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Alkyl esters of gallic acid as anticancer agents: A review
Accepted Manuscript Alkyl esters of gallic acid as anticancer agents: a review Claudriana Locatelli, Fabíola Branco Filippin-Monteiro, Tânia Beatriz Creczynski-Pasa PII:
S0223-5234(12)00664-2
DOI:
10.1016/j.ejmech.2012.10.056
Reference:
EJMECH 5829
To appear in:
European Journal of Medicinal Chemistry
Received Date: 21 August 2012 Revised Date:
29 September 2012
Accepted Date: 3 October 2012
Please cite this article as: C. Locatelli, F.B. Filippin-Monteiro, T.B. Creczynski-Pasa, Alkyl esters of gallic acid as anticancer agents: a review, European Journal of Medicinal Chemistry (2012), doi: 10.1016/ j.ejmech.2012.10.056. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights The antitumor activity of gallates is interconnected to the induction of apoptosis
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The gallates are potential inhibitors of metastasis
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The cell death is related to different mechanisms and it depends on the cell type
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Alkyl esters of gallic acid as anticancer agents: a review
Locatellia,
Fabíola
Branco
Filippin-Monteirob,
Creczynski-Pasab* a
Tânia
Beatriz
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Claudriana
Curso de Farmácia, Universidade do Oeste de Santa Catarina, Videira, SC,
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89560-000, Brazil and Universidade do Alto Vale do Rio do Peixe, Caçador,
b
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SC, 89500-000, Brazil
Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina P.O. Box 476, Florianópolis, SC, 88040-900, Brazil.
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*Corresponding author: Tel: + 55 48 3721 2212; fax: + 55 48 3721 9542
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E-mail: [email protected]; [email protected]
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ABSTRACT
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The current review presents the antitumoral properties of gallic acid and its esters derivatives. Numerous studies have indicated that the alkyl esters are
more effective against tumour cell lines than gallic acid, and that this activity is
related to their hydrophobic moiety. All related studies have shown that the
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antitumor activity is interconnected to the induction of apoptosis by different mechanisms and it depends on the cell type. The results presented in this
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review may help to emphasize that these compounds could be promising as a new alternative for the treatment of cancer, either alone or in combination with other antitumor drugs to potentiate their effects.
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Key words: gallic acid, esters derivatives, antitumoral activity, apoptosis.
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1. Introduction
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In order to achieve efficiency in treatment against cancer, researchers have focused on pharmacological studies with bioactive compounds extracted from
plants or semi-synthetic derivatives from natural compounds. Flavonoids in particular have been receiving a great deal of attention due to their common
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presence in a variety of plants. This special class of compounds consists of a
group of plant pigments widely distributed in nature, which are a complex
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fraction of the constituents of a wide variety of vegetables, fruits and food products like chocolate, tea and wine. These compounds have several important pharmacological properties, especially antitumoral, antiallergic, antiulcer, antioxidant and antiviral activities [1, 2]. Among them, gallic acid (GA),
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a natural plant triphenol, and its derivatives have been extensively evaluated for their antitumor activity against a variety of cell lineages. Some GA derivatives such as methyl, propyl, octyl and dodecyl gallates are widely used in food
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manufacturing as antioxidants, as well as in the pharmaceutical and cosmetic industries. Previous studies have shown that these compounds have potent
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therapeutic properties, including antitumoral, antimicrobial and antiviral, as well as being a potent antioxidant, acting as scavengers of reactive oxygen species (ROS) [3-5].
Recently, our knowledge of GA derivatives has been considerably improved
by the assessment of mechanisms of action in human and murine cell lineages and their promising antitumor activity. Furthermore, our group has described new insights regarding apoptosis involved in the death of melanoma cells. The
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biological activities of GA and their derivatives have been widely investigated. Therefore, this review will focus on the antitumoral properties of GA and its
1.2 Gallic Acid: chemical features
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derivatives.
GA (3,4,5-trihydroxicbenzoic acid) can be obtained under acid hydrolysis of
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hydrolysable tannins. A variety of substituents in the GA acid portion allow the obtainment of esters with a number of analogues with distinct pharmacological
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properties (Table 1). The difference among the esters derivatives is only in the atom carbon number of the aliphatic side chain, giving them different physicochemical characteristics, especially lipophilicity evaluated by the value of partition coefficient (C log P) (Table 1). Chemical changes in the GA
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molecule can modify their pharmacokinetic and pharmacodynamic properties, altering the solubility and the degree of ionization. The names of the compounds were abbreviated according to the atom carbon number of the side
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chain.
Gallic acid and derivatives have shown not only the antitumoral properties
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highlighted here, but also their selective cytotoxicity against a variety of tumour cells with a higher activity than that shown against non-tumoral cell lines [6-8]. These ester derivatives, especially alkyl esters, demonstrated more favourable pharmacological properties, and in many cases these effects were even stronger than those observed for GA itself. For instance, synthetic GA derivatives with eight or more carbon atoms in the side-chain were more efficient than GA in antiviral, antifungal, antioxidant and anticancer activities [5,
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9-11]. These biological activities have been correlated to the amphipathic character of these alkyl ester derivatives. The hydrophobic moiety seems to contribute greatly to the activity, presumably by increasing affinity for cell
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membranes and permeability [12]. Inoue et al. [3] investigated the cytotoxic effect of GA in various cell lines
and showed that the IC50 for HL60RG (human promyelocytic leukemia), P388-
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D1 (mouse lymphoid neoplasm), HeLa (human epithelial carcinoma), dRLh-84
(rat hepatoma), PLC/PRF/5 (human hepatoma), and KB (human epidermoid
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carcinoma) cells were 5.4, 4.8, 6.1, 6.6 and 13.2 µg/ml respectively, while for endothelial cell and fibroblast the IC50 values reached more than 20 µg/ml. These results suggest that GA induced cell death in tumour cells with a relatively high selectivity.
Other authors have also reported that GA and alkyl esters derivatives are
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more effective against tumour cells than against non-tumoral cells. GA alkyl esters derivatives presented low toxicity in vitro. When incubated with slices of rat liver and/or non-tumoral cell line (monkey kidney fibroblasts, VERO cells) the
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compounds were not cytotoxic at concentrations up to 1mM. In addition, the compounds exhibited very low cytotoxicity in normal mouse brain endothelial
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cells [5, 13]. Gallic acid was not cytotoxic to human lymphocytes at concentrations up to 4.17 mM [14]. Additionally, GA seems to be non toxic even when administered at 120 mg/Kg/day in rats [15]. Locatelli et al. [11] showed that tetradecyl gallate did not present toxicity when mice were treated every three days for a period of 28 days with 3.7 mg/Kg body weight. These results showed that alkyl esters derivatives could have potential antitumor activity, since they exhibited low toxicity in vivo and a relative selectivity to tumoral cells.
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2. Antitumoral activity
In vitro and in vivo assays using the alkyl gallates showed activity against several types of tumour cell lines, including cell lines of leukemia,
melanoma, lung cancer and breast cancer. All studies have shown that the
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antitumor activity seems to be related to the induction of apoptosis by
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different mechanisms depending on the cell type.
