Jordana Wietschner
Gallic Acid: Chemical Properties and Cellular Responses
Presented to the S. Daniel Abraham Honors Program In Partial Fulfillment of the Requirements for Completion of the Program
Stern College for Women Yeshiva University April 28th, 2014
Jordana Wietschner Mentor: Dr. Harvey Babich, Biology
Abstract Gallic acid (3,4,5-trihydroxybenzoic acid), a polyphenol common in many plants has pharmaceutical properties with potential health benefits. As with most polyphenols, gallic acid exhibits both antioxidant and prooxidant properties. The connection between gallic acid acting both as a prooxidant and as an apoptosis-inducing agent is ill-defined. The research herein clearly demonstrates a cause-and-effect relationship between gallic acid’s production of hydrogen peroxide (H2O2) and its subsequent induction of apoptosis to human oral carcinoma HSC-2 cells. Using the FOX assay in a cell-free system, it was shown that gallic acid is a strong generator of H2O2. Reduced glutathione (GSH), a thiol-containing a tripeptide, is the main intracellular antioxidant in a cell’s repertoire of defenses against oxidative stress. Using the Intracellular Glutathione Assay, depletion of intracellular GSH, a sign of impending oxidative stress, was observed in HSC-2 cells. The potency of gallic acid to HSC-2 cells was lessened in the presence of scavengers of hydrogen peroxide, such as divalent cobalt. Flow cytometric analyses of HSC-2 cells treated with gallic acid indicated a concentration-dependent response for the induction of apoptosis, which was reversed in the presence of divalent cobalt. These studies showed that the proapoptotic activities of gallic acid to HSC-2 cancer cells were mediated through autooxidation of gallic acid leading to the induction of oxidative stress.
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Table of contents I.
Introduction ..........................................................................................................4
II.
Discussion a. Chemical properties of gallic acid .................................................................4 b. Cellular responses to gallic acid: In Vitro studies ..........................................12 i. Differential sensitivities of normal and cancerous cells to gallic acid ...........................................................................................................12 ii. Mechanism of gallic acid induced cytotoxicity: induction of oxidative stress ..................................................................................................14 iii. Gallic acid: an inducer of apoptosis...................................................21
III.
Conclusion ...........................................................................................................29
IV.
Acknowledgements ..............................................................................................30
V.
References ............................................................................................................31
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Introduction Nutraceutical, a term describing a natural nutrient that possesses pharmaceutical properties, is defined broadly as a food or a chemical constituent of a food that provides medical or health benefits, including the prevention and/or treatment of a disease (Brower et al., 1998). Much effort has been directed to identifying such food items and their active chemical components and to better understand their physiological modes of protection towards promoting human health. One such positive health benefit is the chemotherapeutic effects of these nutraceuticals. Many of today’s “super foods,” contain gallic acid, a naturally occurring polyphenol common in many plants and beverages, including tea, grapes, berries, wine, and even in some hard wood plant species. Interestingly, gallic acid exhibits both anti-oxidative and pro-oxidative qualities, with both qualities contributing to its positive effect on human health. Research on gallic acid has been performed using in vivo test systems with laboratory animal models and in vitro test methods with mammalian cells maintained in culture. This paper will review the chemical properties of gallic acid and will note the modes of bioactivity of developed primarily through in vitro test systems. In regards to the latter, note will be made of my research at Stern College for Women, which correlated the anticancer effects of gallic acid towards carcinoma cells derived from tissues of the human oral cavity with its pro-oxidative nature. Chemical properties of gallic acid Gallic acid (3,4,5-trihydroxybenzoic acid), a polyphenol common in many plantderived foods, occurs freely or complexed with tannins and is isolated upon hydrolysis with tannase. Polyphenols are a class of organic molecules with many phenol groups (a 6-carbon aromatic ring molecule with a hydroxyl group (Figure 1)). Gallic acid, when purified, is a
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colorless or yellow-colored crystal, with the molecular formula of C7H6O5, molecular weight of 170.12 g/mol, water solubility of 1.19 g/100 mL, density of 1.694 g/cm3 at 20o C, and melting point of 260o C.
Figure 1. Molecular structure of gallic acid
Chemically, gallic acid exhibits both antioxidant and pro-oxidant properties. As an antioxidant, gallic acid prevents the oxidation of biologically important molecules such as proteins and DNA, and thus is an inhibitor of oxidative stress. Oxidative stress is characterized by the production of oxygen-containing free radicals (termed reactive oxygen species, ROS) generated by aerobic cellular metabolism. ROS include superoxide free radical (O2-) and hydroxyl free radical (OH.), which can lead to the formation of hydrogen peroxide (H2O2), all which interfere with cellular processes and lead directly to cellular damage. A number of different chemical methodologies are used to describe and to quantify the antioxidant capacity of nutraceuticals with proposed antioxidant capabilities. The most common assays include measurements of the oxygen radical absorbance capacity (ORAC), of radical scavenging by 2,2-diphenyl-1-picrylhydrazyl (DPPH), of the vitamin C equivalent antioxidant capacity (VCEAC), and of superoxide dismutase (SOD)-like activity. The ORAC assay, recommended by the United States Department of Agriculture, measures oxidative
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degradation of a fluorescent molecule after mixture with free radical generators. Antioxidants protect the fluorescent molecule from degeneration; a fluorometer quantifies the fluorescence and therefore the degree of protection afforded by the test compound studied. Antioxidant protection is based on comparative fluorescence in solutions with and without the test agent. Fluorescent intensity decreases with an increased exposure to the generated free radicals. Based on the value obtained by the assay, comparisons can be made between related compounds and their proposed antioxidant capacities. For gallic acid the ORAC value was approximately 2,000 per mol tested sample (x103), which can be compared to an ORAC value of approximately ± 500 for L-ascorbic acid (vitamin C), a known nutraceutical and antioxidant. For ellagic acid, another polyphenolic nutraceutical, the ORAC value was >3,000, indicating a greater antioxidant potential than gallic acid (Ito, 2011). DPPH, a stable free radical, is a scavenger for other radicals and used to measure the radical scavenging activity of antioxidants. Reduction of the rate of a chemical reaction by DPPH, an indicator of the radical nature of that reaction, is used to quantify the antioxidant activity of foods by testing their abilities to act as free radical scavengers (Figure 2). A DPPH solution is mixed with varying concentrations of the test compound and
Figure 2. The structure of DPPH and its reduction
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incubated at room temperature. The odd electron in the DPPH free radical molecule gives the solution a purple color, which changes to yellow upon neutralization. Decolorization is stoichiometric with respect to the number of electrons captured (Miller et al., 2012). Gallic acid had a DPPH value IC50 of 1.9 µM, whereas that of ellagic acid was 1.7; the IC50 values represent the concentration of antioxidant that reduced the free radical concentration by about 50%. As noted with the ORAC assay, the antioxidant potential of gallic acid was less than that of ellagic acid (Ito, 2011). The antioxidant potential of gallic acid was concentration dependent; increasing the concentration of gallic acid from 0.25 to 90 µM progressively increased DPPH scavenging. These values included approximately 80% DPPH scavenging at 60 µM gallic acid, indicative of the strong antioxidant activity (Habtemariam, 2011). An additional assay used to quantify the antioxidant capacity of gallic was is the VCEAC assay, which focuses on expressing the antioxidant capacity of plant foods using vitamin C (mg/100g) as the reference. Kim et al. (2004) determined the VCEAC values of various polyphenols, including a series of benzoic acid derivatives. Within this series, gallic acid ranked as the strongest antioxidant. The sequence of antioxidant potency, as VECAC values (in mg/L), was gallic acid (324.3 ± 4.7), 2,3- dihydroxybenzoic acid (169.6 ± 2.7), 2,4-dihydroxybenzoic acid (165.5 ± 4.0), protocatechuic acid (163.2 ± 2.9), vanillic acid (117.2 ± 4.5), gentisic acid (90.8 ± 4.9), syringic acid (80.4 ± 2.9), 3-hydroxybenzoic acid (53.7 ± 1.7), 4-hydroxybenzoic acid (4.8 ± 0.6), and salicylic acid (1.4 ± 0.4). The high antioxidant capacity of gallic acid was attributed to its 3 hydroxyl groups attached to the aromatic ring.
