Hydrogen sulphide donors selectively potentiate a green tea polyphenol EGCG-induced apoptosis of multiple myeloma cells

Hydrogen sulphide (H2S) is a colourless gas with the odour of rotten eggs and has recently been recognized as a signal mediator in physiological activities related with the regulation of homeostasis, the vascular system and the inflammatory system. Here we show that H2S donors, including sodium hydrogen sulphide (NaHS), GYY 4137 and diallyltrisulfide (DATS), synergistically enhanced the anti-cancer effect of a green tea polyphenol (−)-epigallocatechin-3-O-gallate (EGCG) against multiple myeloma cells without affecting normal cells. NaHS significantly potentiated the anti-cancer effect of EGCG and prolonged survival in a mouse xenograft model. In this mechanism, H2S enhanced apoptotic cell death through cyclic guanosine monophosphate (cGMP)/acid sphingomyelinase pathway induced by EGCG. Moreover, NaHS reduced the enzyme activity of cyclic nucleotide phosphodiesterase that is known as cGMP negative regulator. In conclusion, we identified H2S as a gasotransmitter that potentiates EGCG-induced cancer cell death.

Currently, there is extensive interest in the health benefits of green tea. An epidemiological study demonstrated that green tea consumption was associated with a low risk of hematologic malignancies 1 . The major constituent of green tea, (−)-epigallocatechin-3-O-gallate (EGCG), has been shown to have cancer-preventive and therapeutic effects [2][3][4] . We recently identified a 67-kDa laminin receptor (67LR) as the sensing molecule of EGCG 3,4 . Interestingly, 67LR has been shown to be overexpressed in various types of cancers [4][5][6][7][8] . Indeed, EGCG selectively kills multiple myeloma (MM) cells by targeting cancer-overexpressed 67LR [4][5][6][7] . Moreover, EGCG induces apoptosis by upregulating cyclic guanosine monophosphate (cGMP) in cancer cells, including MM, acute myeloid leukaemia, pancreatic cancer and prostate cancer cells 4 . These results demonstrate the potent and specific anti-cancer activity of EGCG and provide the rationale for its clinical evaluation [7][8][9] . However, the plasma concentration of EGCG is not enough to eradicate cancer cells.
cGMP is well known as an intracellular second messenger that mediates a number of physiological processes including cardiovascular functions, neurotransmission and anti-cancer effect. cGMP is produced by soluble guanylate cyclase (sGC). sGC is the receptor for nitric oxide (NO) that is produced by nitric oxide synthase (NOS) 4, 10-15 . Cyclic nucleotide phosphodiesterases (PDEs) play a major role in cell signalling and are specifically negative regulators of cAMP and cGMP in mammalian tissues. cGMP-PDE is one of the enzymes of the PDE family and is a major inactivator of the intracellular messenger cGMP. Recently, it has been shown that specific PDE inhibitors might be new therapeutic approaches for numerous pathologies [16][17][18] . PDE5 is known to be a cGMP-specific enzyme that degrades cGMP 19 . PDE5 inhibitors have been shown to be effective for the treatment of erectile dysfunction 20 . Moreover, PDE5 inhibitor has been shown to increase cGMP-dependent apoptosis pathway in cancers 4, 5 . There are many recent studies on the discovery of new PDE5 inhibitors for the treatment of numerous pathologies.
Several studies have demonstrated that endogenous H 2 S acts as a gasotransmitter similar to NO and carbon monoxide in the human body 21 . In this study, we used several H 2 S donors, such as a NaHS, GYY 4137 and diallyltrisulfide (DATS). NaHS is an inorganic sulphide salt that has been widely used as H 2 S equivalents in many biological studies. NaHS rapidly releases H 2 S under laboratory conditions. On the other hand, GYY 4137 releases much lesser H 2 S and at a slower rate than NaHS but has shown sustained release of H 2 S in a culture medium. The release mechanism of H 2 S from NaHS and GYY 4127 are known to be by hydrolysis [22][23][24] . DATS is a garlic-derived sulphur compound that produces H 2 S. Release of H 2 S from sulphur compounds is facilitated by increasing the numbers of sulphur atoms and by allyl substituents. DATS, including trisulphides that undergo nucleophilic substitutions at the sulphur atom, then produces H 2 S. The mechanism of release of H 2 S from DATS is by thiol activation in which sulphur atoms react with cell membrane thiol 25 .