Although, alkyl gallates with less than 8 carbon atoms in the side chain seem to present antitumor activity, they are less active than the gallates with 8-14 carbon atoms. It has been reported that GA itself induces apoptosis in human promyelocytic leukemia HL-60 cells [16]; in this case, cell
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proliferation was inhibited with a IC50 value of approximately 300 µM. The effect of GA was reduced by blocking the carboxyl group with methyl, ethyl and propyl groups, but was enhanced with the lauryl group, in which the IC50
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was approximately 33 µM. Additionally, Serrano et al. [6] showed that methyl and propyl esters induced cell death in Wehi- and 231 and L292 cell lines,
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and in human peripheral blood lymphocytes with IC50 less than that of GA. Ishihara and Sakagami [17] have reported cytotoxic activity of GA against
human leukemia (HK-63) cell line. Saleem et al. [18] also reported that GA exhibits cytotoxic potential on HOS-1 cell line. Similarly, GA showed higher cytotoxicity against HSC-2 [19] and HL-60 cell [20] by promoting higher DNA fragmentation rate, compared to the effects on non-tumoral cells. When cytotoxic activity was screened by MTT assay, GA mildly inhibited the
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survival of PC-14 (human lung adenocarcinoma) and MKN45 (human stomach adenocarcinoma) human cancer cell lines [21]. Independently of the cell type, the cytotoxicity of GA, methyl and propyl gallate is mediated by
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ROS generation [3, 22-28] and further biochemical studies demonstrated that ROS generation and intracellular Ca2+ play important roles in the
modulation of early signalling pathway of apoptosis induced by GA [24, 29].
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Previous studies that focused on signalling pathways that lead to apoptosis
in different cell lines, using several pharmacological inhibitors, showed that
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intracellular Ca2+ and ROS are common denominators, although the death signal induced by GA may vary among different cell lines [25, 29]. Isuzugawa et al. [25] suggest that GA promotes changes in intracellular Ca2+ levels and this effect was related to secondary ROS generation and apoptosis induction. The caspase 3 activation was related to the intracellular
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Ca2+ increase, which triggers mitochondrial permeability transition, resulting in the release of cytochrome c into the cytoplasm through a mitochondrial permeability transition (MPT) pore. Thereafter, cytochrome c activates pro-
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caspase-9, an upstream caspase, particularly caspase-3, in combination with apoptotic peptidase activating factor 1 (Apaf-1), followed by the
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activation of downstream caspases to orchestrate the biochemical execution of cells.
Propyl gallate contains a hydrophobic alkyl ester group that helps the
compound to cross the cell membrane and enter into the cytoplasm [23]. Jacobi [23] and Kobayashi [26] proposed that the damage caused by propyl gallate oxidizing the DNA is associated with ROS production. These authors showed that propyl gallate is converted to GA by a cytoplasmic esterase
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releasing H2O2, O2- and the semiquinone reactive species, which are responsible for the DNA damage and consequent apoptosis induction. Reddy et al. [30] and Yeh et al. [31] showed that GA induces caspase-3
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and -8 expression, alteration in Bcl-2/Bax ratio, and an inhibition of tyrosine phosphorylation of BCR/ABL kinase, as well as a downregulation in COX-2
levels in K562 and HL-60 leukemia cell lines. Madlener et al. [32] also
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showed that GA was able to inhibit COX-1 and COX-2 activities in HL-60 cell
lines. This result is interesting because the increased COX-2 expression
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seems to be involved in the development of cancer by triggering cell division and apoptosis inhibition [33, 34].
Considering the phenomenon of metastasis, GA may reduce tumour invasiveness through downregulation of metallopeptidase domain 17
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(ADAM17), p-Erk, and p-Akt. Treatment of U87 cells with 20, 30, and 40 µg/ml of GA for 24 h decreased ADAM17 activity by 47.9%, 43.5%, and 38.9% compared to the control, respectively. These results suggest that GA
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may reduce tumour invasiveness through downregulation of ADAM17, pErk, and p-Akt [13].
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Disintegrin and metalloproteinase domains (ADAMs) are well-known ectodomain sheddases and their domains function as metalloproteinases. The ADAM family belongs to that of Zn-dependent metalloproteinases [35, 36]. ADAM17 is an important member of the ADAM family, and is involved in proteolysis of collagen IV of extracellular matrix (ECM) and the cell surface release of several integrins, suggesting that ADAM17 influences the invasive activity of different cells including glioma cells [37]. Gallic acid, propyl, octyl
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and lauryl gallate significantly downregulate the phosphorylation of members of PTK, PI3K/Akt and Ras/MAPK signal transduction pathways, which have been implicated in cell proliferation, invasion and survival [6, 13, 38]. These
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results suggest that the suppression of ADAM17 by GA may be responsible for the decrease in tumour invasiveness through downregulation of PI3K/AKT and Ras/MAPK pathways[13].
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Additionally, GA suppressed the migration and invasive ability of U-2 OS
(human osteosarcoma) cells, and decreased metalloproteinases (MMPs)
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MMP-2 and MMP-9 expression. These results suggest that the potential signalling pathways of GA to inhibit migration and invasion of these cells may be due to the downregulation of PKC, inhibition of mitogen-activated protein kinase (MAPK) and PI3K/AKT resulting in inhibition of MMP-2 and
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MMP-9 expression [39].
Matrix metalloproteinases (MMPs), zinc-dependent proteolytic enzymes, play an important role in the invasion, metastasis, and angiogenesis of
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cancer cells. Therefore, MMPs are one of the targets for agents to suppress their expression and/or activities, and that could arrest the migration and
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invasion of cancer cells. Gallic acid decreases the MMPs and their associated signalling pathway proteins and MMPs mRNA levels in A375.S2 human melanoma cells. Moreover, the action of GA was involved in the Ras and p-ERK resulting in inhibition of MMP-2 in A375.S2 human melanoma cells. Therefore, these data provide evidence that GA can be a potential drug for cancer therapy, and can markedly inhibit the invasive capability of melanoma cells [40].
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Furthermore,
GA
showed
potent
inhibitory
effects
on
gastric
adenocarcinoma cell migration. The expression of MMP-2/9 of AGS cells was inhibited by 2.0 µM. Multiple proteins are involved in metastasis and in
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cytoskeletal reorganization signal pathway, including Ras, Cdc42, Rac1, RhoA, RhoB, PI3K and p38MAPK, which were also inhibited by GA [41].
It has been reported that the degree of malignancy is directly proportional
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to the expression of ICAM-1 and VCAM-1 [42, 43], the cell adhesion molecules responsible for migration and invasion mechanisms that occur in
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the metastatic tumour. The effect of tetradecyl gallate inhibiting metastasis seems to be associated with the ability of this gallate to inhibit the expression of ICAM-1 and VCAM-1 [11]. Additionally, studies have shown that the growth and progression of most solid cancers are adhesion- and
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angiogenesis- dependent [44, 45].
Moreover, using B16F10-bearing mice, a well-established animal model for cancer metastases, our group found that tetradecyl gallate could reduce
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both tumour growth and metastasis [10, 11], and this effect is probably associated with the increase in NF-κB activity [10]. These results are in
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accordance with those from recent studies, which tested different kinds of drugs, showing that the inhibition of invasion and the motility of tumour cells are related to NF-κB activity, as well as the inhibition of cell adhesion protein expression in vitro [46-48]. Gallic acid decreased both protein and mRNA levels of MMP metalloproteinase-2/9 [39], which are involved in degradation of ECM and play vital roles in cancer cell migration and invasion [48]. Gallic acid and
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propyl gallate also decreased the levels of ERK1/2, which are key molecules in the ERK signalling pathway that have been shown to promote tumour invasion and metastasis, and also decreased the protein levels of AKT/PKB,
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IKK, PKC and JNK [39, 49]. Another proposed mechanism for apoptosis induction by GA is the ratio
between the inhibition of NF-kB activity and downregulation of PI3K/AKT
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pathway. Gallic acid might increase IkB binding to NF-kB, and then suppress the PI3/AKT pathway reducing metastasis on gastric cancer cells [41]. NF-
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kB is a multi-subunit transcription factor involved in cellular responses to viral infection and inflammation. NF-kB is maintained in the cytoplasm through interactions with an inhibitor of NF-kB (IkB). Upon dissociation, NFkB moves into the nucleus and promotes cancer cell proliferation, angiogenesis, and metastasis [27]. Based on these effects, it is possible to
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suppose that GA could also be useful against angiogenesis, but further clarification of the underlying mechanisms is required.
