CAPE is a caffeic acid phenethyl ester derivative which is a naturally occurring hydroxycinnamic acid found in propolis [16
]. It is also known to be highly stable in human plasma [42
]. In our earlier work, we have used tyrosinase as a primary molecular target to bioactivate CAPE to cytotoxic agents [1
]. We also showed that CAPE was metabolized by tyrosinase to CAPE-quinone, depleted GSH, and selectively toxic towards melanoma cells in vitro
and in vivo
In the current work, we have used GST as a second molecular target in melanoma as GST plays an important role in multidrug resistance [9
] due to the over expression in melanoma [2
]. GST is one of the essential targets in the current development of cancer therapy because of resistance to various anticancer agents [13
]. GST also has a vital role in glutathionylation of cellular proteins in cancer, which considered as an important protective mechanism against oxidative stress [44
]. GSTP1 enzyme cooperates with multidrug resistance-associated protein (MRP) to protect the melanoma tumors from the anti-melanoma agents. Synergetic effects of both GSTP1 and MRP lead to melanoma drug resistance [9
]. The important element in the failure of chemotherapy of melanoma is drug resistance [46
]. Melanoma is one of the most aggressive form of skin cancers and resistant to all current modalities of cancer therapy [46
]. Among all resistance mechanisms involved in the melanoma therapy, the over expression of GST and MRP may play critical roles [2
A study by Laio reported that CAPE has anti-metastatic activity in mouse colon carcinoma CT26 cells [48
]. Recently, Chung group showed that CAPE also had inhibitory effects on human hepatocellular carcinoma HepG2 cell line [49
]. Grunderger group showed that CAPE exhibited a significantly greater sensitivity to human tumor cell lines than normal cell lines [50
]. Our previously reported work also showed that CAPE was an anti-melanoma agent [1
]. However, none of these studies demonstrated that CAPE selectively inhibits GST in melanoma cells. Moreover, these previous studies did not investigate the role of tyrosinase in the inhibition of GST by CAPE.
The current study is mainly focused on investigating CAPE as a selective GST inhibitor in the presence of tyrosinase. Such a selective inhibitor will be useful in the treatment of melanoma. Our findings indicate that CAPE-SG conjugate (≥ 10 µM) and CAPE-quinone (≥ 10 µM) significantly inhibit GST whereas CAPE (<50 µM) did not inhibit GST. We have found that CAPE and caffeic acid were weak and reversible inhibitors of GST, whereas CAPE-SG conjugate, CA-SG conjugate [21
], CAPE-quinone, and CA-quinone all were potent inhibitors of GST. It was also found that CAPE-SG conjugate was a reversible competitive and mixed GST inhibitor with respect to CDNB and GSH, respectively (). On the other hand, CAPE-quinone was an irreversible mixed and competitive inhibitor of GST with respect to CDNB and GSH, respectively. For the first time, we also identified a mono glutathione conjugate of CAPE when CAPE was incubated with tyrosinase and glutathione. Previously, we also reported a mono glutathione conjugate of 4-HA [51
] and a mono glutathione conjugate of caffeic acid [52
]. Tyrosine was also investigated as an endogenous substrate of tyrosinase [40
]. Our results indicate that all four compounds CAPE, caffeic acid (a hydrolyzed product of CAPE), 4-HA, and tyrosine were substrates for tyrosinase but not for GST. CAPE-SG conjugate and CAPE-quinone inhibited GST significantly, clearly indicating an additional role for tyrosinase in selective inhibition of GST in melanoma cells by intracellularly bioactivating CAPE (). However, among all tested compounds only CAPE demonstrated significant cytotoxic effects towards SK-MEL-28 cells at low concentrations, which could be due to its high lipid solubility (LogP 3.38) and cell membrane permeability.
Scheme 1 Summary of selective GST inhibition by CAPE in the presence of tyrosinase with respect to CDNB. CAPE can cross biological cell membrane due to its high lipid solubility whereas caffeic acid, an organic anion, cannot cross cell membrane due to its ionization (more ...)
Literature search on previous works has also shown that glutathione conjugates are involved in inhibition of GST [53
]. One recent study revealed that GSH conjugate of doxorubicin inhibited GST activity [53
]. Doxorubicin showed an increased cytotoxicity not only towards MDR cells but also doxorubicin sensitive cells suggesting that glutathione conjugates of drugs such as doxorubicin may play a vital role in the GST expression and hence drug induced cytotoxicity. Lyttle group also reported that a number of compounds which are coupled to the thiol group of glutathione showed significant inhibition of GST activity [54
]. Tyrosinase catalyzes the metabolism of quercetin to quinone and glutathione adducts [55
]. Thilakavathy group reported that the quinone metabolite is more potent in inhibiting GST than quercetin itself [55
]. Previously, Ploemen et al. [41
] also reported that caffeic acid glutathione conjugate as a potent inhibitor of GST. Similarly, our data suggest that the bioactivation of CAPE by tyrosinase leads to the formation of CAPE-quinone, which is a more potent inhibitor of GST than CAPE alone.
In addition, % glutathione consumption mediated by human placenta GST in the absence of tyrosinase was used to investigate if CAPE, CA, 4-HA, and tyrosine were substrates for GST. At 60 min incubation, none of the above compounds found to be a substrate for GST.