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Another method used to determine antioxidant capacity of a compound is to define its superoxide dismutase (SOD)-like activity. The scavenging activities of O2- anions generated by a xanthine-XOD enzymatic reaction were compared for gallic acid and ellagic acid. For gallic acid the IC50 value (μM) was 13, whereas for ellagic acid it was >100, again showing the greater antioxidant capacity of ellagic acid over gallic acid (Ito, 2011). Interestingly, polyphenols, including gallic acid, also exhibit pro-oxidant activity. As opposed to antioxidant activity, which scavenges ROS and inhibits oxidative stress, prooxidants generate ROS and contribute to oxidative stress. When polyphenols are amended to cell culture medium, ROS, principally H2O2, are detected. Elevated levels of H2O2 derived from polyphenols can be toxic to and may reduce the growth of tumor cells (Lee et al. 2004). Lee et al. (2005), in studying the antiproliferative effects of dietary phenolic substances, showed in cell cultured medium gallic acid acted as a pro-oxidant, generating H2O2 in a dose dependent manner. In the summer of 2013, as a research intern working with Biology faculty at SCW and with fellow undergraduates, I researched the pro-oxidant nature of gallic acid (Schuck et al. 2013). The FOX assay was used to determine and quantify the generation of ROS, primarily H2O2, in cell culture medium. This assay is based upon quantifying the oxidation of ferrous ions (Fe2+) to ferric ions (Fe3+) by H2O2 or other ROS produced by a test agent. The resultant Fe3+ ion bonded to xylenol orange and produced a colored complex, measured spectrophotometrically and compared to a standard curve using H2O2. In the initial studies, samples of gallic acid, at concentrations ranging from 0 to 200 µM, were incubated at room temperature for 4 hours in cell culture medium. At the end of the incubation, samples were mixed with methanol and the FOX reagent, vortexed, and incubated for 20 minute at room temperature. The solutions were centrifuged and absorbance
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of the supernatant was recorded at 595 nm against a blank consisting of cell culture medium, methanol, and the FOX reagent. A standard curve of H2O2 from 0 to 200 µmol/L was produced to quantify the level of peroxide generated by gallic acid. Gallic acid was shown to be a strong generator of H2O2. As gallic acid concentration increased, the amount of H2O2 detected in the medium increased (Figure 3). FOX assay 4-Hr incubation in exposure medium
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H2O2 (µmol/L)
140 120 100 80 60 40 20 0 0
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Gallic acid (µM) Figure 3. Generation of hydrogen peroxide, as determined by the FOX assay, in cell culture medium amended with varying concentrations of gallic acid. Hydrogen peroxide was the reference peroxide used to construct the standard curve.
Lipid peroxidation as an indicator of the pro-oxidant nature of gallic acid was also studied. Free radicals are able to extract electrons from cell membranes, thereby damaging the membrane. ROS react with polyunsaturated fatty acids to yield fatty acid radicals. In the presence of O2 a lipid peroxy fatty acid radical forms. This chain reaction occurs and continues until two free radicals interact and generate a non-radical species. These reactions can be quantified by measuring the production of malondialdehyde (MDA), the end product of lipid peroxidation. In the assay, MDA was made to react with thiobarbituric acid (TBA),
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yielding thiobarbituric acid reactive species (TBARS), measured by visible or fluorescence spectrophotometry. Cells were treated with varying concentrations of gallic acid, Fe2+, and a combination of gallic acid and Fe2+. After 3 hours, the agents were aspirated and trichloroacetic acid was added. The cells were then scraped and the resulting suspensions were centrifuged to remove the precipitated proteins. A solution of 1% thiobarbituric acid in NaOH was added to the supernatants, which were heated at 95°C for 20 minutes. Samples were centrifuged again after cooling, the TBARS, occurring as a pink chromagen, were measured at 532 nm. A 3hour exposure to 100 µM gallic acid induced lipid peroxidation, which was moderately potentiated in the presence of Fe2+ (Figure 4).
O.D.532 nm (TBA-reactive substances)
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Figure 4. Gallic acid-induced lipid peroxidation, as mediated by Fe2+.
Fluorescent microscopy was then used to visualize ROS production by gallic acid. Cells, treated with varying concentrations of gallic acid, were treated with dichlorofluorescein diacetate (DCFDA) for 30 minutes at 37ºC. Thereafter, the DCFDA was
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removed and cells were incubated for an additional 30 minutes at 37ºC in warm exposure medium. During this process, the internalized dye was hydrolyzed by intercellular esterases, yielding dichlorofluorescein (DCF). Upon intracellular oxidation of gallic acid, a fluorescent product was noted. The cells were viewed microscopically using a filter set capable of detecting the fluorescent oxidized product (with an absorption maximum of 492 nm) and the fluorescent emission maximum of 517 nm. Minimal fluorescence was detected in control cells (Figure 5A), indicating the background intracellular ROS. Fluorescence, in a concentration-dependent mode, was noted in cells treated with 100 and 200 μM gallic acid (Figure 5B, C), and, as an internal control, in cells treated with 200 µM H2O2 (Figure 5D).
Figure 5. HSC-2 cells were untreated and treated with gallic acid, without or with ascorbic acid, or with hydrogen peroxide, stained with DCFDA, and viewed with a fluorescent microscope. (A) untreated control; (B) 4-hr treatment with 100 µM gallic acid; (C) 4-hr treatment with 200 µM gallic acid; (D) 4-hr treatment with 33 µM hydrogen peroxide (positive oxidant control)
Aside from its antioxidant and prooxidant activities, another important chemical property of gallic acid is lipophilicity, which denotes the ability of a compound to dissolve in nonpolar substances. The lipophilicity of a compound is described by its partition
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coefficient, defined as the ratio of concentrations of a compound in a mixture of two immiscible phases at equilibrium. Most commonly, one solvent is water and the other is octanol. The logarithmic ratio of the concentrations is referred to as log P value, which is useful in determining the ability of a chemical to transverse the phospholipid bilayer of the plasma membrane. The log P value of gallic was calculated as 0.91. Adding functional groups to gallic acid increased its lipophilicity and therefore its log P value. For example, the log P for methyl gallate was 1.54, for ethyl gallate it was 2.07, and for propyl gallate it was 3.13. An increase in the log P value correlated to an increase in cytotoxicity of the chemical gallic acid-derivative (Sergediene et al. 1999). Cellular Responses to Gallic Acid: In Vitro Studies In summer 2013, as a research intern, I performed in vitro studies to determine the response of cancer cells to gallic acid and specifically to demonstrate a correlation between the pro-oxidant properties of gallic acid and its proapototic properties. Research was performed in the laboratory of Dr. Jeffrey Weisburg with Biology faculty at Stern College for Women and with fellow undergraduates. Differential sensitivities of normal and cancerous cells to gallic acid When determining cytotoxicity of gallic acid on cancerous cells, it is also important to determine its cytotoxic effects on normal, healthy cells. While many chemotherapeutics today are successful in killing tumor cells, they can also harm healthy cells. Finding a method of ridding the body of cancerous cells while not harming healthy cells is a welcome alternative. A comparison between the potentially different effects of gallic acid on normal and tumor cells can therefore have positive consequences on the potential of gallic acid usage
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as a chemotherapeutic. Studies conducted by Schuck et al. (2013) evaluated the cytotoxic effects of gallic acid on normal gingival HF-1 fibroblasts and carcinoma HSC-2 cells isolated from the oral cavity. Cytotoxicity was quantified with the neutral red assay, based on the ability of cells to uptake and accumulate neutral red in their lysosomes (Repetto et al. 2008). The HSC-2 carcinoma cells were more sensitive to gallic acid than were the HF-1 fibroblasts, was a 24-hr midpoint cytotoxicity value (NR50) of 80 µM gallic acid for the HSC-2 cells and 175 µM for the HF-1 fibroblasts (Figure 6).
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Figure 6. Comparative 24-hour cytotoxicity of gallic acid to human oral carcinoma HSC-2 cells and to human normal gingival HF-1 fibroblasts.
