Oxygen partial pressure modulates 67-kDa laminin receptor expression, leading to altered activity of the green tea polyphenol, EGCG

Abstract(−)-Epigallocatechin-3-O-gallate (EGCG) exhibits anti-tumor activity mediated via the 67-kDa laminin receptor (67LR). In this study, we found that 67LR protein levels are reduced by exposure to low O2 levels (5%), without affecting the expression of HIF-1α. We also found that EGCG-induced anti-cancer activity is abrogated under low O2 levels (5%) in various cancer cells. Notably, treatment with the proteasome inhibitor, prevented down-regulation of 67LR and restored sensitivity to EGCG under 5% O2. In summary, 67LR expression is highly sensitive to O2 partial pressure, and the activity of EGCG can be regulated in cancer cells by O2 partial pressure

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

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

67-kDa Laminin Receptor-Mediated Cellular Sensing System of Green Tea Polyphenol EGCG and Functional Food Pairing

The body is equipped with a “food factor-sensing system” that senses food factors, such as polyphenols, sulfur-containing compounds, and vitamins, taken into the body, and plays an essential role in manifesting their physiological effects. For example, (−)-epigallocatechin-3-O-gallate (EGCG), the representative catechin in green tea (Camellia sinensi L.), exerts various effects, including anti-cancer, anti-inflammatory, and anti-allergic effects, when sensed by the cell surficial protein 67-kDa laminin receptor (67LR). Here, we focus on three representative effects of EGCG and provide their specific signaling mechanisms, the 67LR-mediated EGCG-sensing systems. Various components present in foods, such as eriodictyol, hesperetin, sulfide, vitamin A, and fatty acids, have been found to act on the food factor-sensing system and affect the functionality of other foods/food factors, such as green tea extract, EGCG, or its O-methylated derivative at different experimental levels, i.e., in vitro, animal models, and/or clinical trials. These phenomena are observed by increasing or decreasing the activity or expression of EGCG-sensing-related molecules. Such functional interaction between food factors is called “functional food pairing”. In this review, we introduce examples of functional food pairings using EGCG

The anti‐cancer effect of epigallocatechin‐3‐O‐gallate against multiple myeloma cells is potentiated by 5,7‐dimethoxyflavone

(−)‐Epigallocatechin‐3‐O‐gallate (EGCG) is one of the major components of green tea polyphenol. Previous studies have shown that EGCG induces cancer‐specific cell death in vitro and in vivo without causing severe side effects. However, the anti‐cancer effect of EGCG alone is limited. 5,7‐dimethoxyflavone (5,7‐DMF), one of the principal functional components of black ginger (Kaempferia parviflora), also exerts anti‐cancer effects. Here, we show that 5,7‐DMF synergistically enhances the anti‐cancer effect of EGCG in multiple myeloma cells by potentiating EGCG‐induced intracellular cyclic guanosine monophosphate (cGMP) production. Moreover, the combination of EGCG and 5,7‐DMF induces apoptotic cell death in multiple myeloma cells, and this is accompanied by activation of the cGMP/acid sphingomyelinase (ASM)/cleaved caspase‐3 pathway. In conclusion, we have shown that 5,7‐DMF enhances the anti‐cancer effect of EGCG by upregulating cGMP in multiple myeloma cells

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