There are several recent studies on the anticancer effects of H 2 S donors [26][27][28] . H 2 S donors exhibit anti-cancer activities. NaHS is reported to have anti-cancer effects through the p38 Mitogen-activated protein kinase (MAPK) signalling pathway in C6 glioma cells 26 . GYY 4137 showed killing effects in seven human cancer cell lines and reduced tumour growth in a xenograft mouse model 22 . Garlic-derived H 2 S donor dialyl disulfide (DADS) enhanced the effect of eicosapentaenoic acid on cancer cell growth 29,30 . However, little is known about the anti-cancer mechanism of H 2 S. H 2 S acts as an inhibitor of PDE that boosts cyclic nucleotide and causes vasorelaxation in vivo 31 . The present study aimed to determine the impact of H 2 S on the effect of anti-cancer agents. Our study assessed the impact of H 2 S on the anti-cancer effects of EGCG. We used NaHS as an H 2 S donor to minimize off-target effects because the mechanisms of NaHS-derived H 2 S release are very simple with few by-product compared with those of other H 2 S donors such as GYY 4137 and DATS.
In this study, we showed that the H 2 S donor NaHS synergically potentiated the anti-MM effect of EGCG through cGMP-PDE inhibition. Furthermore, other H 2 S donors GYY 4137 and DATS also significantly amplified the EGCG-elicited cGMP-dependent apoptosis-inducing signalling pathway. Combination treatment with EGCG and NaHS showed not only extended the survival period but also inhibited tumour growth in a xenograft mouse model. Collectively, our results suggest that the combination of H 2 S donor and EGCG maybe a useful approach for cancer-specific chemotherapy.

Results
H 2 S donors potentiate EGCG-induced cancer-specific cell death. EGCG selectively kills cancer cells by targeting the overexpression of 67LR 2,7,8,32 . However, the killing activity of EGCG at physiological concentration is limited 33 . We assessed the anti-cancer effect of the combination of EGCG and an H 2 S donor NaHS in the three MM cell lines. NaHS pretreatment significantly potentiated the anti-MM effect of EGCG, with 50% inhibitory concentrations (IC 50 ) values of 7.6 µM (U266), 3.3 µM (ARH77) and 5.4 µM (MPC-11) in the cell lines, while the IC 50 values for EGCG were 21.3 µM (U266), 25.8 µM (ARH77) and 21.6 µM (MPC-11) in the cell lines ( Fig. 1a,b). H 2 S is a gasotransmitter that regulates blood pressure 34 and penile erection 35 . However, little is known about its anti-cancer effect. The IC 50 of NaHS was 88 µM (U266), 154.3 µM (ARH77) and 158 µM (MPC-11) in the cell lines ( Supplementary Fig. 1). The isobologram plot is a well-used method for evaluating synergy based on the dose-response relationship of individual drugs. A straight line was noted on plotting the IC 50 doses of EGCG and NaHS on the x-and y-axes, respectively. Isobologram analysis of growth-inhibition curves revealed that the combination of EGCG and NaHS was not simply additived but was synergistic in all the three cell lines (Fig. 1a,b). The same results were obtained for other H 2 S donors including GYY 4137 and DATS (Fig. 1c,d).
Selective toxicity is the most important factor in the anti-cancer effect. We found that all three H 2 S donors (NaHS, GYY 4137 and DATS) and EGCG showed a significant anti-MM effect without any negative effect to normal human peripheral blood mononuclear cells (PBMCs) (Fig. 1e). Collectively, the H 2 S donors synergistically potentiated the anti-MM effect of EGCG without any negative effect to normal cells.

Combination of EGCG and NaHS induces apoptosis in MM cells. EGCG induces apoptotic cell death
in MM cells 4,7 . To investigate whether the combination of EGCG and NaHS induces apoptosis in MM cells, cells were treated with a combination of EGCG and NaHS and were stained with Annexin V-Alexa Fluor 488. These combination significantly potentiated apoptosis induction in the MM cell lines U266, ARH77 (human MM) and MPC-11 (mouse myeloma) cells (Fig. 2a,b). Furthermore, we found that the level of cleaved caspase-3, a key mediator in apoptosis, significantly increased in EGCG/NaHS-treated human MM cells (Fig. 2c). Taken together, these findings suggest that the H 2 S donor NaHS potentiates apoptosis-inducing activity of EGCG in MM cells.