Similar research has shown that gallic acid was more cytotoxic to cancerous cell as opposed to normal cells. Russel et al. (2011) found human prostate cancer LNCap cells to be more sensitive to gallic acid than normal prostate epithelial cells and Inoue et al. (1995) and Isuzugawa et al. (2001) noted rat hepatoma dRLH-84, human epithelioid carcinoma, and human hepatoma PLC/PRF/5 cancerous cells were less tolerant of exposures to gallic acid than were normal cells, including rat primary cultured hepatocytes, keratinocytes, macrophages, endothelial cells, and normal fibroblasts. Nair et al. (2007) observed that, in 13
general, normal cells are more resistant to polyphenols than are cancer cells. The reason normal cells have greater resistance to gallic acid, when acting as a prooxidant, than cancer cells may reflect differences in their intracellular defenses against oxidative stress, as exemplified by the higher intracellular levels of catalase in normal cells (Isuzugawa et al., 2001). Catalase, an enzyme, catalyzes the decomposition of H2O2 to H2O and O2, thereby detoxifying intracellular H2O2 generated by gallic acid. Tumor cells contain depressed levels of catalase due to their lower rate of catalase transcription, resulting from increased methylation of the gene encoding for catalase (Sun et al., 1993). Cancer cells, therefore, were more vulnerable to ROS toxicity and more likely to undergo apoptosis. Another reason, proposed by Schumacker et al. (2006), explained that, in general, cancer cells possess more metabolic activity than normal cells and therefore may have greater basal levels of mitochondrial-derived ROS. The additional ROS generated by gallic acid could therefore overwhelm cellular defense mechanisms against oxidative stress, leading to an increase in cytotoxicity. Nair et al. (2007), in a study using human colon HT-29 cancer cells and human prostate cancer PC-3 cells, noted that cancerous cells required overexpression of certain signaling molecules, such as epidermal growth factor receptor (EGFR) and NF-κB transcription factor, to maintain cell proliferation and survival, whereas normal cells do not exhibit an overexpression of signaling molecules. Polyphenols are inhibitory to many of these signal molecules and cancer cells, with their greater number of intracellular targets, exhibit greater sensitivity to polyphenols, including gallic acid, than normal cells. Mechanism of Gallic Acid-Induced Cytotoxicity: Induction of Oxidative Stress After observing that gallic acid was cytotoxic to cancer cells, Schuck et al. (2013) determined the mechanism of its cytotoxicity. My portion of that study showed that gallic 14
acid induced oxidative stress, which subsequently led to apoptosis in HSC-2 cells. Oxidative stress reflects the imbalance between a cell’s production of ROS and cellular defense against these potentially toxic substances. ROS are produced as a by-product of mitochondrial aerobic cellular respiration and include H2O2, O2.- , and (OH.), among others. These oxygen derivatives are all strong oxidizing agents and can therefore damage cells by inactivating enzymes and other proteins, damaging DNA and RNA, and causing lipid peroxidation. To protect itself against the toxic effects of ROS, the cell employs various defense mechanisms and ROS scavengers, including intracellular glutathione, enzymes such as superoxide dismutase and peroxidase, and other scavengers including pyruvate and divalent copper. If the ROS overwhelm the cell, however, and the defense mechanisms therefore cannot properly protect the cell, oxidative stress occurs, which can cause damage the cell or result in apoptosis. One important intracellular defense is the presence of reduced glutathione (GSH) (figure 7).
Figure 7. Molecular structure of glutathione.
GSH, a tripeptide, contains a thiol group from its cysteine amino acid, which aids in its reduction potential by donating an electron to other unstable molecules, such as ROS. In the process, GSH is converted to glutathione disulfide (GSSG), its oxidized form, via glutathione peroxidase. GSSG can then be reduced to GSH through the use of glutathione reductase,
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using NADPH as an electron donor. The overall process allows GSH to be continuously recycled and used. Glutathione therefore protects the cell against ROS by neutralizing them. An increased ratio of GSSG to GSH, signifying the overuse of GSH is thus an indicator of oxidative stress. GSH is the main cellular defense against ROS (Circu et al. 2010) and testing for the intracellular levels is thereby a determinant of intracellular ROS. Other cellular defenses include the actions of various enzymes such as superoxide dismutase, catalase, and peroxidase. Superoxide dismutase catalyzes the dismutation - a redox reaction in which a substance is both oxidized and reduced to form two different products - of O2.- to molecular oxygen (O2) and H2O2, which can be degraded by catalase. Catalase, a tetramer with four porphyrin heme groups, is a common enzyme found in nearly all organisms that are exposed to oxygen and aids in the decomposition of H2O2 to O2 and water (H2O) (Boon et al., 2007). Peroxidase can also be used to catalyze the reduction of H2O2 to H2O (figure 8).
Figure 8. The action of superoxide dismutase, catalase, and peroxidase (Todar, 2008).
Additional scavengers of ROS include pyruvate and divalent cobalt (Co2+). Pyruvate, as well as Co2+, has been found to be a scavenger of H2O2 (Long et al., 2009). Pyruvate is oxidatively decarboxylated by H2O2 to yield acetate, carbon dioxide (CO2), and H2O; Co2+ catalytically decomposes H2O2 to H2O and O2 (Babich et al., 2011). Although the cell employs these various protective measures against ROS, generated ROS may reach elevated levels and eventually overwhelm cellular defenses and induce
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oxidative stress. The FOX assay, performed during my internship and also by Lee et al. (2005), showed that gallic acid generated H2O2, confirming that gallic acid itself actually produced this ROS. Schuck et al. (2013) focused on the quantity of H2O2 produced and whether the level generated was sufficient to cause oxidative stress and cytotoxicity to HSC2 cells. While the addition of gallic acid resulted in generation of H2O2, studies indicated that levels of H2O2 were lowered by co-incubations with catalase, pyruvate, and divalent copper, all scavengers of H2O2. HSC-2 cells were treated with cytotoxic concentrations of gallic acid in the presence of catalase, pyruvate, and Co2+. Upon co-incubation of gallic acid with these
Percent of control (NR assay)
H2O2 scavengers, the toxicity of gallic acid was lessened (Figure 9).
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HSC-2 cells 24-hr exposure
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* * 75 µM gallic acid 100 µM gallic acid 125 µM gallic acid
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Catalase Pyruvate Cobalt chloride (200 Units/ml) (110 mg/L) (250 µM)
Figure 9. Cytotoxicity of gallic acid to HSC-2 cells in the presence of scavengers of hydrogen peroxide as compared to control.
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These studies indicated that gallic acid-induced cytotoxicity occurred through the generation of H2O2 since cytotoxicity was lessened in the presence of scavengers of H2O2. Similar studies by Chen et al. (2009) found that catalase lessened the toxicity of gallic acid to human prostate DU145 cancer cells. Studies have also shown that exposure to gallic acid caused an increase in the production of ROS, which subsequently results in a decreased level of intracellular GSH and subsequent cytotoxicity due to a decrease in cellular defenses. HSC-2 cells, maintained in growth medium in tissue culture plates, were washed with phosphate buffered saline (PBS) and then treated with 25 to 150 µM of gallic acid. Thereafter, cells were then washed again with PBS, lysed with Triton X-100, and proteins were precipitated with sulfosalicylic acid. After cells were harvested by scraping and centrifugation, GSH levels were analyzed by determining the oxidation of GSH (in a 6 mM solution of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and prepared in a phosphate buffer/EDTA (pH 7.5) to GSSG, with a stoichiometric formation of 5-thio-2-nitrobenzoic acid, a yellow chromagen measured spectrophotometrically at 412 nm. My studies showed that a gradual increase in the concentration of gallic acid from 25 to 150 µM resulted in progressively reduced levels of intracellular GSH (figure 10). Similar studies showing a decrease in intracellular GSH with an increase in concentration of gallic acid were performed using human pulmonary adenocarcinoma Calu-6 cells (You et al., 2011) and human primary vein endothelial cells (Park et al., 2012).
