Vitamin A Enhances Antitumor Effect of a Green Tea Polyphenol on Melanoma by Upregulating the Polyphenol Sensing Molecule 67-kDa Laminin Receptor

BACKGROUND: Green tea consumption has been shown to have cancer preventive qualities. Among the constituents of green tea, (-)-Epigallocatechin-3-O-gallate (EGCG) is the most effective at inhibiting carcinogenesis. However, the concentrations of EGCG that are required to elicit the anticancer effects in a variety of cancer cell types are much higher than the peak plasma concentration that occurs after drinking an equivalent of 2-3 cups of green tea. To obtain the anticancer effects of EGCG when consumed at a reasonable concentration in daily life, we investigated the combination effect of EGCG and food ingredient that may enhance the anticancer activity of EGCG on subcutaneous tumor growth in C57BL/6N mice challenged with B16 melanoma cells. METHODOLOGY/PRINCIPAL FINDINGS: All-trans-retinoic acid (ATRA) enhanced the expression of the 67-kDa laminin receptor (67LR) and increased EGCG-induced cell growth inhibition in B16 melanoma cells. The cell growth inhibition seen with the combined EGCG and ATRA treatment was abolished by treatment with an anti-67LR antibody. In addition, the combined EGCG and ATRA treatment significantly suppressed the melanoma tumor growth in mice. Expression of 67LR in the tumor increased upon oral administration of ATRA or a combined treatment of EGCG and ATRA treatment. Furthermore, RNAi-mediated silencing of the retinoic acid receptor (RAR) alpha attenuated the ATRA-induced enhancement of 67LR expression in the melanoma cells. An RAR agonist enhanced the expression levels of 67LR and increased EGCG-induced cell growth inhibition. CONCLUSIONS/SIGNIFICANCE: Our findings provide a molecular basis for the combination effect seen with dietary components, and indicate that ATRA may be a beneficial food component for cancer prevention when combined with EGCG

RAR agonist induced EGCG-elicited cell growth inhibition through 67LR upregulation.

<p><b>A</b>) Cells were treated with or without 0.1 µM ATRA or 0.1 µM TTNPB in DMEM supplemented with 1% FCS for 48 h and were analyzed by Western blot analysis. Levels of 67LR expression were normalized to β-Actin. Band intensities were quantified using NIH Image J software. <b>B</b>) Anti-67 LR antibody conjugated with Alexa Fluor 488 was used at a dilution of 1∶100. Photographs were taken under Keyence BZ-8100 fluorescence microscope. <b>C</b>) Cells were treated with 0.1 µM ATRA or RARα agonist, 0.1 µM TTNPB in DMEM supplemented with 1% FCS for 48 h, then treated with 0.5 µM of EGCG for 48 h. Data shown are means ± S.D. for three samples. Data containing asterisk marks are significantly different from the values in control at **<i>p</i><0.01, ***<i>p</i><0.001. <b>D</b>) B16 cells were treated with 0.1 µM TTNPB in DMEM supplemented with 1% FCS for 48 h, then the cells were treated with either anti-67LR (MLuC5) or control antibody (mouse IgM) for 2 h, and the cells were added 0.5 µM of EGCG for 48 h. Cells proliferation was assessed by the WST-1 reagent. Cell number was measured as 430 nm absorbance and shown as relative of control. Data shown are means ± S.D. for three samples. Data containing asterisk marks are significantly different from the values in control at ***<i>p</i><0.001.</p

ATRA induces the EGCG activity through the enhancing of EGCG binding to 67LR.

<p><b>A</b>) EGCG binding to the surface of B16 cells treated with (red line) or without (black line) ATRA monitored by surface plasmon resonance. EGCG was injected at a concentration of 5 µM for the indicated time interval (+ EGCG). <b>B</b>) B16 cells treated with 0.1 µM ATRA in DMEM supplemented with 1% FCS for 48 h. Then, the cells were treated with either anti-67LR (MLuC5) or control antibody (mouse IgM) for 2 h and then the cells were added to 0.5 µM EGCG for 48 h. Cell proliferation was assessed by the WST-1 reagent. Cell number was measured as 430 nm absorbance and shown as relative of control. Data shown are means ± S.D. for three samples. Data containing asterisk marks are significantly different from the values in control at ***<i>p</i><0.001.</p

ATRA enhances the expression of 67LR and cell growth inhibitory activity of EGCG.

<p><b>A</b>) Structure of EGCG and ATRA. <b>B</b>) 67LR protein levels in B16 cells exposed to the indicated concentrations of ATRA for 48 h were analyzed by Western blot analysis. Levels of 67LR expression were detected with anti 67LR serum, and were normalized to β-Actin. Band intensities were quantified using NIH Image J software. <b>C</b>) Anti-67LR antibody conjugated with Alexa Fluor 488 (1 mg/ml) was used at a dilution of 1∶100. Photographs were taken under Keyence BZ-8001 fluorescence microscope. <b>D</b>) Cells were counted after treatment with or without 0.5 µM EGCG and/or 0.1 µM ATRA in DMEM supplemented with 1% FCS for 48 h and 96 h each. Cell proliferation was evaluated by counting the number of cells using a Counlter Counter. Data shown are means ± S.D. for three samples. Data containing asterisk marks are significantly different from the values in control at ***<i>p</i><0.001.</p

ATRA enhances the 67LR expression via RARα.

<p><b>A</b>) RARα knockdown in B16 cells stably transfected with RARα shRNA expression vector was confirmed by Western blot analysis. <b>B</b>) B16 cells stably transfected with the control shRNA or the RARα shRNA expression vector were treated with or without 0.1 µM ATRA in DMEM supplemented with 1% FCS for 48 h. Levels of 67LR expression were analyzed by Western blot analysis and were normalized to β-Actin. Band intensities were quantified using NIH Image J software.</p

Tumor growth and expression levels of 67LR after the treatment ATRA and EGCG <i>in vivo</i>.

<p>C57BL/6N mice were subcutaneosly inoculated with B16 cells. The administration of 0.1% EGCG, ATRA (10.5 mg/kg), and combination of EGCG and ATRA was started 1 day before the cell inoculation. <b>A</b>) We excised tumors from mice 22 days after cell inoculation and photographed them. <b>B</b>) Tumor weights are represented as the mean ± S.E. of five mice. Logarithmic transformation for them were analyzed using an one-way ANOVA coupled with a Dunnet test in which *<i>p</i><0.05 was the minimum requirement for a statistically significant difference from control group. <b>C</b>) Tumor volumes was measured in two dimensions and calculated as follows: lengh/2× width². Each data point represents the mean ± S.E. of tumor volumes from five animals and they are significantly different from control group at *<i>p</i><0.05 (Mann-Whitney <i>U</i> test). <b>D</b>) Representative Western blot analyses of 67LR from each individual mouse. Levels of these proteins expression were normalized to β-Actin. Band intensities were quantified using NIH Image J software.</p