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Nevertheless, GA has been reported to be partially responsible for the anti-angiogenesis activities of Rubus leaf extract in vitro [50]. At the
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concentrations of 30 and 40 µg/ml, GA dramatically inhibited cell migration to 29% and 30% in U87 cells, and 51% and 31% in U251n cells, respectively [13].
Neoangiogenesis, the growth of new capillaries in response to pro-
angiogenic factors secreted by glioma cells due to a lack of oxygen and nutrients, plays a crucial role in tumour growth [51, 52]. Whereas GA and propyl gallate inhibit the expression of NF-kB on gastric cancer cells, the
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octyl, decyl and dodecyl gallates have the ability to increase the expression of NF-kB in melanoma cells [9, 10]. Although the NF-kB activation may promote the transcription of both anti- and proapoptotic proteins, it was
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reported that its activation occurs in pro-oxidant conditions [53]. Based on that, it is possible to infer that the NF-kB induction by esters of GA demonstrated in several studies would be directly related to the induction of
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ROS generation by these compounds [54].
Madlener at al. [32] suggest that GA inhibit ribonucleotide reductase,
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resulting in a significant alteration of the dNTP (deoxynucleoside triphosphate) pool balance, rapidly blocking DNA synthesis and leading to disruption of the cell cycle and apoptosis induction. Propyl gallate in its turn was able to induce apoptosis in THP-1 but not in HL-60 or Jurkat cells. In THP-1, propyl gallate induced marked apoptotic bubbling and chromatin
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condensation [49].
Caspase activation induces cell death and up-regulation of Bax, Bcl-2
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and p53, which is an important via of the apoptosis process. Propyl gallate mediated the cleavage of caspases 3, 8 and 9 and affected Bax, Bcl-2 and
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p53 levels in treated THP-1 cells [49]. Octyl and dodecyl gallates promoted activation of caspase 3 in mouse B-cell lymphoma line Wehi-231 and in B16F10 melanoma cell line [38, 54]. Gallic acid, octyl and dodecyl gallate induced a decrease in mitochondrial
membrane potential [54, 55], as well as an increase in Bax expression and a decrease in Bcl-2 expression. This inhibitory effect of octyl and dodecyl
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gallate on Bcl-2 expression is particularly important because it is known that this protein is involved in the elevated resistance to apoptosis [38, 54]. Glutathione (GSH) depletion and ROS production are two important
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biochemical events in the apoptotic process. Several gallates (GA, propyl, octyl, decyl, lauryl and tetradecyl gallate) induced GSH decrease and ROS
increase [9, 10, 28, 54-56] however, GSH depletion is not dependent on
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intracellular ROS production. GSH depletion induced by gallates is
associated with a decrease in enzyme activity and in protein expression of γ-
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glutamyl cysteine synthase (γ-GCS) [10, 49].
The receptor Fas has been shown to be an important mediator of apoptotic cell death and its activation by Fas ligand (Fas-L) induces apoptosis [57]. Propyl gallate induced a marked increase in Fas expression
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and a significant increase in Fas-L level [49] however octyl and dodecyl gallate did not alter the Fas receptor level [54]. Another important effect of octyl and dodecyl gallates is the ability to
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inhibit the drug efflux pump P-gp. Alkyl gallates are not expulsed from the cells increasing the cellular accumulation, although this effect is dependent
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on their alkyl chain lengths [58, 59]. Results reported by Calcabrini et al. [60] showed that dodecyl gallate
induces a reduction of cell survival and cell cycle alterations in MCF7 (human breast cancer), MCF7 ADR (human breast cancer multidrugresistant) and MDA-MB-231 cells (mutant p53 breast cancer). An evident p21 up regulation was observed in these three breast cancer cell lines, followed by apoptotic cell death. Both p21 up regulation and apoptosis
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induction were regulated, at least in part, by ERK1/2 activation. Interestingly, a stronger cytotoxic effect was observed on multidrug-resistant MCF7 ADR cells as well as on MDA-MB-231 carrying mutations in the p53 gene, which
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in theory should be more resistant to cytotoxic treatments and apoptosis. The induction of death caused by dodecyl gallate in MCF7 cells may be directly related to the ability of this gallate to inhibit the Pgp.
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Studies reported by Tammela et al. [61] support the idea that gallates containing more than eight carbon atoms in the acid moiety esterified GA
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have a better interaction with cell membrane, resulting in enhanced cytotoxic effect.
The Table 2 and 3 show the IC50 values for cell cytotoxicity of GA and
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gallates, respectively and the respective mechanism of cell death induction.
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3. Conclusion In an attempt to summarize the findings described, a scheme was elaborated (Figure 1) showing the different mechanisms of induction of
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apoptosis mediated by gallic acid and its alkyl esters derivatives. Most of the results presented in this review were obtained with GA, and some studies
with octyl and dodecyl gallates, in different cell lines, are particularly
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interesting. By analysing the results presented in the work carried out with
the gallates, we suppose that the GA and propyl gallate induce apoptosis by
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extrinsic pathway through the activation of cell death receptor Fas. The gallates with eight or more carbon atoms appear to induce apoptosis by the intrinsic via. The studies indicate that alkyl esters are more effective than GA for apoptosis induction, and this activity has been correlated to the amphipathic character of these alkyl ester derivatives, which is related to affinity
and
cell
membrane
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increasing
permeability.
Studies
have
demonstrated that the antitumoral effects of the GA and gallates are related to the capability of ROS generation and intracellular Ca2+ increase, which
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triggers caspase activation and cytochrome c release into the cytoplasm through a mitochondrial permeability transition pore. Another effect
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promoted by gallates is the capability to alter Bcl-2/Bax ratio and inhibit tyrosine phosphorylation of BCR/ABL kinase, resulting in alteration in mitochondrial potential and induction of apoptosis. Gallates with eight or more carbon atoms in the side chain promote decrease in ATP levels and trigger alteration in mitochondrial potential. A study with propyl gallate also showed a similar effect because this compound inhibited the activity of succinate dehydrogenase, enzyme associated with Krebs cycle. The
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increase of ROS levels promotes the increase of catalase (CAT) and superoxide dismutase (SOD) activities and the reduction of GSH levels, but reduction of GSH levels may be associated with the capability of the gallates
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to decrease the activity and expression of γ-glutamyl cysteine synthase. The gallates also seem to promote an increase of p53 and p21 expression, resulting in cell cycle changes such as increasing the G1/S phase and cell
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death. Other important effects related to the octyl, dodecyl and tetradecyl gallates are the ability of these compounds to inhibit Pgp, as well as cell
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migration and adhesion, by inhibiting the expression of ICAM-1 and VCAM-1 adhesion proteins. The inhibition of ICAM-1 and VCAM-1 is particularly important because it decreases the metastasis capability of the tumour cells. Gallic acid, in turn, appears to inhibit the expression of metaloproteases 2/9 leading to the reduction of cell migration and metastasis. The ability of these
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gallates to block Pgp could be applied to decrease the resistance of the tumour cells to the action of various drugs. Regardless of the mechanism of action of these promised gallates, the final effect is apoptosis induction,
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preferentially in tumour cells rather than in non-tumour cells, it is therefore necessary to conduct in vivo studies (preclinical) which show the
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effectiveness of tumour therapy in compounds, since most of the studies were only carried out in vitro.