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Gallic acid (µM) Figure 10. Effect of a 4-hr exposure to gallic acid on the intracellular levels of reduced glutathione in HSC-2 cells
Cells were also exposed to gallic acid in the presence of 250 µM Co2+, a scavenger of H2O2. Levels of intracellular GSH were unaffected by gallic acid in the presence of the H2O2 scavenger. HSC-2 cells were also treated with various substances that reduced the levels of GSH to confirm that the pro-oxidant nature of gallic acid was responsible for growth-inhibitory effects. With lower levels of GSH, ROS can more easily overwhelm the cell and cause oxidative stress. Each depleter of GSH interfered with a different aspect of GSH-GSSG cycling. The redox cycle of GSH/GSSG employs many enzymes to synthesize the production of GSH and subsequently aid in its oxidation GSSG and recycling to GSH. Intracellularly, GSH is produced in two steps: the first step controlled by ɣ-glutamylcysteine synthetase, which catalyzes the ATP-dependent condensation of cysteine and glutamate to form the dipeptide, ɣ-glutamylcysteine; the second step is controlled by glutathione
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synthetase, which catalyzes the condensation of ɣ-glutamylcysteine and glycine, forming GSH (Njålsson, 2005). Glutathione-S-transferase can then aid in catalyzing the conjugation of electrophilic substrates to GSH (Sheehan et al., 2001). Subsequently, GSH is oxidized to GSSG via glutathione peroxidase. Finally, GSSG is reduced back to GSH using glutathione reductase. GSH-depleting reagents used included 1-chloro-2,4-dinitrobenzene (CDNB), DLbuthionine-[S,R]-sulfoximine (BSO), and bis-(2-chloroethyl)-N-nitrosourea (BCNU). CDNB inactivated glutathione S-transferase, BSO inhibited glutamylcysteine synthetase, and BCNU inhibited glutathione reducatse (Babich et al., 2011). Pretreatment with CDNB or BCNU and co-exposure with BSO potentiated the toxicity of gallic acid to the HSC-2 cells, as these treatments lessened the intracellular level of GSH (Figure 11).
Percent of control (NR assay)
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25 µM gallic acid 50 µM gallic acid 75 µM gallic acid 100 µM gallic acid
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Figure 11. Cytotoxicity of gallic acid toward HSC-2 cells in the presence of depleters of intracellular reduced glutathione.
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As noted earlier, gallic acid also induced lipid peroxidation in HSC-2 cells, another indicator of cytotoxicity by ROS. This was verified, as a three-hour exposure of HSC-2 cells to gallic acid (100 µM) induced lipid peroxidation, which was moderately potentiated in the presence of ferrous iron (Fe2+) (Schuck et al., 2013). Gallic Acid: an Inducer of Apoptosis Apoptosis, the process of programmed cell death, is generally regulated in an organism as a whole, working to balance cellular proliferation with cellular destruction, while maintaining constant cell numbers in somatic tissues. It also functions to rid the body of genetically damaged and potentially malignant cells by their removal before they multiply. In the process of apoptosis, chromosomal DNA is fragmented, chromatin condenses, the nucleus dissociates into small pieces, and the cytoskeleton is disrupted. Eventually, the cell shrinks and is broken into various membrane-enclosed fragments. Apoptotic cells and their cellular fragments are recognized and phagocytosed by macrophages, thereby removing them from the somatic tissues. Apoptosis occurs through a series of cellular pathways. One such method includes mitochondria sending out a signal to induce apoptosis, triggering the inactivation of a molecule termed the inhibitor of apoptosis proteins (IAP). Once inactivated, apoptosis occurs through caspases, which are proteases containing a cysteine residue at their active sites and that cleave substrate proteins after aspartic acid residues. A cascade chain of initiator and effector caspases cleave various cellular target proteins, ultimately leading to cell death. Apoptotic protease activating factor (Apaf-1) and cytochrome c, released by mitochondria, bind to an apoptosome, a multi-subunit complex, which activates caspase-9 to cleave and thereby activate the effector caspase-3. The effector caspase then cleaves
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different cellular proteins, including nuclear lamins, cytoskeletal proteins, and an inhibitor of deoxyribonuclease (DNase), ultimately leading to cell death (Cooper et al., 2007). Genes, occurring either as antiapoptotic or proapoptotic activators, regulate apoptosis in cells. The triggering of apoptosis is based on the ratio of activity between the proapoptotic and antiapoptotic gene products. Proapoptotic genes include Bax, Bak, and BH3 and antiapoptotic genes include Bcl-2. When activated, Bax and Bak form oligomers in the mitochondrial outer membrane, leading to the release of cytochrome c and to caspase activation. Apoptosis can also be induced directly by activating certain receptors on the target cell. One such cell surface receptor is the Fas receptor. The Fas receptor activates an initiator caspase, caspase-8, which can then initiate downstream effector caspases. Studies have shown that gallic acid induced apoptosis by activating the Fas protein receptor and initiating the caspase cascade (Hsu et al., 2006). Studies with HeLa cells determined that apoptosis induction with gallic acid included the upregulation of Bax and the downregulation of Bcl-2 genes (You et al., 2010). Apoptosis induced by gallic acid was correlated with elevated levels of ROS, leading to increased oxidative stress (Chen et al., 2009). Increased oxidative stress may lead to decreased levels of GSH and thereby reduce cellular antioxidant defenses against ROS damage. Poot et al. (1995) observed that the induction of apoptosis was inversely related to the cellular GSH content. Using flow cytometry, my research was directed to correlating the prooxidant property of gallic acid with its subsequent induction of apoptosis in carcinoma HSC-2 cells. In addition, Western blotting was employed to visualize poly(ADP-ribose)-polymerase
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(PARP) fragments, an indicator of caspase activities, and microscopy to visualize morphological cellular alterations characteristic of apoptosis. Flow cytometry is a technique that can directly differentiate and measure the number of cells that are alive, dead, or undergoing apoptosis. In this technique, the cells were suspended in fluid and passed through an electronic detection apparatus. The cells, passed single file through a light beam and detected by a flow cytometer, had been treated with a DNA binding dye with differential permeability for viable, apoptotic, and nonviable cells. Data collection and analysis was performed using a computer. In my studies, HSC-2 cells were untreated (control) or treated with varying concentrations of gallic acid for 24 hours. The cells were then washed and diluted to a concentration slightly less than 5 x 105 cells/mL. A 20 L sample of cells and 380 L of Guava ViaCount Reagent were placed on ice in the dark for 5 minutes. A Guava Easycyte Miniflow Cytometer was used to determine cell viability, apoptosis, and cell death. As seen in Figure 12, gallic acid induced apoptosis in a concentration-dependent manner.
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(A)
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Figure 12. A) Representative FACS profiles of the viability and apoptosis of HSC-2 cells after a 20-hr treatment with varying concentrations of gallic acid. Viable cells are located on the left side of each panel; apoptotic cells between the two lines; and dead cells on the right side of the panel. Panel 1 is untreated cells, panel 2 is cells treated with 75 µM gallic acid; panel 3 is cells treated with 100 µM gallic acid; panel 4 is cells treated with 125 µM gallic acid; and panel 5 is cells treated with 150 µM gallic acid. (B) Cytometric analysis, using Guava ViaCount Reagent, of a 20-hr exposure of HSC-2 cells to increasing concentrations of gallic acid. Percentage of viable, apoptotic, and dead cells were quantified by the flow cytometer.
Gallic acid-induced apoptosis was apparently mediated by oxidative stress, as the number of apoptotic and dead cells were reduced in the presence of Co2+, a scavenger of H2O2 (Figure 13).
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HSC-2 carcinoma cells 20-hr exposure
Percentage of total
80
60
40
20
Apoptotic cells
Dead cells
0
0
75 125 0 75 125 0 75 Gallic acid (µM) + 250 µM CoCl 2
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Figure 13. (A) Representative FACS profiles of viabile and apoptotic HSC-2 cells after a 20-hr treatment with 250 µM CoCl2, alone (control) and in the presence of 75 and 125 µM gallic acid. Viable cells are located on the left side of each panel; apoptotic cells between the two lines; and dead cells on the right side of the panel. Panel 1 is control cells, panel 2 is cells treated with CoCl2 and 75 µM gallic acid; and panel 3 is cells treated with CoCl2 and 125 µM gallic acid. (B) Cytometric analysis, using Guava ViaCount Reagent, of a 20-hr exposure of HSC-2 cells to 250 µM CoCl2, alone (control) and in the presence of 75 and 125 µM gallic acid. Percentage of viable, apoptotic, and dead cells were quantified by the flow cytometer.
