, 3.2% ± 0.23%, 6.5% ± 0.23%, and 11.7% ± 0.41% after treatment with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® 0.5 μM, respectively, at 24 hours (Figure 3B). The fraction of the annexin V-positive cells increased 1.7-fold for cells added with EGCG and 5.8-fold for cells added with pNG compared with that of cells added with EGCG-pNG (Figure 3A). The fraction of the annexin V-positive and PI-negative cells increased 2.03-fold for cells added with EGCG and 6.0-fold for cells added with pNG compared with that of cells added with EGCG-pNG (Figure 3B). It appears that EGCG-pNG has an enhanced effect on B16F10 apoptotic cell death. Taxol®-induced apoptosis, as the positive control, was observed through the increasing fraction of the annexin V-positive cells and the fraction of the annexin V-positive and PI-negative cells.


Improving anticancer efficacy of (-)-epigallocatechin-3-gallate gold nanoparticles in murine B16F10 melanoma cells.

Chen CC, Hsieh DS, Huang KJ, Chan YL, Hong PD, Yeh MK, Wu CJ - Drug Des Devel Ther (2014)

Enhanced apoptosis induction of EGCG by pNG in B16F10 cells.Notes: (A) The fractions of annexin V-positive B16F10 cells were 3.59%±0.30%, 12.63%±0.61%, 20.93%±0.55%, and 25.7%±0.54%, after treatment with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® (Sigma-Aldrich; St Louis, MO, USA) 0.5 μM, respectively, at 24 hours; (B) the fractions of annexin V-positive and PI-negative B16F10 cells were 1.0% ± 0.17%, 3.2% ± 0.23%, 6.5% ± 0.23%, and 11.7% ± 0.41%, after treatment with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® 0.5 μM, respectively, at 24 hours; (C) cells treated with EGCG-pNG for 24 hours expressed more green fluorescence than those treated with EGCG (magnification ×200). Depolarized mitochondria are indicated by green fluorescence (JC-10 monomer), and polarized mitochondria are indicated by orange fluorescence (aggregated JC-10). Cell nuclei are indicated by blue fluorescence coupled with Hoechst 33342 staining. Scale bar, 10 μm; (D) cells treated with EGCG-pNG for 24 hours showed a significant increase of caspase-3, -8, and -9 activity compared with those treated with EGCG; (E) initiation of time-dependent apoptotic activation by EGCG-pNG treatment via the caspase pathway in B16F10 melanoma cells. Data shown are mean ± standard deviation for three samples. Data containing asterisks are significantly different from the control values at *P<0.05; **P<0.01; ***P<0.001.Abbreviations: PI, propidium iodide; EGCG, (–)-epigallocatechin-3-gallate; pNG, physical nanogold; P2.5, pNG 2.5 ppm; E50, EGCG 50 μM; E50-P2.5, EGCG-pNG 50 μM:2.5 ppm; ZVAD, Z-VAD-FMK, N-Benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone.
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Show All Figures
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f3-dddt-8-459: Enhanced apoptosis induction of EGCG by pNG in B16F10 cells.Notes: (A) The fractions of annexin V-positive B16F10 cells were 3.59%±0.30%, 12.63%±0.61%, 20.93%±0.55%, and 25.7%±0.54%, after treatment with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® (Sigma-Aldrich; St Louis, MO, USA) 0.5 μM, respectively, at 24 hours; (B) the fractions of annexin V-positive and PI-negative B16F10 cells were 1.0% ± 0.17%, 3.2% ± 0.23%, 6.5% ± 0.23%, and 11.7% ± 0.41%, after treatment with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® 0.5 μM, respectively, at 24 hours; (C) cells treated with EGCG-pNG for 24 hours expressed more green fluorescence than those treated with EGCG (magnification ×200). Depolarized mitochondria are indicated by green fluorescence (JC-10 monomer), and polarized mitochondria are indicated by orange fluorescence (aggregated JC-10). Cell nuclei are indicated by blue fluorescence coupled with Hoechst 33342 staining. Scale bar, 10 μm; (D) cells treated with EGCG-pNG for 24 hours showed a significant increase of caspase-3, -8, and -9 activity compared with those treated with EGCG; (E) initiation of time-dependent apoptotic activation by EGCG-pNG treatment via the caspase pathway in B16F10 melanoma cells. Data shown are mean ± standard deviation for three samples. Data containing asterisks are significantly different from the control values at *P<0.05; **P<0.01; ***P<0.001.Abbreviations: PI, propidium iodide; EGCG, (–)-epigallocatechin-3-gallate; pNG, physical nanogold; P2.5, pNG 2.5 ppm; E50, EGCG 50 μM; E50-P2.5, EGCG-pNG 50 μM:2.5 ppm; ZVAD, Z-VAD-FMK, N-Benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone.
Mentions: EGCG has been widely reported to induce apoptosis in various cancer cells.32–34 To further determine whether the enhanced cytotoxicity of EGCG-pNG in B16F10 cells also occurred via the same mechanism, B16F10 cells were first treated with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® 0.5 μM, respectively, for 24 hours. Annexin V and PI staining for apoptotic bodies were evaluated by flow cytometry analysis with 10,000 cells. The fractions of annexin V-positive B16F10 cells were 3.59% ± 0.30%, 12.63% ± 0.61%, 20.93% ± 0.55%, and 25.7% ± 0.54% after treatment with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® 0.5 μM, respectively, at 24 hours (Figure 3A). The fractions of annexin V-positive and PI-negative B16F10 cells were 1.0% ± 0.17%, 3.2% ± 0.23%, 6.5% ± 0.23%, and 11.7% ± 0.41% after treatment with pNG 2.5 ppm, EGCG 50 μM, EGCG-pNG 50 μM:2.5 ppm, and Taxol® 0.5 μM, respectively, at 24 hours (Figure 3B). The fraction of the annexin V-positive cells increased 1.7-fold for cells added with EGCG and 5.8-fold for cells added with pNG compared with that of cells added with EGCG-pNG (Figure 3A). The fraction of the annexin V-positive and PI-negative cells increased 2.03-fold for cells added with EGCG and 6.0-fold for cells added with pNG compared with that of cells added with EGCG-pNG (Figure 3B). It appears that EGCG-pNG has an enhanced effect on B16F10 apoptotic cell death. Taxol®-induced apoptosis, as the positive control, was observed through the increasing fraction of the annexin V-positive cells and the fraction of the annexin V-positive and PI-negative cells.