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Acknowledgments The studies developed by our group were supported by grants (and a PNPDfellowship for Fabiola B. Filippin-Monteiro, PhD) from CNPq (Conselho Nacional
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de Desenvolvimento Científico e Tecnológico), CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior) and FAPESC (Fundação de Amparo à Pesquisa de Santa Catarina). The authors wish to thank the group of
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Prof. Ricardo J. Nunes and Prof. Rosendo A. Yunes for synthetizing the gallates
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for our studies.
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Figure caption
Figure 1. Scheme with the probable targets of gallic acid and its ester derivatives. Gallic acid was named GA and the names of the gallates were
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abbreviated according to the atom carbon number in the lateral chain, as
showed in the Table 1. Other abbreviations: PgP, P-glycoprotein (multi drug
resistance protein related); Fas, death receptor; FasL, death receptor ligand; ADAM, disintegrin and metalloproteinase domains; ErK, extracellular signal-
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regulated kinases; AkT/PKB, protein kinase B; MMP, matrix metalloproteinase; COX, cyclooxygenase; NF-kB nuclear factor kappa-light-chain-enhancer of activated B cells; IkB, NF-kB regulatory protein; ICAM, intercellular adhesion
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molecule; VCAM, vascular adhesion molecule; PARP, poly ADP-ribose polymerase; SOD, superoxide dismutase; CAT, catalase; ROS, reactive oxygen species; BCL-2, anti-apoptotic protein ; Bax, pro-apoptotic member of the Bcl-2 protein family; GSH reduced glutathione; γ GCS, γ glutamyl synthase; p21, cell cycle regulator protein; p53, genome reparative protein; G1/S phase, pos-
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mitotic cell cycle phase; ∆Ψm, mitochondrial membrane potential;
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Table 1. Chemical structures and C log P values of gallic acid n-alkyl ester derivatives.
OH
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OH
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HO
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COOR
Gallates
-R
C log P
GA
Gallic acid
-H
0.89
G1
Methyl
-CH3
0.92
G2
Ethyl
-(CH2)-CH3
1.27
G3
Propyl
-(CH2)2-CH3
1.73
G4
Butyl
-(CH2)3-CH3
2.13
G5
Pentyl
-(CH2)4-CH3
2.53
Hexyl
-(CH2)5-CH3
2.92
Heptyl
-(CH2)6-CH3
2.32
Octyl
-(CH2)7-CH3
3.72
Decyl
-(CH2)9-CH3
4.51
G10
Undecyl
-(CH2)10-CH3
4.90
Dodecyl
-(CH2)11-CH3
5.30
Tetradecyl
-(CH2)13-CH3
6.09
G16
Hexadecyl
-(CH2)15-CH3
6.89
G18
Octadecyl
-(CH2)17-CH3
7.68
G6 G7 G8
G12
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G14
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G10
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Abbreviation
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Cell line
IC50 (µM)
HL60
300
HL60RG dRLh-84
317 364
PLC/PRF/5 HLE
387
P388-D1
282
HeLa
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GA
358
Calu-6
10-50
A549
100-200
Wehi231
40
L929
250
U251n/U87
nd
K562
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33
IR-K562
4.75
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U251n/U87/ HUH-7/KB nd – not determined
Action
nd
References
↑H2O2; ↑OH; ↑ O2 ;↑ Ca ; ↑ PARP cleavage; ↑Release of cytochrome c; ↑ Caspase 3; ↑ Caspase 9; ↓ COX-1; ↓ COX-2; DNA damage; ↑catalase expression ↓ GSH; ↓ ∆Ψm; ↓ expression and activity of ADAM17; ↓ p-Erk; ↓ p-Akt; ↑ p21; ↑ p27; ↑ Bax; ↓ Cyclin D1/2/3/E; ↑ FasL; ↓ BcL-2; ↓ tyrosine BCR/ALB kinase phosphorylation; ↓ NF-kB activity
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Gallates
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Table 2. Values of IC50 of gallic acid (GA) in different cell lines, and the mechanism related to cell death induction.
-
2+
3; 6; 9; 10 12; 13; 14; 26 27; 29; 31; 36; 40; 42; 51
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G12
L929
40
HeLa
800
Wehi 231
1.5
L929
1
↑ ROS; ↓ GT cyt and mit; ↓ γ-GCS activity; ↓ ATP;
B16F10
45
↓ Adhesion cell; ↑NF-kB activation;
9; 10; 14; 42;
KB-C2
nd 22
↑ caspase 3; ↓ ∆Ψm;↓ Bcl-2; ↑ Bax;
55; 59
L1210 CEM
50
B16F10
82
L1210
12.2
CEM
9.8
MCF7/MCF7/ADR/KB-C2
nd
MDA-MB-231
1
Wehi 231
1
L929
1
B16F10 HL60 L1210 CEM B16F10
G14
L1210 CEM
nd - not determined
14; 23; 24;
12
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↑ SOD; ↓ ∆Ψm; ↑Fas; ↑FasL; ↑Caspase 3; ↑Caspase 8; ↑Caspase 9; ↑p53; ↑ERK; p38 activation; ↓ Nrf-2; ↓ GSH; ↓ γ-GCS
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G10
↑H2O2; ↑.OH; ↑semiquinone radical; DNA damage; ↑TBARS; ↓succinate dehydrogenase activity; ↑ Catalase;
Wehi231
26; 27; 50; 56
Blocking P-gp; ↑ NF-kB; ↓ NF-kB activity
↑ ROS, ↓ GT cyt and mit, ↓ γ-GCS activity, ↓ ATP;
9; 10
↓ Adhesion cell, ↑NF-kB activation; ↓ GSH mit, ↓ GSH cit
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G8
References
IC50 (µM) nd
↑ G1 phase; ↑ S phase; ↑ G2/M phases; ↑ p53; ↑ p21 ↓ cyclin D1; ↓ Bcl-2; ↑ phosphorylation of Erk1/2;
6; 9; 10; 12;
↓ protein tyrosine phosphorylation; ↓ ∆Ψm;
31; 36; 39; 42;
48
↑release cytochrome c; ↑ caspase 3; ↑ cleavage PARP; ↑ ROS;
55; 59; 61
33
↓ GT cyt and mit; ↓ γ-GCS activity; ↓ ATP; ↓ Adhesion cell;
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G3
Action
Cell line HL60/GM05757/THP-1
10.9
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Gallates
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Table 3. Values of IC50 of gallates in different cell lines, and the mechanism related to cell death induction.