Another study to determine if gallic acid induced apoptosis in the HSC-2 cells was the observation of poly(ADP-ribose) polymerase (PARP) fragments. PARP catalyzes the formation of poly(ADP-ribose), a post-translational modification involved in various cellular functions, including surveillance of genome integrity (Isabelle et al., 2010). Proteolytic inactivation and cleavage of PARP by caspases, in particular by caspase-3, is a useful monitor of the induction of apoptosis. PARP fragmentation in untreated cells and in cells treated with gallic acid for 24 hours was determined. After the 24-hour incubation, the cells
27
were lysed with RIPA buffer, centrifuged to remove cellular debris, and protein concentration of the cell lysates was quantified with a BCA Protein Assay Kit. Equivalent levels of total protein from each sample were separated by SDS-PAGE electrophoresis, electroblotted to nitrocellulose membranes, and blocked with 5% dry milk in Tris-buffered saline containing 0.1% Tween 20. Membranes, probed with polyclonal anti-PARP to detect full-length and cleaved PARP, were incubated with the appropriate peroxidase-conjugated secondary antibodies and were developed using an ECL detection kit. PARP cleavage products were detected via Western blot analysis only in those protein lysates of HSC-2 cells exposed to gallic acid. Again, oxidative stress induced by gallic acid was shown, as in the presence of CoCl2, gallic acid failed to trigger apoptosis (Figure 14).
Figure 14. Immunoblot analysis of PARP cleavage in HSC-2 cells untreated and treated with increasing concentrations of gallic acid (GA) after a 20-hr exposure in exposure medium without and with 250 µM ClCl 2. Cellular proteins, separated by 10% SDS-PAGE, were transferred to nitrocellulose and probed with an antibody specific for full length PARP and one specific for the ~85 kD C-terminal cleaved fragment of PARP, which were identified by comparison to a standard molecular weight marker. An anti-actin antibody was the loading control.
Induction of apoptosis by gallic acid, as mediated by oxidative stress, was also shown microscopically. Cells treated with gallic acid and in the presence of pyruvate and Co2+, both scavengers of ROS, were visualized via microscopy. Control HSC-2 cells had a large
28
cytoplasm and nucleus, whereas for cells treated with gallic acid, the cytoplasmic mass was reduced, the nucleus was hypercondensed, and cell blebbing was evident - all morphological signs of apoptosis. In cells cotreated with gallic acid and either pyruvate or Co2+, however, these cellular indications of apoptosis were not observed (Figure 15).
A
B
C
D
Figure 15. Brightfield microscopy of HSC-2 cells untreated and treated with gallic acid for 24-hr, in the absence and presence of scavengers of hydrogen peroxide. (A) Control, untreated cells; (B) treated with 125 µM gallic acid, alone (C) treated with 125 µM gallic acid in the presence of 110 mg/L pyruvate; and (D) treated with 125 µM gallic acid in the presence of 250 µM CoCl2.
Conclusion Research has indicated gallic acid’s role as an antioxidant and/or as a prooxidant. The FOX assay, used to determine and quantify the generation of ROS (primarily
29
H2O2) in cell culture medium, displayed an increase in the production of H2O2 in cell culture medium amended with increasing concentrations of gallic acid. Studies conducted in our lab have shown that gallic acid is more cytotoxic to HSC-2 cells than to normal cells. Similar studies have determined this to be a characteristic of other cancerous cell lines as well. Gallic acid, therefore, has the potential to be used as a less harmful chemotherapeutic agent than past cancer drugs. Exposure to gallic acid caused a decrease in levels of intracellular glutathione, increased lipophilicity, and an increase in intracellular ROS levels. The induction of apoptosis was noted in a concentration-dependent manner in cells exposed to gallic acid. In the presence of scavengers of ROS such as Co2+ or reagents that deplete intracellular glutathione levels, however, the previously described effects were not observed. These studies demonstrated a correlation between the autooxidation of gallic acid, which generates H2O2 to induce oxidative stress, and the subsequent initiation of apoptosis in cancer cells. Acknowledgments I would like to express my gratitude toward Dr. Harvey Babich for his constant help and motivation in researching and writing this paper. I would also like to thank Dr. Jeffrey Weisburg for his support and guidance in the laboratory when conducting the research presented. Lastly, I would like the express my appreciation to the S. Daniel Abraham Honors Program at Stern College for Women, Yeshiva University for support, in part, of this research.
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References Babich, H., Schuck, A. G., Weisburg, J. H., & Zuckerbraun, H. L. (2011). Research strategies in the study of the pro-oxidant nature of polyphenol nutraceuticals. Journal of toxicology, 2011. Boon EM, Downs A, Marcey D. Catalase: H2O2: H2O2 Oxidoreductase. Catalase Structural Tutorial Text. (2007) Brand-Williams, W., Cuvelier, M. E., & Berset, C. L. W. T. (1995). Use of a free radical method to evaluate antioxidant activity. LWT-Food Science and Technology, 28(1), 25-30. Brower, V. (1998). Nutraceuticals: poised for a healthy slice of the healthcare market?. Nature biotechnology, 16, 728-732. Chen, H. M., Wu, Y. C., Chia, Y. C., Chang, F. R., Hsu, H. K., Hsieh, Y. C., Yuan, S. S. (2009). Gallic acid, a major component of Toona sinensis leaf extracts, contains a ROSmediated anti-cancer activity in human prostate cancer cells. Cancer letters, 286(2), 161-171. Circu, M. L., & Aw, T. Y. (2010). Reactive oxygen species, cellular redox systems, and apoptosis. Free Radical Biology and Medicine, 48(6), 749-762. Cooper, G. M., & Hausman, R. E. (2007). Add Chapter. The cell: a molecular approach (4th ed., p. 790-791). Washington, D.C.: ASM Press. Habtemariam, S. (2011). Methyl-3-O-methyl gallate and gallic acid from the leaves of Peltiphyllum peltatum: isolation and comparative antioxidant, prooxidant, and cytotoxic effects in neuronal cells. Journal of medicinal food, 14(11), 1412-1418. Hsu, C. L., Lo, W. H., & Yen, G. C. (2007). Gallic acid induces apoptosis in 3T3-L1 preadipocytes via a Fas-and mitochondrial-mediated pathway. Journal of agricultural and food chemistry, 55(18), 7359-7365. Inoue, M., Suzuki, R., Sakaguchi, N., Li, Z., Takeda, T., Ogihara, Y., ... & Chen, Y. (1995). Selective induction of cell death in cancer cells by gallic acid. Biological and Pharmaceutical Bulletin, 18(11), 1526-1530. Isabelle, M., Moreel, X., Gagné, J. P., Rouleau, M., Ethier, C., Gagné, P., ... & Poirier, G. G. (2010). Investigation of PARP-1, PARP-2, and PARG interactomes by affinity-purification mass spectrometry. Proteome science, 8(1), 22. Isuzugawa, K., Inoue, M., & Ogihara, Y. (2001). Catalase contents in cells determine sensitivity to the apoptosis inducer gallic acid. Biological & pharmaceutical bulletin, 24(9), 1022-1026. Ito, H. (2011). Metabolites of the ellagitannin geraniin and their antioxidant activities. Planta Medica-Natural Products and MedicinalPlant Research, 77(11), 1110. 31
Kim, D. O., & Lee, C. Y. (2004). Comprehensive study on vitamin C equivalent antioxidant capacity (VCEAC) of various polyphenolics in scavenging a free radical and its structural relationship. Critical reviews in food science and nutrition, 44(4), 253-273. Lee, K. W., Hur, H. J., Lee, H. J., & Lee, C. Y. (2005). Antiproliferative effects of dietary phenolic substances and hydrogen peroxide. Journal of agricultural and food chemistry, 53(6), 1990-1995. Lee, K. W., Lee, H. J., & Lee, C. Y. (2004). Vitamins, phytochemicals, diets, and their implementation in cancer chemoprevention. Critical reviews in food science and nutrition, 44(6), 437-452. Long, L. H., & Halliwell, B. (2009). Artefacts in cell culture: pyruvate as a scavenger of hydrogen peroxide generated by ascorbate or epigallocatechin gallate in cell culture media. Biochemical and biophysical research communications, 388(4), 700-704. Prakash, A., Rigelhof, F., & MIller, E. (2001). Antioxidant activity. Medallion Laboratories Analytical Progress, 19(2), 1-4. Njälsson, R., & Norgren, S. (2005). Physiological and pathological aspects of GSH metabolism. Acta paediatrica, 94(2), 132-137. Repetto, G., del Peso, A., & Zurita, J. L. (2008). Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nature protocols, 3(7), 1125-1131. Russell, L. H., Mazzio, E., Badisa, R. B., Zhu, Z. P., Agharahimi, M., Millington, D. J., & Goodman, C. B. (2011). Differential cytotoxicity of triphala and its phenolic constituent gallic acid on human prostate cancer LNCap and normal cells. Anticancer research, 31(11), 3739-3745. Sheehan, D., Meade, G., Foley, V., & Dowd, C. (2001). Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Biochem. J, 360, 1-16. Schuck, A. G., Weisburg, J. H., Esan, H., Robin, E. F., Bersson, A. R., Weitschner, J. R., Lahasky, T., Zuckerbraun H. L., & Babich, H. (2013). Cytotoxic and proapoptotic activities of gallic acid to human oral cancer HSC-2 cells. Oxidants and Antioxidants in Medical Science, 2(4), 265-274. Schumacker, P. T. (2006). Reactive oxygen species in cancer cells: live by the sword, die by the sword. Cancer cell, 10(3), 175-176. Sergediene, E., Jönsson, K., Szymusiak, H., Tyrakowska, B., & Rietjens, I. M. (1999). Prooxidant toxicity of polyphenolic antioxidants to HL-60 cells: description of quantitative structure-activity relationships. FEBS letters, 462(3), 392-396.
