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.

(À)-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.5,7dimethoxyflavone (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.
Cyclic nucleotide phosphodiesterases (PDEs) play a pivotal role in cGMP-or/and cAMP-mediated cell signaling and are well known as inactivators of cGMP or/and cAMP in mammalian tissues.PDE5 is one of the cGMP-specific enzymes that degrade intracellular messenger cGMP production [18][19][20][21].Recently, it has been shown that inhibition of PDE enzyme activity might be a new therapeutic approach for various pathologies [18][19][20].PDE5 inhibitors have shown positive effects for numerous pathologies, including erectile dysfunction [22].Moreover, inhibition of PDE5 has shown to enhance cGMP-dependent cancer apoptosis pathway in several cancer cells [6,7].
In this study, we showed that 5,7-dimethoxyflavone synergistically potentiated the anti-cancer effect of EGCG, accompanied by the enhancement of EGCGinduced cGMP upregulation in multiple myeloma cells.
Analyzed apoptotic cell death 2 9 10 4 cellsÁmL À1 of U266 cells were cultured in 1% FBS-RPMI 1640 at 100% humidity and 5% CO 2 , at 37 °C.Apoptotic cells were determined using a flow cytometric test.Annexin-V + cells were evaluated by combining early Annexin-V + propidium iodide-and late Annexin-V + propidium iodide + cells after treatment with EGCG or/and 5,7-DMF.The analysis was performed after 96 h using a Verse TM system from BD (Bergen county, NJ, USA).

Quantitative reverse transcription PCR (qRT-PCR)
Applied Biosystems with 40 cycles of 15 s at 95 °C, 60 s at 60 °C according to the manufacturer's instructions.The data were analyzed using v2.1 STEPONE software from Applied Biosystems.

cGMP assays
Measurements of intracellular cGMP production were carried out using the TRFRET cGMP assay kit (Perkin-Elmer) following the manufacturer's protocol.The cells were treated with EGCG and/or 5,7-DMF for 3 h in 96-well plates, and the plate assessment was performed using the EnVision TM Plate Reader from Perkin-Elmer (Waltham, MA, USA).

ASM activity measurement
U266 cells were lysed with lysis buffer and incubated for 1 h at 4 °C, followed by centrifugation for 20 min at 15 000 g.The resulting supernatant was incubated with substrate buffer, including 200 mM sodium acetate, 1% Triton X-100, and 400 pmol BODIPY-C12 sphingomyelin in dH 2 O, for 18 h at 37 °C.BODIPY-C12-sphingomyelin was obtained from Sigma-Aldrich.

Statistical analysis
Our data are indicated as mean AE SEM.Calculation the IC 50 values and isobologram methods were determined by using the GRAPHPAD software (San Diego, CA, USA).Tukey's test was used to assess the significance of differences between experimental variables.Statistical analyses were performed using KYPLOT software (Kyens Lab, Tokyo, Japan).

5,7-DMF synergistically potentiates cell death effect of EGCG in U266 cells
We showed based on the chemical structure of EGCG and 5,7-DMF (Fig. 1A,B).EGCG selectively induced apoptosis in cancer cells by targeting the 67LR [8,9,27].However, the physiological concentration of EGCG is limited in this effect [28].We assessed the anti-multiple myeloma effect of the combination of 5,7-DMF and EGCG in U266 cells.We showed 5 or 10 lM 5,7-DMF significantly potentiated the multiple myeloma killing activity of EGCG, with 50% inhibitory concentrations (IC 50 ) values of 10.26 or 5.228 lM, while the IC 50 values for EGCG were 16.47 lM, and for 5,7-DMF were 49.15 lM in U266 cells (Fig. 1CÀF).Isobologram analysis is a well-known analysis for evaluating synergy effects based on the dose-response manner of each drug.A straight line was observed between the y-axis (representing 5,7-DMF at 49.15 lM) and x-axis (representing EGCG at 16.47 lM), respectively, on plotting the IC50 values on each axis.The isobologram plot of cell viability curves showed that the combination of EGCG and 5,7-DMF was not just additive but had a synergistic effect in U266 cells (Fig. 1G).
Combination of EGCG and 5,7-DMF induces apoptotic cell death EGCG has been shown to induce apoptosis in multiple myeloma cells [6,9].To investigate whether the combination of EGCG and 5,7-DMF induces apoptosis, we treated U266 cells with EGCG and/or 5,7-DMF.Our results demonstrate that 10 lM 5,7-DMF significantly potentiated the apoptosis-inducing effect of EGCG, whereas treatment with 5 lM EGCG or 10 lM 5,7-DMF alone did not induce apoptosis in U266 cells (Fig. 2A).Additionally, we found that the level of cleaved caspase-3, a key mediator of apoptosis, increased significantly in U266 cells treated with the combination of EGCG and 5,7-DMF (Fig. 2B).Taken together, these results suggested that the 5,7-DMF potentiates apoptosis inducing effect of EGCG in U266 cells.

5,7-DMF potentiates anti-cancer effect of cGMP inducer bay 41-2272
To estimate whether intracellular cGMP production is sufficient to induce cell death in multiple myeloma, we assessed the effect of 5,7-DMF and/or Bay 41-2272 in U266 cells (Fig. 3).We found that 5,7-DMF did not induce cell death at concentrations ranging from 0 to 25 lM (Fig. 3A).Bay 41-2272 induced cell death in a dose-dependent manner in U266 cells, with IC50 values for Bay 41-2272 of 7.301 lM (Fig. 3B).Moreover, we demonstrated that 5,7-DMF potentiated Bay 41-2272induced cell death, with IC50 values of 1.376 lM (Fig. 3C).Isobologram analysis of cell viability curves showed that the combination effect of EGCG and Bay 41-2272 was not just additive but was synergistic (Fig. 3D).These results suggested that 5,7-DMF enhanced cGMP-inducer induced cell death in U266 cells.