NaHS amplifies cGMP-dependent apoptosis signalling pathway. Previously, we reported that EGCG initiated apoptosis by activating the cGMP/Protein Kinase C delta (PKCδ)/Acid sphingomyelinase (ASM) axis in MM cells 4,32,36 . To examine the role of the cGMP-dependent cell death pathway in EGCG and NaHS combination-induced cell death, we evaluated the involvement of ASM, a downstream mediator of cGMP-elicited cell death, in MM cells. The combination of EGCG and NaHS upregulated ASM activity in MM cell lines U266, ARH77 and MPC-11 cells (Fig. 3a). In addition, the ASM inhibitor desipramine (Des) abrogated the anti-MM effect of the combination treatment of EGCG and NaHS in U266, ARH77 and MPC-11 cells (Fig. 3b). To determine the effect of NaHS on the upstream signal of cGMP, we evaluated the effect of NaHS on EGCG-elicited phosphorylation of endothelial NOS (eNOS) at Ser1177. EGCG elicited phosphorylation of eNOS at Ser1177, but NaHS did not affect the EGCG-induced phosphorylation of eNOS at Ser1177 (Fig. 3c).
PDE is a well-established negative regulator of cGMP 17,18 . Importantly, we found that NaHS reduced the enzyme activity of cGMP-PDE in U266 cells (Fig. 3d), although it did not affect the protein level of PDE5A ( Supplementary Fig. 2).
Moreover, the combination of EGCG and NaHS increased cGMP levels in MM cells (Supplementary Fig. 3). H 2 S can be endogenously produced in mammalian cells. We assessed the effect of EGCG on H 2 S production and cystathionine γ-lyase (CSE) expression, the major enzyme involved in H 2 S production 37 . EGCG did not affect both H 2 S production and CSE expression in MM cells ( Supplementary Figs 4 and 5).
NaHS synergically potentiated the anti-MM effect of the NO-independent cGMP inducer Bay 41-2272 (Fig. 3e,f). To determine the effect of an H 2 S donor on cGMP-dependent cell death signalling, we assessed the effect of NaHS on Bay 41-2272-induced ASM activation. NaHS potentiated Bay 41-2272-elicited ASM activation in all three MM cell lines (Fig. 3g).
Taken together, these results suggest that NaHS potentiated EGCG-induced cell death through enhancement of cGMP-dependent cell death signalling in MM cells.

NaHS potentiates the anti-MM effect of EGCG in vivo.
To evaluate the anti-cancer activity of the combination of EGCG and NaHS in a xenograft mouse model, MPC-11 cells were injected subcutaneously into 5-week-old BALB/c female mice. After the appearance of palpable tumours, the mice were administered intraperitoneal (i.p.) injections of EGCG (15 mg/kg/day) and/or NaHS (10 mg/kg/day) every 2 days. The combination of EGCG and NaHS resulted in significant suppression of tumour growth in the mouse model, while EGCG or NaHS alone did not show any effect on tumour growth (Fig. 4a,b). Moreover, log-rank analyses of the Kaplan-Meier survival curves showed significantly better survival among mice treated with the combination of EGCG and NaHS than among mice treated with PBS (control group), EGCG alone, or NaHS alone (Fig. 4c).
We evaluated the serum ALT/AST levels during NaHS treatment. BALB/c mice were administered intraperitoneal injections of NaHS (2.5 mg/kg/day, 5 mg/kg/day and 10 mg/kg/day) or PBS every 2 days. NaHS did not increase the serum ALT/AST levels when compared with the levels in the control group (Fig. 4d). In addition, there were no significant changes in mice tissue weights (heart, kidney, liver, spleen and lung) and mice body  Supplementary Information  (Supplementary Fig. 7). Data are presented as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001.
EGCG and NaHS amplify the tumour apoptosis in mouse xenograft model. We confirmed the effect of the combination of EGCG and NaHS in a tumour-bearing model based on immunofluorescence analysis. Neither EGCG (15 mg/kg/day) nor NaHS (10 mg/kg/day) caused significant induction of cleaved caspase-3 levels in tumour tissues compared with that in control group. However, the combination of EGCG and NaHS (i.p.) increased cleaved caspase-3 levels (Fig. 5a). Moreover, the combination of EGCG and NaHS significantly upregulated the ASM activity (Fig. 5b). On the other hand, EGCG elicited phosphorylation of eNOS at Ser1177, which is an upstream signal of cGMP, but NaHS did not affect the phosphorylation of eNOS at Ser1177 (Fig. 5c). We showed schematic representation indicating that H 2 S donor potentiates EGCG-induced cell death pathway (Fig. 5d).