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Sun, Y., Colburn, N. H., & Oberley, L. W. (1993). Depression of catalase gene expression after immortalization and transformation of mouse liver cells. Carcinogenesis, 14(8), 15051510. Todar, K. (2008) Online Textbook of Bacteriology. Online Textbook of Bacteriology. Retrieved April 26, 2014, from http://textbookofbacteriology.net/index.htm You, B. R., Moon, H. J., Han, Y. H., & Park, W. H. (2010). Gallic acid inhibits the growth of HeLa cervical cancer cells via apoptosis and/or necrosis. Food and chemical toxicology, 48(5), 1334-1340.
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Presented to the S. Daniel Abraham Honors Program In Partial Fulfillment of the Requirements for Completion of the Program
Stern College for Women Yeshiva University April 28th, 2014
Jordana Wietschner Mentor: Dr. Harvey Babich, Biology
Abstract Gallic acid (3,4,5-trihydroxybenzoic acid), a polyphenol common in many plants has pharmaceutical properties with potential health benefits. As with most polyphenols, gallic acid exhibits both antioxidant and prooxidant properties. The connection between gallic acid acting both as a prooxidant and as an apoptosis-inducing agent is ill-defined. The research herein clearly demonstrates a cause-and-effect relationship between gallic acid’s production of hydrogen peroxide (H2O2) and its subsequent induction of apoptosis to human oral carcinoma HSC-2 cells. Using the FOX assay in a cell-free system, it was shown that gallic acid is a strong generator of H2O2. Reduced glutathione (GSH), a thiol-containing a tripeptide, is the main intracellular antioxidant in a cell’s repertoire of defenses against oxidative stress. Using the Intracellular Glutathione Assay, depletion of intracellular GSH, a sign of impending oxidative stress, was observed in HSC-2 cells. The potency of gallic acid to HSC-2 cells was lessened in the presence of scavengers of hydrogen peroxide, such as divalent cobalt. Flow cytometric analyses of HSC-2 cells treated with gallic acid indicated a concentration-dependent response for the induction of apoptosis, which was reversed in the presence of divalent cobalt. These studies showed that the proapoptotic activities of gallic acid to HSC-2 cancer cells were mediated through autooxidation of gallic acid leading to the induction of oxidative stress.
2
Table of contents I.
Introduction ..........................................................................................................4
II.
Discussion a. Chemical properties of gallic acid .................................................................4 b. Cellular responses to gallic acid: In Vitro studies ..........................................12 i. Differential sensitivities of normal and cancerous cells to gallic acid ...........................................................................................................12 ii. Mechanism of gallic acid induced cytotoxicity: induction of oxidative stress ..................................................................................................14 iii. Gallic acid: an inducer of apoptosis...................................................21
III.
Conclusion ...........................................................................................................29
IV.
Acknowledgements ..............................................................................................30
V.
References ............................................................................................................31
3
Introduction Nutraceutical, a term describing a natural nutrient that possesses pharmaceutical properties, is defined broadly as a food or a chemical constituent of a food that provides medical or health benefits, including the prevention and/or treatment of a disease (Brower et al., 1998). Much effort has been directed to identifying such food items and their active chemical components and to better understand their physiological modes of protection towards promoting human health. One such positive health benefit is the chemotherapeutic effects of these nutraceuticals. Many of today’s “super foods,” contain gallic acid, a naturally occurring polyphenol common in many plants and beverages, including tea, grapes, berries, wine, and even in some hard wood plant species. Interestingly, gallic acid exhibits both anti-oxidative and pro-oxidative qualities, with both qualities contributing to its positive effect on human health. Research on gallic acid has been performed using in vivo test systems with laboratory animal models and in vitro test methods with mammalian cells maintained in culture. This paper will review the chemical properties of gallic acid and will note the modes of bioactivity of developed primarily through in vitro test systems. In regards to the latter, note will be made of my research at Stern College for Women, which correlated the anticancer effects of gallic acid towards carcinoma cells derived from tissues of the human oral cavity with its pro-oxidative nature. Chemical properties of gallic acid Gallic acid (3,4,5-trihydroxybenzoic acid), a polyphenol common in many plantderived foods, occurs freely or complexed with tannins and is isolated upon hydrolysis with tannase. Polyphenols are a class of organic molecules with many phenol groups (a 6-carbon aromatic ring molecule with a hydroxyl group (Figure 1)). Gallic acid, when purified, is a
4
colorless or yellow-colored crystal, with the molecular formula of C7H6O5, molecular weight of 170.12 g/mol, water solubility of 1.19 g/100 mL, density of 1.694 g/cm3 at 20o C, and melting point of 260o C.