47.4 60
↑NF-kB activation; ↓ Bcl-2; ↑ Bax; Blocking P-gp; ↓ GSH mit and cit
8.5
↑ ROS; ↓ GT cyt and mit; ↓ γ-GCS activity; ↓ ATP;
>50
↓ICAM-1; ↓VCAM-1; ↓ GSH mit; ↓ GSH cit; DNA damage
9; 10; 11
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S0223-5234(12)00664-2
DOI:
10.1016/j.ejmech.2012.10.056
Reference:
EJMECH 5829
To appear in:
European Journal of Medicinal Chemistry
Received Date: 21 August 2012 Revised Date:
29 September 2012
Accepted Date: 3 October 2012
Please cite this article as: C. Locatelli, F.B. Filippin-Monteiro, T.B. Creczynski-Pasa, Alkyl esters of gallic acid as anticancer agents: a review, European Journal of Medicinal Chemistry (2012), doi: 10.1016/ j.ejmech.2012.10.056. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Highlights The antitumor activity of gallates is interconnected to the induction of apoptosis
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The gallates are potential inhibitors of metastasis
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The cell death is related to different mechanisms and it depends on the cell type
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Alkyl esters of gallic acid as anticancer agents: a review
Locatellia,
Fabíola
Branco
Filippin-Monteirob,
Creczynski-Pasab* a
Tânia
Beatriz
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Claudriana
Curso de Farmácia, Universidade do Oeste de Santa Catarina, Videira, SC,
SC
89560-000, Brazil and Universidade do Alto Vale do Rio do Peixe, Caçador,
b
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SC, 89500-000, Brazil
Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina P.O. Box 476, Florianópolis, SC, 88040-900, Brazil.
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*Corresponding author: Tel: + 55 48 3721 2212; fax: + 55 48 3721 9542
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E-mail: [email protected]; [email protected]
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ABSTRACT
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The current review presents the antitumoral properties of gallic acid and its esters derivatives. Numerous studies have indicated that the alkyl esters are
more effective against tumour cell lines than gallic acid, and that this activity is
related to their hydrophobic moiety. All related studies have shown that the
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antitumor activity is interconnected to the induction of apoptosis by different mechanisms and it depends on the cell type. The results presented in this
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review may help to emphasize that these compounds could be promising as a new alternative for the treatment of cancer, either alone or in combination with other antitumor drugs to potentiate their effects.
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Key words: gallic acid, esters derivatives, antitumoral activity, apoptosis.
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1. Introduction
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In order to achieve efficiency in treatment against cancer, researchers have focused on pharmacological studies with bioactive compounds extracted from
plants or semi-synthetic derivatives from natural compounds. Flavonoids in particular have been receiving a great deal of attention due to their common
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presence in a variety of plants. This special class of compounds consists of a
group of plant pigments widely distributed in nature, which are a complex
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fraction of the constituents of a wide variety of vegetables, fruits and food products like chocolate, tea and wine. These compounds have several important pharmacological properties, especially antitumoral, antiallergic, antiulcer, antioxidant and antiviral activities [1, 2]. Among them, gallic acid (GA),
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a natural plant triphenol, and its derivatives have been extensively evaluated for their antitumor activity against a variety of cell lineages. Some GA derivatives such as methyl, propyl, octyl and dodecyl gallates are widely used in food
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manufacturing as antioxidants, as well as in the pharmaceutical and cosmetic industries. Previous studies have shown that these compounds have potent
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therapeutic properties, including antitumoral, antimicrobial and antiviral, as well as being a potent antioxidant, acting as scavengers of reactive oxygen species (ROS) [3-5].
Recently, our knowledge of GA derivatives has been considerably improved
by the assessment of mechanisms of action in human and murine cell lineages and their promising antitumor activity. Furthermore, our group has described new insights regarding apoptosis involved in the death of melanoma cells. The
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biological activities of GA and their derivatives have been widely investigated. Therefore, this review will focus on the antitumoral properties of GA and its
1.2 Gallic Acid: chemical features
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derivatives.
GA (3,4,5-trihydroxicbenzoic acid) can be obtained under acid hydrolysis of
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hydrolysable tannins. A variety of substituents in the GA acid portion allow the obtainment of esters with a number of analogues with distinct pharmacological
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properties (Table 1). The difference among the esters derivatives is only in the atom carbon number of the aliphatic side chain, giving them different physicochemical characteristics, especially lipophilicity evaluated by the value of partition coefficient (C log P) (Table 1). Chemical changes in the GA
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molecule can modify their pharmacokinetic and pharmacodynamic properties, altering the solubility and the degree of ionization. The names of the compounds were abbreviated according to the atom carbon number of the side
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chain.
Gallic acid and derivatives have shown not only the antitumoral properties
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highlighted here, but also their selective cytotoxicity against a variety of tumour cells with a higher activity than that shown against non-tumoral cell lines [6-8]. These ester derivatives, especially alkyl esters, demonstrated more favourable pharmacological properties, and in many cases these effects were even stronger than those observed for GA itself. For instance, synthetic GA derivatives with eight or more carbon atoms in the side-chain were more efficient than GA in antiviral, antifungal, antioxidant and anticancer activities [5,
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9-11]. These biological activities have been correlated to the amphipathic character of these alkyl ester derivatives. The hydrophobic moiety seems to contribute greatly to the activity, presumably by increasing affinity for cell
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membranes and permeability [12]. Inoue et al. [3] investigated the cytotoxic effect of GA in various cell lines
and showed that the IC50 for HL60RG (human promyelocytic leukemia), P388-
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D1 (mouse lymphoid neoplasm), HeLa (human epithelial carcinoma), dRLh-84
(rat hepatoma), PLC/PRF/5 (human hepatoma), and KB (human epidermoid
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carcinoma) cells were 5.4, 4.8, 6.1, 6.6 and 13.2 µg/ml respectively, while for endothelial cell and fibroblast the IC50 values reached more than 20 µg/ml. These results suggest that GA induced cell death in tumour cells with a relatively high selectivity.
Other authors have also reported that GA and alkyl esters derivatives are
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more effective against tumour cells than against non-tumoral cells. GA alkyl esters derivatives presented low toxicity in vitro. When incubated with slices of rat liver and/or non-tumoral cell line (monkey kidney fibroblasts, VERO cells) the
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compounds were not cytotoxic at concentrations up to 1mM. In addition, the compounds exhibited very low cytotoxicity in normal mouse brain endothelial
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cells [5, 13]. Gallic acid was not cytotoxic to human lymphocytes at concentrations up to 4.17 mM [14]. Additionally, GA seems to be non toxic even when administered at 120 mg/Kg/day in rats [15]. Locatelli et al. [11] showed that tetradecyl gallate did not present toxicity when mice were treated every three days for a period of 28 days with 3.7 mg/Kg body weight. These results showed that alkyl esters derivatives could have potential antitumor activity, since they exhibited low toxicity in vivo and a relative selectivity to tumoral cells.
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2. Antitumoral activity
In vitro and in vivo assays using the alkyl gallates showed activity against several types of tumour cell lines, including cell lines of leukemia,
melanoma, lung cancer and breast cancer. All studies have shown that the
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antitumor activity seems to be related to the induction of apoptosis by
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different mechanisms depending on the cell type.
