Epigallocatechin-3-gallate affects the growth of LNCaP cells via membrane fluidity and distribution of cellular zinc

Abstract

Objective: To evaluate effects of epigallocatechin-3-gallate (EGCG) on the viability, membrane properties, and zinc distribution, with and without the presence of Zn(2+), in human prostate carcinoma LNCaP cells.

Methods: We examined changes in cellular morphology and membrane fluidity of LNCaP cells, distribution of cellular zinc, and the incorporated portion of EGCG after treatments with EGCG, Zn(2+), and EGCG+Zn(2+).

Results: We observed an alteration in cellular morphology and a decrease in membrane fluidity of LNCaP cells after treatment with EGCG or Zn(2+). The proportion of EGCG incorporated into liposomes treated with the mixture of EGCG and Zn(2+) at the ratio of 1:1 was 90.57%, which was significantly higher than that treated with EGCG alone (30.33%). Electron spin resonance (ESR) studies and determination of fatty acids showed that the effects of EGCG on the membrane fluidity of LNCaP were decreased by Zn(2+). EGCG accelerated the accumulation of zinc in the mitochondria and cytosol as observed by atomic absorption spectrometer.

Conclusion: These results show that EGCG interacted with cell membrane, decreased the membrane fluidity of LNCaP cells, and accelerated zinc accumulation in the mitochondria and cytosol, which could be the mechanism by which EGCG inhibits proliferation of LNCaP cells. In addition, high concentrations of Zn(2+) could attenuate the actions elicited by EGCG.

Fig. 1

Cytotoxicity of EGCG and Zn²⁺ on LNCaP cells by MTT assay (n=7) (a) Treatments with different concentrations of EGCG only; (b) Treatments with different concentrations of Zn²⁺ only

Fig. 1

Cytotoxicity of EGCG and Zn²⁺ on LNCaP cells by MTT assay (n=7) (a) Treatments with different concentrations of EGCG only; (b) Treatments with different concentrations of Zn²⁺ only

Fig. 2

Cytotoxicity of the interactions of EGCG with Zn²⁺ on LNCaP cells by MTT assay (n=7) (a) After different concentrations of Zn²⁺ were added, LNCaP cells were incubated for 2 h at 37 °C, and then mixed with 80 μmol/L EGCG for 24 h; (b) After 80 μmol/L EGCG was added, LNCaP cells were incubated for 2 h at 37 °C, then mixed with different concentrations of Zn²⁺ for 24 h; (c) After 80 μmol/L EGCG and different concentrations of Zn²⁺ were added at the same time, LNCaP cells were incubated for 24 h at 37 °C

Fig. 3

Microscopic images of LNCaP cells after exposure for 24 h (a) Normal LNCaP cells; (b) LNCaP cells treated with 80 μmol/L EGCG; (c) LNCaP cells treated with 80 μmol/L Zn²⁺; (d) LNCaP cells treated with the mixture of 80 μmol/L EGCG and 80 μmol/L Zn²⁺

Fig. 3

Microscopic images of LNCaP cells after exposure for 24 h (a) Normal LNCaP cells; (b) LNCaP cells treated with 80 μmol/L EGCG; (c) LNCaP cells treated with 80 μmol/L Zn²⁺; (d) LNCaP cells treated with the mixture of 80 μmol/L EGCG and 80 μmol/L Zn²⁺

Fig. 3

Microscopic images of LNCaP cells after exposure for 24 h (a) Normal LNCaP cells; (b) LNCaP cells treated with 80 μmol/L EGCG; (c) LNCaP cells treated with 80 μmol/L Zn²⁺; (d) LNCaP cells treated with the mixture of 80 μmol/L EGCG and 80 μmol/L Zn²⁺

Fig. 3

Microscopic images of LNCaP cells after exposure for 24 h (a) Normal LNCaP cells; (b) LNCaP cells treated with 80 μmol/L EGCG; (c) LNCaP cells treated with 80 μmol/L Zn²⁺; (d) LNCaP cells treated with the mixture of 80 μmol/L EGCG and 80 μmol/L Zn²⁺

Fig. 4

Typical ESR spectra of 5-SASL (a) and 16-SASL (b) in the membranes of LNCaP cells at 25 °C The spectra were recorded at 9.852 GHz at 25 °C, microwave power of 20 mW, modulation amplitude of 4.0 G, center field strength of 3509 G, and scan range of 60 G. The order parameters (S) were calculated from the ESR spectra by using the equation: S=0.5407(T _//−T _⊥)/a ₀, where a ₀=(T _//+2T _⊥)/3. The rotation correlation time (t) was calculated from the ESR spectra by using the equation: t=6.51×10⁻¹⁰ΔH ₀[(h ₀/h ₋₁)−(h ₀/h ₊₁)]S, where ΔH ₀ was the center line width. T _//, T _⊥, h ₀, h ₋₁, and h ₊₁ were measured from the ESR spectra as indicated in the figure

Fig. 4

Typical ESR spectra of 5-SASL (a) and 16-SASL (b) in the membranes of LNCaP cells at 25 °C The spectra were recorded at 9.852 GHz at 25 °C, microwave power of 20 mW, modulation amplitude of 4.0 G, center field strength of 3509 G, and scan range of 60 G. The order parameters (S) were calculated from the ESR spectra by using the equation: S=0.5407(T _//−T _⊥)/a ₀, where a ₀=(T _//+2T _⊥)/3. The rotation correlation time (t) was calculated from the ESR spectra by using the equation: t=6.51×10⁻¹⁰ΔH ₀[(h ₀/h ₋₁)−(h ₀/h ₊₁)]S, where ΔH ₀ was the center line width. T _//, T _⊥, h ₀, h ₋₁, and h ₊₁ were measured from the ESR spectra as indicated in the figure

Fig. 5

Effects of EGCG and Zn²⁺ on the amount of EGCG incorporated into the liposomes (a) Different concentrations of EGCG were added into the liposomal solution; (b) 200 μmol/L EGCG and different concentrations of Zn²⁺ (0, 100, 200, 400 μmol/L) were added into the liposomal solution. Then the liposomal solution was incubated at 20 °C for 20 min, centrifuged and determined by HPLC. The ratio (%) was calculated by dividing the amount of EGCG incorporated by the amount of EGCG added. Data were expressed as mean±SE of at least three times. Significant difference from the minimum concentration: ^* P<0.05, ^** P<0.01

Fig. 5

Effects of EGCG and Zn²⁺ on the amount of EGCG incorporated into the liposomes (a) Different concentrations of EGCG were added into the liposomal solution; (b) 200 μmol/L EGCG and different concentrations of Zn²⁺ (0, 100, 200, 400 μmol/L) were added into the liposomal solution. Then the liposomal solution was incubated at 20 °C for 20 min, centrifuged and determined by HPLC. The ratio (%) was calculated by dividing the amount of EGCG incorporated by the amount of EGCG added. Data were expressed as mean±SE of at least three times. Significant difference from the minimum concentration: ^* P<0.05, ^** P<0.01

Fig. 6

Content of Zn²⁺ in the subcellular organelles of LNCaP cells The subcellular organelles were isolated by a refrigerated centrifuge and measured by electrothermal atomic absorption spectrometry. Cells were treated with 80 μmol/L EGCG for 24 h and cells without treatment were considered as control. Data were expressed as mean±SE of at least three times. Significant difference from minimum concentration: ^* P<0.05, ^** P<0.01