5,7-DMF potentiates EGCG-induced apoptotic cell death through activation of cGMP pathway
To evaluate the effect of 5,7-DMF on the upstream signal of cGMP, we determined its effect on 5,7-DMF on EGCG-derived eNOS phosphorylation.We observed that EGCG induced eNOS phosphorylation at Ser1177, but 5,7-DMF did not affect it (Fig. 4A).We showed that 10 lM EGCG induced cGMP production in U266 cells, while 0-5 lM EGCG did not have a significant effect (Fig. 4B).Moreover, our data showed that 5,7-DMF potentiated the EGCG-induced cGMP production in U266 cells (Fig. 4C).These finding suggested that 5,7-DMF may reduce the cGMP-PDE enzyme activity in U266 cells.We showed 5,7-DMF synergically potentiated the ASM activity of EGCG (Fig. 4D).To determine the effect of 5,7-DMF on cGMP-dependent signaling in multiple myeloma cell death, we assessed its effect on the BAY 41-2272induced activation of ASM, which is known to be downstream of cGMP.5,7-DMF potentiated BAY 41-2272-elicited activation of ASM in U266 cells (Fig. 4E).We showed a schematic representation indicating that 5,7-DMF potentiates the EGCG-induced apoptotic cell death in human multiple myeloma U266 cells (Fig. 4F).
Taken together, these results suggested that 5,7-DMF potentiates EGCG-induced cell death in multiple myeloma cells, accompanied by an enhancement of cGMP-dependent multiple myeloma cell death signaling in U266 cells.

5,7-DMF enhanced anti-cancer effect of EGCG through 67LR without affecting expression levels of 67LR
Because anti-67LR antibody treatment is widely used to assess the role of 67LR in EGCG-induced cell death [6], we assess the role of 67LR in the anti-cancer effect of the combination by using anti-67LR antibody.Our result showed that anti-67LR antibody pretreatment significantly attenuated the effect of the combination (Fig. 5A).Those results showed that 67LR is involved in the anti-cancer effect of the combination.Moreover, we assessed the effect of EGCG/5,7-DMF on the expression level of 67LR.Our result showed that the EGCG and/or 5,7-DMF did not affect the 67LR expression levels (mRNA/protein; Fig. 5B,C).Taken together, these results suggested that the combination of EGCG and 5,7-DMF induced cell death through 67LR without affecting expression levels of 67LR.

Discussion
cGMP is a second messenger that involved in penile erection and vascular homeostasis.Clinical studies have shown that several direct or indirect intracellular cGMP producers are effective in treating many types of diseases, such as pulmonary hypertension, erectile dysfunction, and heart failure [19][20][21][22].Interestingly, EGCG-induced cGMP plays a key role in inducing cell death in several types of cancer, including multiple myeloma cells [6].However, the effect of EGCG at physiological concentrations is limited [28].Here we demonstrated that 5,7-DMF synergistically potentiates EGCG-induced apoptotic cell death accompanied with intracellular cGMP production at physiological concentrations of EGCG in U266 cells.
Previous study has reported the anti-cancer effects of 5,7-DMF, a component derived from black ginger (K.parviflora) [26].5,7-DMF from black ginger acts as an inhibitor of PDE5 activity, a specific negative regulator of cGMP [23,29].However, the concentrations of 5,7-DMF used in that study were relatively high, with an IC50 value of 25 lM.
We showed that a single treatment of 5 lM EGCG or 10 lM 5,7-DMF did not affect the cGMP level, while the combination of 5 lM EGCG and 10 lM 5,7-DMF upregulated intracellular cGMP production in multiple myeloma cells.These results suggest that 5,7-DMF potentiates EGCG-induced multiple myeloma cell death accompanied with enhancing the cGMP induction activity of EGCG.Nitric oxide (NO) is produced by NOS, followed by NO regulates sGC to induce intracellular cGMP levels [12][13][14][15].Previously, we assessed the role of NO/cGMP signaling activation in EGCG-induced apoptosis mechanisms in cancer cells [6,30].We also showed that EGCG increased NO production in cancer cells including multiple myeloma cells [6].EGCG also increased phosphorylation levels of eNOS at Ser1177, which upregulates its enzyme activity [6,30].We confirmed that EGCG induced eNOS phosphorylation at Ser1177, while a single treatment of 5,7-DMF did not appear to induce eNOS phosphorylation at Ser1177.These results suggest that 5,7-DMF potentiates EGCG-induced multiple myeloma cell death by enhancing the downstream effects of NO, but it does not affect the upstream effects of NO, such as phosphorylation of eNOS at Ser1177 in U266 cells.
Previously, we reported that activation of ASM is a major downstream mechanism of EGCG-induced cell death, including multiple myeloma cells [9].This ASM activation is involved in several mechanisms of cancer cell apoptosis induced by cisplatin, oxidative stress, and ultraviolet light [31,32].ASM activation is an indispensable mediator in cGMP-initiated apoptosis [6,9].Here, we showed that 5,7-DMF potentiated the multiple myeloma-killing activity of EGCG at physiological concentrations, accompanied with the activation of the cGMP/ASM/cleaved caspase-3 pathway.We suggested that the cGMP/ASM mechanisms may contribute to these effects.Taken together, we suggested that this mechanism and our using concentrations are worthy of particular consideration.Moreover, in combination of EGCG and 5,7-DMF have high potential and could be effective for chemotherapy for treating multiple myeloma.

Fig. 1 .
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 AE SEM (n = 3), Tukey's test, *P < 0.05, ***P < 0.001.