Although, alkyl gallates with less than 8 carbon atoms in the side chain seem to present antitumor activity, they are less active than the gallates with 8-14 carbon atoms. It has been reported that GA itself induces apoptosis in human promyelocytic leukemia HL-60 cells [16]; in this case, cell
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proliferation was inhibited with a IC50 value of approximately 300 µM. The effect of GA was reduced by blocking the carboxyl group with methyl, ethyl and propyl groups, but was enhanced with the lauryl group, in which the IC50
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was approximately 33 µM. Additionally, Serrano et al. [6] showed that methyl and propyl esters induced cell death in Wehi- and 231 and L292 cell lines,
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and in human peripheral blood lymphocytes with IC50 less than that of GA. Ishihara and Sakagami [17] have reported cytotoxic activity of GA against
human leukemia (HK-63) cell line. Saleem et al. [18] also reported that GA exhibits cytotoxic potential on HOS-1 cell line. Similarly, GA showed higher cytotoxicity against HSC-2 [19] and HL-60 cell [20] by promoting higher DNA fragmentation rate, compared to the effects on non-tumoral cells. When cytotoxic activity was screened by MTT assay, GA mildly inhibited the
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survival of PC-14 (human lung adenocarcinoma) and MKN45 (human stomach adenocarcinoma) human cancer cell lines [21]. Independently of the cell type, the cytotoxicity of GA, methyl and propyl gallate is mediated by
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ROS generation [3, 22-28] and further biochemical studies demonstrated that ROS generation and intracellular Ca2+ play important roles in the
modulation of early signalling pathway of apoptosis induced by GA [24, 29].
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Previous studies that focused on signalling pathways that lead to apoptosis
in different cell lines, using several pharmacological inhibitors, showed that
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intracellular Ca2+ and ROS are common denominators, although the death signal induced by GA may vary among different cell lines [25, 29]. Isuzugawa et al. [25] suggest that GA promotes changes in intracellular Ca2+ levels and this effect was related to secondary ROS generation and apoptosis induction. The caspase 3 activation was related to the intracellular
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Ca2+ increase, which triggers mitochondrial permeability transition, resulting in the release of cytochrome c into the cytoplasm through a mitochondrial permeability transition (MPT) pore. Thereafter, cytochrome c activates pro-
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caspase-9, an upstream caspase, particularly caspase-3, in combination with apoptotic peptidase activating factor 1 (Apaf-1), followed by the
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activation of downstream caspases to orchestrate the biochemical execution of cells.
Propyl gallate contains a hydrophobic alkyl ester group that helps the
compound to cross the cell membrane and enter into the cytoplasm [23]. Jacobi [23] and Kobayashi [26] proposed that the damage caused by propyl gallate oxidizing the DNA is associated with ROS production. These authors showed that propyl gallate is converted to GA by a cytoplasmic esterase
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releasing H2O2, O2- and the semiquinone reactive species, which are responsible for the DNA damage and consequent apoptosis induction. Reddy et al. [30] and Yeh et al. [31] showed that GA induces caspase-3
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and -8 expression, alteration in Bcl-2/Bax ratio, and an inhibition of tyrosine phosphorylation of BCR/ABL kinase, as well as a downregulation in COX-2
levels in K562 and HL-60 leukemia cell lines. Madlener et al. [32] also
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showed that GA was able to inhibit COX-1 and COX-2 activities in HL-60 cell
lines. This result is interesting because the increased COX-2 expression
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seems to be involved in the development of cancer by triggering cell division and apoptosis inhibition [33, 34].
Considering the phenomenon of metastasis, GA may reduce tumour invasiveness through downregulation of metallopeptidase domain 17
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(ADAM17), p-Erk, and p-Akt. Treatment of U87 cells with 20, 30, and 40 µg/ml of GA for 24 h decreased ADAM17 activity by 47.9%, 43.5%, and 38.9% compared to the control, respectively. These results suggest that GA
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may reduce tumour invasiveness through downregulation of ADAM17, pErk, and p-Akt [13].
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Disintegrin and metalloproteinase domains (ADAMs) are well-known ectodomain sheddases and their domains function as metalloproteinases. The ADAM family belongs to that of Zn-dependent metalloproteinases [35, 36]. ADAM17 is an important member of the ADAM family, and is involved in proteolysis of collagen IV of extracellular matrix (ECM) and the cell surface release of several integrins, suggesting that ADAM17 influences the invasive activity of different cells including glioma cells [37]. Gallic acid, propyl, octyl
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and lauryl gallate significantly downregulate the phosphorylation of members of PTK, PI3K/Akt and Ras/MAPK signal transduction pathways, which have been implicated in cell proliferation, invasion and survival [6, 13, 38]. These
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results suggest that the suppression of ADAM17 by GA may be responsible for the decrease in tumour invasiveness through downregulation of PI3K/AKT and Ras/MAPK pathways[13].
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Additionally, GA suppressed the migration and invasive ability of U-2 OS
(human osteosarcoma) cells, and decreased metalloproteinases (MMPs)
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MMP-2 and MMP-9 expression. These results suggest that the potential signalling pathways of GA to inhibit migration and invasion of these cells may be due to the downregulation of PKC, inhibition of mitogen-activated protein kinase (MAPK) and PI3K/AKT resulting in inhibition of MMP-2 and
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MMP-9 expression [39].
Matrix metalloproteinases (MMPs), zinc-dependent proteolytic enzymes, play an important role in the invasion, metastasis, and angiogenesis of
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cancer cells. Therefore, MMPs are one of the targets for agents to suppress their expression and/or activities, and that could arrest the migration and
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invasion of cancer cells. Gallic acid decreases the MMPs and their associated signalling pathway proteins and MMPs mRNA levels in A375.S2 human melanoma cells. Moreover, the action of GA was involved in the Ras and p-ERK resulting in inhibition of MMP-2 in A375.S2 human melanoma cells. Therefore, these data provide evidence that GA can be a potential drug for cancer therapy, and can markedly inhibit the invasive capability of melanoma cells [40].
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Furthermore,
GA
showed
potent
inhibitory
effects
on
gastric
adenocarcinoma cell migration. The expression of MMP-2/9 of AGS cells was inhibited by 2.0 µM. Multiple proteins are involved in metastasis and in
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cytoskeletal reorganization signal pathway, including Ras, Cdc42, Rac1, RhoA, RhoB, PI3K and p38MAPK, which were also inhibited by GA [41].
It has been reported that the degree of malignancy is directly proportional
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to the expression of ICAM-1 and VCAM-1 [42, 43], the cell adhesion molecules responsible for migration and invasion mechanisms that occur in
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the metastatic tumour. The effect of tetradecyl gallate inhibiting metastasis seems to be associated with the ability of this gallate to inhibit the expression of ICAM-1 and VCAM-1 [11]. Additionally, studies have shown that the growth and progression of most solid cancers are adhesion- and
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angiogenesis- dependent [44, 45].
Moreover, using B16F10-bearing mice, a well-established animal model for cancer metastases, our group found that tetradecyl gallate could reduce
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both tumour growth and metastasis [10, 11], and this effect is probably associated with the increase in NF-κB activity [10]. These results are in
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accordance with those from recent studies, which tested different kinds of drugs, showing that the inhibition of invasion and the motility of tumour cells are related to NF-κB activity, as well as the inhibition of cell adhesion protein expression in vitro [46-48]. Gallic acid decreased both protein and mRNA levels of MMP metalloproteinase-2/9 [39], which are involved in degradation of ECM and play vital roles in cancer cell migration and invasion [48]. Gallic acid and
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propyl gallate also decreased the levels of ERK1/2, which are key molecules in the ERK signalling pathway that have been shown to promote tumour invasion and metastasis, and also decreased the protein levels of AKT/PKB,
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IKK, PKC and JNK [39, 49]. Another proposed mechanism for apoptosis induction by GA is the ratio
between the inhibition of NF-kB activity and downregulation of PI3K/AKT
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pathway. Gallic acid might increase IkB binding to NF-kB, and then suppress the PI3/AKT pathway reducing metastasis on gastric cancer cells [41]. NF-
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kB is a multi-subunit transcription factor involved in cellular responses to viral infection and inflammation. NF-kB is maintained in the cytoplasm through interactions with an inhibitor of NF-kB (IkB). Upon dissociation, NFkB moves into the nucleus and promotes cancer cell proliferation, angiogenesis, and metastasis [27]. Based on these effects, it is possible to
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suppose that GA could also be useful against angiogenesis, but further clarification of the underlying mechanisms is required.