Figure 1. Molecular structure of gallic acid
Chemically, gallic acid exhibits both antioxidant and pro-oxidant properties. As an antioxidant, gallic acid prevents the oxidation of biologically important molecules such as proteins and DNA, and thus is an inhibitor of oxidative stress. Oxidative stress is characterized by the production of oxygen-containing free radicals (termed reactive oxygen species, ROS) generated by aerobic cellular metabolism. ROS include superoxide free radical (O2-) and hydroxyl free radical (OH.), which can lead to the formation of hydrogen peroxide (H2O2), all which interfere with cellular processes and lead directly to cellular damage. A number of different chemical methodologies are used to describe and to quantify the antioxidant capacity of nutraceuticals with proposed antioxidant capabilities. The most common assays include measurements of the oxygen radical absorbance capacity (ORAC), of radical scavenging by 2,2-diphenyl-1-picrylhydrazyl (DPPH), of the vitamin C equivalent antioxidant capacity (VCEAC), and of superoxide dismutase (SOD)-like activity. The ORAC assay, recommended by the United States Department of Agriculture, measures oxidative
5
degradation of a fluorescent molecule after mixture with free radical generators. Antioxidants protect the fluorescent molecule from degeneration; a fluorometer quantifies the fluorescence and therefore the degree of protection afforded by the test compound studied. Antioxidant protection is based on comparative fluorescence in solutions with and without the test agent. Fluorescent intensity decreases with an increased exposure to the generated free radicals. Based on the value obtained by the assay, comparisons can be made between related compounds and their proposed antioxidant capacities. For gallic acid the ORAC value was approximately 2,000 per mol tested sample (x103), which can be compared to an ORAC value of approximately ± 500 for L-ascorbic acid (vitamin C), a known nutraceutical and antioxidant. For ellagic acid, another polyphenolic nutraceutical, the ORAC value was >3,000, indicating a greater antioxidant potential than gallic acid (Ito, 2011). DPPH, a stable free radical, is a scavenger for other radicals and used to measure the radical scavenging activity of antioxidants. Reduction of the rate of a chemical reaction by DPPH, an indicator of the radical nature of that reaction, is used to quantify the antioxidant activity of foods by testing their abilities to act as free radical scavengers (Figure 2). A DPPH solution is mixed with varying concentrations of the test compound and
Figure 2. The structure of DPPH and its reduction
6
incubated at room temperature. The odd electron in the DPPH free radical molecule gives the solution a purple color, which changes to yellow upon neutralization. Decolorization is stoichiometric with respect to the number of electrons captured (Miller et al., 2012). Gallic acid had a DPPH value IC50 of 1.9 µM, whereas that of ellagic acid was 1.7; the IC50 values represent the concentration of antioxidant that reduced the free radical concentration by about 50%. As noted with the ORAC assay, the antioxidant potential of gallic acid was less than that of ellagic acid (Ito, 2011). The antioxidant potential of gallic acid was concentration dependent; increasing the concentration of gallic acid from 0.25 to 90 µM progressively increased DPPH scavenging. These values included approximately 80% DPPH scavenging at 60 µM gallic acid, indicative of the strong antioxidant activity (Habtemariam, 2011). An additional assay used to quantify the antioxidant capacity of gallic was is the VCEAC assay, which focuses on expressing the antioxidant capacity of plant foods using vitamin C (mg/100g) as the reference. Kim et al. (2004) determined the VCEAC values of various polyphenols, including a series of benzoic acid derivatives. Within this series, gallic acid ranked as the strongest antioxidant. The sequence of antioxidant potency, as VECAC values (in mg/L), was gallic acid (324.3 ± 4.7), 2,3- dihydroxybenzoic acid (169.6 ± 2.7), 2,4-dihydroxybenzoic acid (165.5 ± 4.0), protocatechuic acid (163.2 ± 2.9), vanillic acid (117.2 ± 4.5), gentisic acid (90.8 ± 4.9), syringic acid (80.4 ± 2.9), 3-hydroxybenzoic acid (53.7 ± 1.7), 4-hydroxybenzoic acid (4.8 ± 0.6), and salicylic acid (1.4 ± 0.4). The high antioxidant capacity of gallic acid was attributed to its 3 hydroxyl groups attached to the aromatic ring.
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Another method used to determine antioxidant capacity of a compound is to define its superoxide dismutase (SOD)-like activity. The scavenging activities of O2- anions generated by a xanthine-XOD enzymatic reaction were compared for gallic acid and ellagic acid. For gallic acid the IC50 value (μM) was 13, whereas for ellagic acid it was >100, again showing the greater antioxidant capacity of ellagic acid over gallic acid (Ito, 2011). Interestingly, polyphenols, including gallic acid, also exhibit pro-oxidant activity. As opposed to antioxidant activity, which scavenges ROS and inhibits oxidative stress, prooxidants generate ROS and contribute to oxidative stress. When polyphenols are amended to cell culture medium, ROS, principally H2O2, are detected. Elevated levels of H2O2 derived from polyphenols can be toxic to and may reduce the growth of tumor cells (Lee et al. 2004). Lee et al. (2005), in studying the antiproliferative effects of dietary phenolic substances, showed in cell cultured medium gallic acid acted as a pro-oxidant, generating H2O2 in a dose dependent manner. In the summer of 2013, as a research intern working with Biology faculty at SCW and with fellow undergraduates, I researched the pro-oxidant nature of gallic acid (Schuck et al. 2013). The FOX assay was used to determine and quantify the generation of ROS, primarily H2O2, in cell culture medium. This assay is based upon quantifying the oxidation of ferrous ions (Fe2+) to ferric ions (Fe3+) by H2O2 or other ROS produced by a test agent. The resultant Fe3+ ion bonded to xylenol orange and produced a colored complex, measured spectrophotometrically and compared to a standard curve using H2O2. In the initial studies, samples of gallic acid, at concentrations ranging from 0 to 200 µM, were incubated at room temperature for 4 hours in cell culture medium. At the end of the incubation, samples were mixed with methanol and the FOX reagent, vortexed, and incubated for 20 minute at room temperature. The solutions were centrifuged and absorbance
8
of the supernatant was recorded at 595 nm against a blank consisting of cell culture medium, methanol, and the FOX reagent. A standard curve of H2O2 from 0 to 200 µmol/L was produced to quantify the level of peroxide generated by gallic acid. Gallic acid was shown to be a strong generator of H2O2. As gallic acid concentration increased, the amount of H2O2 detected in the medium increased (Figure 3). FOX assay 4-Hr incubation in exposure medium
160
H2O2 (µmol/L)
140 120 100 80 60 40 20 0 0
50
100
150
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Gallic acid (µM) Figure 3. Generation of hydrogen peroxide, as determined by the FOX assay, in cell culture medium amended with varying concentrations of gallic acid. Hydrogen peroxide was the reference peroxide used to construct the standard curve.
Lipid peroxidation as an indicator of the pro-oxidant nature of gallic acid was also studied. Free radicals are able to extract electrons from cell membranes, thereby damaging the membrane. ROS react with polyunsaturated fatty acids to yield fatty acid radicals. In the presence of O2 a lipid peroxy fatty acid radical forms. This chain reaction occurs and continues until two free radicals interact and generate a non-radical species. These reactions can be quantified by measuring the production of malondialdehyde (MDA), the end product of lipid peroxidation. In the assay, MDA was made to react with thiobarbituric acid (TBA),
9
yielding thiobarbituric acid reactive species (TBARS), measured by visible or fluorescence spectrophotometry. Cells were treated with varying concentrations of gallic acid, Fe2+, and a combination of gallic acid and Fe2+. After 3 hours, the agents were aspirated and trichloroacetic acid was added. The cells were then scraped and the resulting suspensions were centrifuged to remove the precipitated proteins. A solution of 1% thiobarbituric acid in NaOH was added to the supernatants, which were heated at 95°C for 20 minutes. Samples were centrifuged again after cooling, the TBARS, occurring as a pink chromagen, were measured at 532 nm. A 3hour exposure to 100 µM gallic acid induced lipid peroxidation, which was moderately potentiated in the presence of Fe2+ (Figure 4).
O.D.532 nm (TBA-reactive substances)
0.06
HSC-2 cells 3-Hr exposure
*
0.05
* 0.04
** 0.03
0.02
0.01
0.00
control
100 µM GA
2 mM Fe2+ Fe2+ + GA
Figure 4. Gallic acid-induced lipid peroxidation, as mediated by Fe2+.
Fluorescent microscopy was then used to visualize ROS production by gallic acid. Cells, treated with varying concentrations of gallic acid, were treated with dichlorofluorescein diacetate (DCFDA) for 30 minutes at 37ºC. Thereafter, the DCFDA was
10
removed and cells were incubated for an additional 30 minutes at 37ºC in warm exposure medium. During this process, the internalized dye was hydrolyzed by intercellular esterases, yielding dichlorofluorescein (DCF). Upon intracellular oxidation of gallic acid, a fluorescent product was noted. The cells were viewed microscopically using a filter set capable of detecting the fluorescent oxidized product (with an absorption maximum of 492 nm) and the fluorescent emission maximum of 517 nm. Minimal fluorescence was detected in control cells (Figure 5A), indicating the background intracellular ROS. Fluorescence, in a concentration-dependent mode, was noted in cells treated with 100 and 200 μM gallic acid (Figure 5B, C), and, as an internal control, in cells treated with 200 µM H2O2 (Figure 5D).
