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

Abstract

(-)-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.

Keywords: (−)-epigallocatechin-3-O-gallate; 5,7-dimethoxyflavone; acid sphingomyelinase; cyclic guanosine monophosphate; multiple myeloma.

Fig. 1

5,7‐DMF synergistically potentiates cell death effect of EGCG in U266 cells. (A, B) Based on chemical structure of EGCG and 5,7‐DMF. (C–F) The human multiple myeloma cell line U266 was cultured with or without 5,7‐DMF and/or EGCG at the indicated concentration for 96 h, and the viability of U266 was measured by trypan blue staining assay. (G) The combination effect of EGCG and 5,7‐DMF was measured by isobologram analysis in U266 cells. Data are presented as mean ± SEM (n = 3), Tukey's test, *P < 0.05, ***P < 0.001.

Fig. 2

Combination of EGCG and 5,7‐DMF induces apoptotic cell death. (A) Apoptotic cells were stained with Annexin V–Alexa Fluor 488 and PI in U266 cells. (B) Cells were treated with or without EGCG (5 μm) in the presence or absence of 5,7‐DMF (10 μm) for 96 h, and cleaved caspase‐3 levels were assessed using western blot analysis. Data are presented as mean ± SEM (n = 3), Tukey's test, ***P < 0.001.

Fig. 3

5,7‐DMF potentiates intracellular cGMP activator Bay 41‐2272‐induced apoptotic cell death. (A–C) U266 cells were cultured with/without 5,7‐DMF and/or Bay 41‐2272 at the indicated concentration for 96 h, and viability was measured by trypan blue staining assay. (D) The synergy effect of Bay 41‐2272 and 5,7‐DMF is measured by isobologram analysis. Data are presented as mean ± SEM (n = 3), Tukey's test, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4

5,7‐DMF potentiates EGCG‐induced apoptotic cell death through activation of cGMP pathway. (A) U266 cells were treated with 5 μm EGCG and/or 10 μm 5,7‐DMF for 3 h, and eNOS phosphorylation at Ser1177 was assessed using western blot analysis (n = 3). (B) Intracellular cGMP production induced by EGCG was evaluated in U266 cells (n = 4). Cells were treated with EGCG for 3 h, and intracellular cGMP production was determined. (C) Intracellular cGMP production was determined in cells treated with 5 μm EGCG or/and 10 μm 5,7‐DMF for 3 h (n = 4). (D) ASM activity was determined by TLC analysis in cells treated with 5 μm EGCG or/and 10 μm 5,7‐DMF (n = 3). (E) ASM activity was assessed using TLC analysis in cells treated with 1 μm Bay 41‐2272 or/and 10 μm 5,7‐DMF (n = 3). (F) The schematic diagram shows the signaling pathways of EGCG and 5,7‐DMF in multiple myeloma cells. Data are presented as mean ± SEM (n = 3). Tukey's test, **P < 0.01, ***P < 0.001.

Fig. 5

5,7‐DMF induced a cell death effect of EGCG through 67LR without affecting expression levels of 67LR. (A) U266 cells were treated with control IgM antibody or anti‐67LR antibody (MLuC5) for 2 h and then the cells were added to 5 μm EGCG and/or 10 μm 5,7‐DMF for 96 h. Cell viability was assessed by trypan blue staining assay (n = 3). (B) U266 cells were treated with 5 μm EGCG and/or 10 μm 5,7‐DMF for 3 h, and 67LR mRNA expression level was assessed using real‐time PCR (n = 3). (C) U266 cells were treated with 5 μm EGCG and/or 10 μm 5,7‐DMF for 3 h, and 67LR protein level was measured using western blot analysis (n = 3). Data are presented as mean ± SEM (n = 3), Tukey's test, **P < 0.01, ***P < 0.001.