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Nevertheless, GA has been reported to be partially responsible for the anti-angiogenesis activities of Rubus leaf extract in vitro [50]. At the
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concentrations of 30 and 40 µg/ml, GA dramatically inhibited cell migration to 29% and 30% in U87 cells, and 51% and 31% in U251n cells, respectively [13].
Neoangiogenesis, the growth of new capillaries in response to pro-
angiogenic factors secreted by glioma cells due to a lack of oxygen and nutrients, plays a crucial role in tumour growth [51, 52]. Whereas GA and propyl gallate inhibit the expression of NF-kB on gastric cancer cells, the
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octyl, decyl and dodecyl gallates have the ability to increase the expression of NF-kB in melanoma cells [9, 10]. Although the NF-kB activation may promote the transcription of both anti- and proapoptotic proteins, it was
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reported that its activation occurs in pro-oxidant conditions [53]. Based on that, it is possible to infer that the NF-kB induction by esters of GA demonstrated in several studies would be directly related to the induction of
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ROS generation by these compounds [54].
Madlener at al. [32] suggest that GA inhibit ribonucleotide reductase,
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resulting in a significant alteration of the dNTP (deoxynucleoside triphosphate) pool balance, rapidly blocking DNA synthesis and leading to disruption of the cell cycle and apoptosis induction. Propyl gallate in its turn was able to induce apoptosis in THP-1 but not in HL-60 or Jurkat cells. In THP-1, propyl gallate induced marked apoptotic bubbling and chromatin
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condensation [49].
Caspase activation induces cell death and up-regulation of Bax, Bcl-2
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and p53, which is an important via of the apoptosis process. Propyl gallate mediated the cleavage of caspases 3, 8 and 9 and affected Bax, Bcl-2 and
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p53 levels in treated THP-1 cells [49]. Octyl and dodecyl gallates promoted activation of caspase 3 in mouse B-cell lymphoma line Wehi-231 and in B16F10 melanoma cell line [38, 54]. Gallic acid, octyl and dodecyl gallate induced a decrease in mitochondrial
membrane potential [54, 55], as well as an increase in Bax expression and a decrease in Bcl-2 expression. This inhibitory effect of octyl and dodecyl
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gallate on Bcl-2 expression is particularly important because it is known that this protein is involved in the elevated resistance to apoptosis [38, 54]. Glutathione (GSH) depletion and ROS production are two important
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biochemical events in the apoptotic process. Several gallates (GA, propyl, octyl, decyl, lauryl and tetradecyl gallate) induced GSH decrease and ROS
increase [9, 10, 28, 54-56] however, GSH depletion is not dependent on
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intracellular ROS production. GSH depletion induced by gallates is
associated with a decrease in enzyme activity and in protein expression of γ-
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glutamyl cysteine synthase (γ-GCS) [10, 49].
The receptor Fas has been shown to be an important mediator of apoptotic cell death and its activation by Fas ligand (Fas-L) induces apoptosis [57]. Propyl gallate induced a marked increase in Fas expression
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and a significant increase in Fas-L level [49] however octyl and dodecyl gallate did not alter the Fas receptor level [54]. Another important effect of octyl and dodecyl gallates is the ability to
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inhibit the drug efflux pump P-gp. Alkyl gallates are not expulsed from the cells increasing the cellular accumulation, although this effect is dependent
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on their alkyl chain lengths [58, 59]. Results reported by Calcabrini et al. [60] showed that dodecyl gallate
induces a reduction of cell survival and cell cycle alterations in MCF7 (human breast cancer), MCF7 ADR (human breast cancer multidrugresistant) and MDA-MB-231 cells (mutant p53 breast cancer). An evident p21 up regulation was observed in these three breast cancer cell lines, followed by apoptotic cell death. Both p21 up regulation and apoptosis
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induction were regulated, at least in part, by ERK1/2 activation. Interestingly, a stronger cytotoxic effect was observed on multidrug-resistant MCF7 ADR cells as well as on MDA-MB-231 carrying mutations in the p53 gene, which
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in theory should be more resistant to cytotoxic treatments and apoptosis. The induction of death caused by dodecyl gallate in MCF7 cells may be directly related to the ability of this gallate to inhibit the Pgp.
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Studies reported by Tammela et al. [61] support the idea that gallates containing more than eight carbon atoms in the acid moiety esterified GA
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have a better interaction with cell membrane, resulting in enhanced cytotoxic effect.
The Table 2 and 3 show the IC50 values for cell cytotoxicity of GA and
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gallates, respectively and the respective mechanism of cell death induction.
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3. Conclusion In an attempt to summarize the findings described, a scheme was elaborated (Figure 1) showing the different mechanisms of induction of
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apoptosis mediated by gallic acid and its alkyl esters derivatives. Most of the results presented in this review were obtained with GA, and some studies
with octyl and dodecyl gallates, in different cell lines, are particularly
SC
interesting. By analysing the results presented in the work carried out with
the gallates, we suppose that the GA and propyl gallate induce apoptosis by
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extrinsic pathway through the activation of cell death receptor Fas. The gallates with eight or more carbon atoms appear to induce apoptosis by the intrinsic via. The studies indicate that alkyl esters are more effective than GA for apoptosis induction, and this activity has been correlated to the amphipathic character of these alkyl ester derivatives, which is related to affinity
and
cell
membrane
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increasing
permeability.
Studies
have
demonstrated that the antitumoral effects of the GA and gallates are related to the capability of ROS generation and intracellular Ca2+ increase, which
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triggers caspase activation and cytochrome c release into the cytoplasm through a mitochondrial permeability transition pore. Another effect
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promoted by gallates is the capability to alter Bcl-2/Bax ratio and inhibit tyrosine phosphorylation of BCR/ABL kinase, resulting in alteration in mitochondrial potential and induction of apoptosis. Gallates with eight or more carbon atoms in the side chain promote decrease in ATP levels and trigger alteration in mitochondrial potential. A study with propyl gallate also showed a similar effect because this compound inhibited the activity of succinate dehydrogenase, enzyme associated with Krebs cycle. The
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increase of ROS levels promotes the increase of catalase (CAT) and superoxide dismutase (SOD) activities and the reduction of GSH levels, but reduction of GSH levels may be associated with the capability of the gallates
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to decrease the activity and expression of γ-glutamyl cysteine synthase. The gallates also seem to promote an increase of p53 and p21 expression, resulting in cell cycle changes such as increasing the G1/S phase and cell
SC
death. Other important effects related to the octyl, dodecyl and tetradecyl gallates are the ability of these compounds to inhibit Pgp, as well as cell
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migration and adhesion, by inhibiting the expression of ICAM-1 and VCAM-1 adhesion proteins. The inhibition of ICAM-1 and VCAM-1 is particularly important because it decreases the metastasis capability of the tumour cells. Gallic acid, in turn, appears to inhibit the expression of metaloproteases 2/9 leading to the reduction of cell migration and metastasis. The ability of these
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gallates to block Pgp could be applied to decrease the resistance of the tumour cells to the action of various drugs. Regardless of the mechanism of action of these promised gallates, the final effect is apoptosis induction,
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preferentially in tumour cells rather than in non-tumour cells, it is therefore necessary to conduct in vivo studies (preclinical) which show the
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effectiveness of tumour therapy in compounds, since most of the studies were only carried out in vitro.