Bottom Line: (-)-Epigallocatechin-3-gallate (EGCG), the major bioactive constituent in green tea, has been reported to effectively inhibit the formation and development of tumors.To maximize the effectiveness of EGCG, we attached it to nanogold particles (EGCG-pNG) in various ratios to examine in vitro cytotoxicity and in vivo anti-cancer activity.EGCG-pNG showed improved anti-cancer efficacy in B16F10 murine melanoma cells; the cytotoxic effect in the melanoma cells treated with EGCG-pNG was 4.91 times higher than those treated with EGCG.

View Article: PubMed Central - PubMed

Affiliation: Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan, Republic of China ; Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China.

ABSTRACT
(-)-Epigallocatechin-3-gallate (EGCG), the major bioactive constituent in green tea, has been reported to effectively inhibit the formation and development of tumors. To maximize the effectiveness of EGCG, we attached it to nanogold particles (EGCG-pNG) in various ratios to examine in vitro cytotoxicity and in vivo anti-cancer activity. EGCG-pNG showed improved anti-cancer efficacy in B16F10 murine melanoma cells; the cytotoxic effect in the melanoma cells treated with EGCG-pNG was 4.91 times higher than those treated with EGCG. The enhancement is achieved through mitochondrial pathway-mediated apoptosis as determined by annexin V assay, JC-10 staining, and caspase-3, -8, -9 activity assay. Moreover, EGCG-pNG was 1.66 times more potent than EGCG for inhibition of tumor growth in a murine melanoma model. In the hemolysis assay, the pNG surface conjugated with EGCG is most likely the key factor that contributes to the decreased release of hemoglobin from human red blood cells.

Show MeSH
Related in: MedlinePlus