Ethacrynic acid (EA) is known as a GST inhibitor [58
] because of its α,β-unsaturated carbonyl functional group [30
]. EA can inhibit cell proliferation at higher concentrations, and is also able to enhance the cytotoxicity of many anticancer agents [59
]. Because our data suggested that CAPE acted as a selective GST inhibitor in melanoma cells due to its bioactivation with tyrosinase, the co-incubation of EA with CAPE did not enhance CAPE induced toxicity in SK-MEL-28 melanoma cells, which express tyrosinase () [28
Computational docking of EA into the dimer interface of GST was previously reported and tested experimentally [36
]. Our molecular docking studies also showed that CAPE and EA bind to the active site of GST (), suggesting that EA and CAPE may act as GST inhibitors. The binding modes of both CAPE-quinone and CAPE-SG conjugates may differ from that of the un-conjugated CAPE molecule. These molecular modeling suggest that CAPE and CAPE-SG conjugate may share the same binding sites with EA and EA-SG conjugate on GST.
The GST inhibition results suggested that the nature of GST inhibition by CAPE and CAPE-quinone are mixed whereas the nature of GST inhibition by CAPE-SG and EA are competitive with respect to CDNB (0.2–1 mM) ( and ). With respect to GSH, the nature of GST inhibition by CAPE and CAPE-SG is different from ethacrynic acid (EA) and CAPE-quinone. GST inhibition studies at 0.2–1 mM concentration of GSH () suggested that CAPE and CAPE-SG are mixed inhibitors of GST with respect to GSH whereas CAPE-quinone and EA are competitive and non-competitive inhibitors of GST with respect to GSH, respectively. Awasthi et al. has suggested that EA is a non-competitive inhibitor of GST with Ki
of 11.5 µM whereas EA–SG conjugate is a competitive inhibitor of GST inhibitor of GST with Ki
of 1.5 µM with respect to both CDNB and GSH [38
The over-expression of GST is not always conferring a significant protection from the anticancer agents. GST must be co-expressed with MRPs to protect cells from anticancer agents [8
]. It was previously reported that the detoxification of anti-cancer agents is a combined effect of both GSTs and MRPs [8
]. This study also investigated the role of MRP in CAPE induced toxicity in human SK-MEL-28 melanoma cells. Our results showed that CAPE induced toxicity in SK-MEL-28 cells are enhanced by BSO, a glutathione biosynthesis inhibitor [61
] (data not shown), and MK-571, a selective MRP inhibitor [17
] and probenecid, a non-selective MRP inhibitor [18
]. An increase in toxicity of CAPE in the presence of BSO in SK-MEL-28 sounds reasonable because glutathione depletion plays a vital role in CAPE toxicity towards melanoma cells as reported previously [1
]. Most anticancer agents become resistant due to high levels of GST and high levels of glutathione in tumor cells, which makes anti-neoplastic agents inactive. Melanoma is one of the most chemo-resistant cancers, which expresses high levels of GST [2
] and high levels of MRP [13
]. MK-571, a selective MRP inhibitor [17
], and probenecid, a non-selective MRP inhibitor [18
], increased CAPE induced cell toxicity, apoptosis, and significantly decreased mitochondrial membrane potential in SK-MEL-28 melanoma cells when co-incubated with CAPE. However, the effect of probenecid, a non-selective MRP inhibitor, was more significant than MK-571, a selective MRP inhibitor, suggesting other transporters may also play a role in CAPE induced toxicity. Previous literature also suggested that GST and MRP can act in synergy to protect cancer cells from toxicity of anticancer agents [62
In summary, for the first time, we showed that CAPE acts as a selective inhibitor of GST in the presence of tyrosinase. Our investigation describes tyrosinase-catalyzed activation of CAPE to a quinone intermediate that reacts spontaneously with GSH to form a CAPE-SG conjugate that competitively and reversibly inhibits GST with respect to CDNB, while CAPE alone is not inhibitory. The CAPE quinone also irreversibly inhibits GST in the absence of GSH, likely by inactivation via alkylation of a key cysteine residue on GST. The irreversible inhibition by CAPE of GST was shown in cell lysates, but not in intact cells, which contain GSH in mM levels [63
]. Similar results were found with caffeic acid, albeit with less potent dose–response, and in agreement with results with caffeic acid reported by Ploemen et al. [41
]. GST and MRP play vital roles in the protection of cancer cells against cancer therapy. The toxicity of CAPE was enhanced by MK-571, a selective MRP inhibitor, and probenecid, a non-specific MRP inhibitor, suggesting MRP as a CAPE resistance factor, presumably via efflux of the CAPE-SG conjugate.
Because tyrosinase is expressed in melanoma, this may allow selective inhibition of GST as a secondary target in melanoma cells compared to non-melanoma cells that do not express tyrosinase, in addition to the direct toxicity of CAPE due to the selective activation and cytotoxicity of the CAPE quinone. Therefore, bioactivated CAPE and its GSH conjugates selectively inhibit GST in the presence of tyrosinase. It is expected that these metabolites selectively inhibit GST in melanoma cells as they contain tyrosinase. Here, the selective inhibition refers to selective inhibition of GST in melanoma cells versus non-melanoma tissue or cells. In addition to selective GST inhibition in melanoma cells, CAPE and its derivatives may also mediate a variety of biologically and toxicologically relevant processes and reactions, which were not investigated in this study.
Our study suggests that tyrosinase plays a major role in the bioactivation of CAPE, which leads to significant and selective inhibition of GST in human SK-MEL-28 melanoma cells. It also suggests a role for MRP in the biochemical mechanism of CAPE induced toxicity in melanoma cell line.