Discussion
The gasotransmitter NO is produced by eNOS and regulates sGC for cGMP production [10][11][12][13] . The cGMP axis plays a key role in MM-specific cell death and mediates the MM-killing activity of EGCG 4 . However, the activity of EGCG at physiological concentrations is limited in MM cells 33 .
Here we showed that H 2 S donors, including NaHS, GYY 4137 and DATS, potentiate EGCG-induced cell death in MM cells. Moreover, the combination of EGCG and H 2 S donor extended the survival time and reduced the tumour volume in a xenograft mouse model.
Several studies have reported the growth-suppressing and apoptosis-inducing effects of H 2 S or H 2 S donors through the Bax, Blc-X and p38 MAPK signalling pathways [26][27][28][29] . In our study, three different H 2 S inducers strongly potentiated EGCG-induced cell death at very low concentrations (H 2 S donor 10 μM in vitro; NaHS, 10 mg/kg body weight in vivo) than those that are enough to induce anti-cancer effects (NaHS, approximately 200-1000 μM in vitro; GYY4131, 100-300 mg/kg/day). In this study, we observed that the H 2 S donor NaHS suppressed the enzyme activity of cGMP-PDE, the negative regulator of cGMP. Moreover, NaHS potentiated EGCG-induced cGMP production level.
Activation of the ASM axis is an essential mediator in cGMP-initiated apoptosis 4,7 . Our data also showed that the ASM activity and cleaved caspase-3 levels increased in tumour tissues of mice treated with combination of EGCG and NaHS compared with those in control group. In contrast, compared with control group, EGCG or NaHS alone did not affect both ASM activity and cleaved caspase-3 levels. Furthermore, we confirmed that the H 2 S donor potentiated ASM activation induced by Bay 41-2272, a NO-independent sGC activator. The EGCG-induced eNOS/cGMP/ASM/caspase-3 axis plays an important role in MM cell death 4 . Single NaHS treatment does not appear to induce phosphorylation of eNOS at Ser1177 in vitro and in vivo. We also confirmed that NaHS did not have an impact on EGCG-elicited phosphorylation of eNOS at Ser1177. These results suggest that H 2 S donor potentiates the anti-MM effect of EGCG by enhancing the downstream of cGMP but not affecting eNOS phosphorylation at Ser1177. Moreover, H 2 S is known as an inhibitor of PDE5 activity 31 . Furthermore, EGCG did not affect H 2 S production in MM cells. These results suggest that intracellular H 2 S produced by NaHS potentiates EGCG-induced anti-MM effect via inhibition of cGMP-PDE enzyme activity.
We also showed that H 2 S donors dramatically potentiated the anti-cancer effects of EGCG at a physiological concentration without affecting normal PBMCs. With regard to efficient chemotherapy, novel therapeutic drugs with different mechanisms and highly selective toxicity are required. Hepatotoxicity is a well-known adverse effect of high-dose EGCG 38 and, in some cases, elevation of the transaminases ALT and AST has been observed in clinical trials of EGCG 39 . Importantly, the combination of EGCG and NaHS did not increase the serum ALT/AST levels, suggesting that an H 2 S donor could be a potent candidate to enhance the pharmacological effect of EGCG without enhancing its adverse effects.
In conclusion, H 2 S donor potentiates the anti-MM effect of EGCG at a physiological concentration. The combination of EGCG and an H 2 S donor could provide a novel strategy for MM treatment without affecting normal PBMCs. Moreover, our data suggest that H 2 S may potentiate the downstream mediators of EGCG-induced apoptosis along with the inhibition of cGMP-PDE enzyme activity. PDE5 overexpressed in many types of cancer cells, such as a chronic lymphocytic leukaemia, acute myeloid leukaemia, stomach cancer, pancreatic cancer, prostate cancer, breast cancer and MM cells 4,5,32 . EGCG and PDE5 inhibitor in combination also induce apoptotic cell death in these cancer cells 4,5,32 . Taken together, we suggest that H 2 S donor and EGCG in combination could be effective for chemotherapy and have high potential for use in various types of cancers including MM.
MM cells were seeded into 24-well plates at a seeding density of 5 × 10 4 cells/well and were then treated with drugs at various concentrations or indicated periods in RPMI 1640 medium supplemented with 1% FBS, 200 U/ ml catalase and 5 U/ml superoxide dismutase (SOD) (Sigma-Aldrich).