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Acknowledgments The studies developed by our group were supported by grants (and a PNPDfellowship for Fabiola B. Filippin-Monteiro, PhD) from CNPq (Conselho Nacional
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de Desenvolvimento Científico e Tecnológico), CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior) and FAPESC (Fundação de Amparo à Pesquisa de Santa Catarina). The authors wish to thank the group of
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Prof. Ricardo J. Nunes and Prof. Rosendo A. Yunes for synthetizing the gallates
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for our studies.
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AC C
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effects
of
gallic
acid
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flavonoids and alkyl gallates in Caco-2 cells and in phospholipid vesicles,
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Figure caption
Figure 1. Scheme with the probable targets of gallic acid and its ester derivatives. Gallic acid was named GA and the names of the gallates were
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abbreviated according to the atom carbon number in the lateral chain, as
showed in the Table 1. Other abbreviations: PgP, P-glycoprotein (multi drug
resistance protein related); Fas, death receptor; FasL, death receptor ligand; ADAM, disintegrin and metalloproteinase domains; ErK, extracellular signal-
SC
regulated kinases; AkT/PKB, protein kinase B; MMP, matrix metalloproteinase; COX, cyclooxygenase; NF-kB nuclear factor kappa-light-chain-enhancer of activated B cells; IkB, NF-kB regulatory protein; ICAM, intercellular adhesion
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molecule; VCAM, vascular adhesion molecule; PARP, poly ADP-ribose polymerase; SOD, superoxide dismutase; CAT, catalase; ROS, reactive oxygen species; BCL-2, anti-apoptotic protein ; Bax, pro-apoptotic member of the Bcl-2 protein family; GSH reduced glutathione; γ GCS, γ glutamyl synthase; p21, cell cycle regulator protein; p53, genome reparative protein; G1/S phase, pos-
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EP
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mitotic cell cycle phase; ∆Ψm, mitochondrial membrane potential;
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Table 1. Chemical structures and C log P values of gallic acid n-alkyl ester derivatives.
OH
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OH
SC
HO
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COOR
Gallates
-R
C log P
GA
Gallic acid
-H
0.89
G1
Methyl
-CH3
0.92
G2
Ethyl
-(CH2)-CH3
1.27
G3
Propyl
-(CH2)2-CH3
1.73
G4
Butyl
-(CH2)3-CH3
2.13
G5
Pentyl
-(CH2)4-CH3
2.53
Hexyl
-(CH2)5-CH3
2.92
Heptyl
-(CH2)6-CH3
2.32
Octyl
-(CH2)7-CH3
3.72
Decyl
-(CH2)9-CH3
4.51
G10
Undecyl
-(CH2)10-CH3
4.90
Dodecyl
-(CH2)11-CH3
5.30
Tetradecyl
-(CH2)13-CH3
6.09
G16
Hexadecyl
-(CH2)15-CH3
6.89
G18
Octadecyl
-(CH2)17-CH3
7.68
G6 G7 G8
G12
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G14
EP
G10
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Abbreviation
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Cell line
IC50 (µM)
HL60
300
HL60RG dRLh-84
317 364
PLC/PRF/5 HLE
387
P388-D1
282
HeLa
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GA
358
Calu-6
10-50
A549
100-200
Wehi231
40
L929
250
U251n/U87
nd
K562
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33
IR-K562
4.75
AC C
EP
U251n/U87/ HUH-7/KB nd – not determined
Action
nd
References
↑H2O2; ↑OH; ↑ O2 ;↑ Ca ; ↑ PARP cleavage; ↑Release of cytochrome c; ↑ Caspase 3; ↑ Caspase 9; ↓ COX-1; ↓ COX-2; DNA damage; ↑catalase expression ↓ GSH; ↓ ∆Ψm; ↓ expression and activity of ADAM17; ↓ p-Erk; ↓ p-Akt; ↑ p21; ↑ p27; ↑ Bax; ↓ Cyclin D1/2/3/E; ↑ FasL; ↓ BcL-2; ↓ tyrosine BCR/ALB kinase phosphorylation; ↓ NF-kB activity
SC
Gallates
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Table 2. Values of IC50 of gallic acid (GA) in different cell lines, and the mechanism related to cell death induction.
-
2+
3; 6; 9; 10 12; 13; 14; 26 27; 29; 31; 36; 40; 42; 51
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G12
L929
40
HeLa
800
Wehi 231
1.5
L929
1
↑ ROS; ↓ GT cyt and mit; ↓ γ-GCS activity; ↓ ATP;
B16F10
45
↓ Adhesion cell; ↑NF-kB activation;
9; 10; 14; 42;
KB-C2
nd 22
↑ caspase 3; ↓ ∆Ψm;↓ Bcl-2; ↑ Bax;
55; 59
L1210 CEM
50
B16F10
82
L1210
12.2
CEM
9.8
MCF7/MCF7/ADR/KB-C2
nd
MDA-MB-231
1
Wehi 231
1
L929
1
B16F10 HL60 L1210 CEM B16F10
G14
L1210 CEM
nd - not determined
14; 23; 24;
12
SC
↑ SOD; ↓ ∆Ψm; ↑Fas; ↑FasL; ↑Caspase 3; ↑Caspase 8; ↑Caspase 9; ↑p53; ↑ERK; p38 activation; ↓ Nrf-2; ↓ GSH; ↓ γ-GCS
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G10
↑H2O2; ↑.OH; ↑semiquinone radical; DNA damage; ↑TBARS; ↓succinate dehydrogenase activity; ↑ Catalase;
Wehi231
26; 27; 50; 56
Blocking P-gp; ↑ NF-kB; ↓ NF-kB activity
↑ ROS, ↓ GT cyt and mit, ↓ γ-GCS activity, ↓ ATP;
9; 10
↓ Adhesion cell, ↑NF-kB activation; ↓ GSH mit, ↓ GSH cit
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G8
References
IC50 (µM) nd
↑ G1 phase; ↑ S phase; ↑ G2/M phases; ↑ p53; ↑ p21 ↓ cyclin D1; ↓ Bcl-2; ↑ phosphorylation of Erk1/2;
6; 9; 10; 12;
↓ protein tyrosine phosphorylation; ↓ ∆Ψm;
31; 36; 39; 42;
48
↑release cytochrome c; ↑ caspase 3; ↑ cleavage PARP; ↑ ROS;
55; 59; 61
33
↓ GT cyt and mit; ↓ γ-GCS activity; ↓ ATP; ↓ Adhesion cell;
EP
G3
Action
Cell line HL60/GM05757/THP-1
10.9
AC C
Gallates
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Table 3. Values of IC50 of gallates in different cell lines, and the mechanism related to cell death induction.
47.4 60
↑NF-kB activation; ↓ Bcl-2; ↑ Bax; Blocking P-gp; ↓ GSH mit and cit
8.5
↑ ROS; ↓ GT cyt and mit; ↓ γ-GCS activity; ↓ ATP;
>50
↓ICAM-1; ↓VCAM-1; ↓ GSH mit; ↓ GSH cit; DNA damage
9; 10; 11
AC C
EP
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SC
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