Figure 5. HSC-2 cells were untreated and treated with gallic acid, without or with ascorbic acid, or with hydrogen peroxide, stained with DCFDA, and viewed with a fluorescent microscope. (A) untreated control; (B) 4-hr treatment with 100 µM gallic acid; (C) 4-hr treatment with 200 µM gallic acid; (D) 4-hr treatment with 33 µM hydrogen peroxide (positive oxidant control)
Aside from its antioxidant and prooxidant activities, another important chemical property of gallic acid is lipophilicity, which denotes the ability of a compound to dissolve in nonpolar substances. The lipophilicity of a compound is described by its partition
11
coefficient, defined as the ratio of concentrations of a compound in a mixture of two immiscible phases at equilibrium. Most commonly, one solvent is water and the other is octanol. The logarithmic ratio of the concentrations is referred to as log P value, which is useful in determining the ability of a chemical to transverse the phospholipid bilayer of the plasma membrane. The log P value of gallic was calculated as 0.91. Adding functional groups to gallic acid increased its lipophilicity and therefore its log P value. For example, the log P for methyl gallate was 1.54, for ethyl gallate it was 2.07, and for propyl gallate it was 3.13. An increase in the log P value correlated to an increase in cytotoxicity of the chemical gallic acid-derivative (Sergediene et al. 1999). Cellular Responses to Gallic Acid: In Vitro Studies In summer 2013, as a research intern, I performed in vitro studies to determine the response of cancer cells to gallic acid and specifically to demonstrate a correlation between the pro-oxidant properties of gallic acid and its proapototic properties. Research was performed in the laboratory of Dr. Jeffrey Weisburg with Biology faculty at Stern College for Women and with fellow undergraduates. Differential sensitivities of normal and cancerous cells to gallic acid When determining cytotoxicity of gallic acid on cancerous cells, it is also important to determine its cytotoxic effects on normal, healthy cells. While many chemotherapeutics today are successful in killing tumor cells, they can also harm healthy cells. Finding a method of ridding the body of cancerous cells while not harming healthy cells is a welcome alternative. A comparison between the potentially different effects of gallic acid on normal and tumor cells can therefore have positive consequences on the potential of gallic acid usage
12
as a chemotherapeutic. Studies conducted by Schuck et al. (2013) evaluated the cytotoxic effects of gallic acid on normal gingival HF-1 fibroblasts and carcinoma HSC-2 cells isolated from the oral cavity. Cytotoxicity was quantified with the neutral red assay, based on the ability of cells to uptake and accumulate neutral red in their lysosomes (Repetto et al. 2008). The HSC-2 carcinoma cells were more sensitive to gallic acid than were the HF-1 fibroblasts, was a 24-hr midpoint cytotoxicity value (NR50) of 80 µM gallic acid for the HSC-2 cells and 175 µM for the HF-1 fibroblasts (Figure 6).
Percent of control (NR assay)
100
*
80
*
HF-1 fibroblasts *
60
*
* *
40
*
* *
20
HSC-2 carcinoma cells
24-hr exposure 0 0
50
100
150
200
Gallic acid (µM)
Figure 6. Comparative 24-hour cytotoxicity of gallic acid to human oral carcinoma HSC-2 cells and to human normal gingival HF-1 fibroblasts.
Similar research has shown that gallic acid was more cytotoxic to cancerous cell as opposed to normal cells. Russel et al. (2011) found human prostate cancer LNCap cells to be more sensitive to gallic acid than normal prostate epithelial cells and Inoue et al. (1995) and Isuzugawa et al. (2001) noted rat hepatoma dRLH-84, human epithelioid carcinoma, and human hepatoma PLC/PRF/5 cancerous cells were less tolerant of exposures to gallic acid than were normal cells, including rat primary cultured hepatocytes, keratinocytes, macrophages, endothelial cells, and normal fibroblasts. Nair et al. (2007) observed that, in 13
general, normal cells are more resistant to polyphenols than are cancer cells. The reason normal cells have greater resistance to gallic acid, when acting as a prooxidant, than cancer cells may reflect differences in their intracellular defenses against oxidative stress, as exemplified by the higher intracellular levels of catalase in normal cells (Isuzugawa et al., 2001). Catalase, an enzyme, catalyzes the decomposition of H2O2 to H2O and O2, thereby detoxifying intracellular H2O2 generated by gallic acid. Tumor cells contain depressed levels of catalase due to their lower rate of catalase transcription, resulting from increased methylation of the gene encoding for catalase (Sun et al., 1993). Cancer cells, therefore, were more vulnerable to ROS toxicity and more likely to undergo apoptosis. Another reason, proposed by Schumacker et al. (2006), explained that, in general, cancer cells possess more metabolic activity than normal cells and therefore may have greater basal levels of mitochondrial-derived ROS. The additional ROS generated by gallic acid could therefore overwhelm cellular defense mechanisms against oxidative stress, leading to an increase in cytotoxicity. Nair et al. (2007), in a study using human colon HT-29 cancer cells and human prostate cancer PC-3 cells, noted that cancerous cells required overexpression of certain signaling molecules, such as epidermal growth factor receptor (EGFR) and NF-κB transcription factor, to maintain cell proliferation and survival, whereas normal cells do not exhibit an overexpression of signaling molecules. Polyphenols are inhibitory to many of these signal molecules and cancer cells, with their greater number of intracellular targets, exhibit greater sensitivity to polyphenols, including gallic acid, than normal cells. Mechanism of Gallic Acid-Induced Cytotoxicity: Induction of Oxidative Stress After observing that gallic acid was cytotoxic to cancer cells, Schuck et al. (2013) determined the mechanism of its cytotoxicity. My portion of that study showed that gallic 14
acid induced oxidative stress, which subsequently led to apoptosis in HSC-2 cells. Oxidative stress reflects the imbalance between a cell’s production of ROS and cellular defense against these potentially toxic substances. ROS are produced as a by-product of mitochondrial aerobic cellular respiration and include H2O2, O2.- , and (OH.), among others. These oxygen derivatives are all strong oxidizing agents and can therefore damage cells by inactivating enzymes and other proteins, damaging DNA and RNA, and causing lipid peroxidation. To protect itself against the toxic effects of ROS, the cell employs various defense mechanisms and ROS scavengers, including intracellular glutathione, enzymes such as superoxide dismutase and peroxidase, and other scavengers including pyruvate and divalent copper. If the ROS overwhelm the cell, however, and the defense mechanisms therefore cannot properly protect the cell, oxidative stress occurs, which can cause damage the cell or result in apoptosis. One important intracellular defense is the presence of reduced glutathione (GSH) (figure 7).
Figure 7. Molecular structure of glutathione.
GSH, a tripeptide, contains a thiol group from its cysteine amino acid, which aids in its reduction potential by donating an electron to other unstable molecules, such as ROS. In the process, GSH is converted to glutathione disulfide (GSSG), its oxidized form, via glutathione peroxidase. GSSG can then be reduced to GSH through the use of glutathione reductase,
15
using NADPH as an electron donor. The overall process allows GSH to be continuously recycled and used. Glutathione therefore protects the cell against ROS by neutralizing them. An increased ratio of GSSG to GSH, signifying the overuse of GSH is thus an indicator of oxidative stress. GSH is the main cellular defense against ROS (Circu et al. 2010) and testing for the intracellular levels is thereby a determinant of intracellular ROS. Other cellular defenses include the actions of various enzymes such as superoxide dismutase, catalase, and peroxidase. Superoxide dismutase catalyzes the dismutation - a redox reaction in which a substance is both oxidized and reduced to form two different products - of O2.- to molecular oxygen (O2) and H2O2, which can be degraded by catalase. Catalase, a tetramer with four porphyrin heme groups, is a common enzyme found in nearly all organisms that are exposed to oxygen and aids in the decomposition of H2O2 to O2 and water (H2O) (Boon et al., 2007). Peroxidase can also be used to catalyze the reduction of H2O2 to H2O (figure 8).
Figure 8. The action of superoxide dismutase, catalase, and peroxidase (Todar, 2008).
Additional scavengers of ROS include pyruvate and divalent cobalt (Co2+). Pyruvate, as well as Co2+, has been found to be a scavenger of H2O2 (Long et al., 2009). Pyruvate is oxidatively decarboxylated by H2O2 to yield acetate, carbon dioxide (CO2), and H2O; Co2+ catalytically decomposes H2O2 to H2O and O2 (Babich et al., 2011). Although the cell employs these various protective measures against ROS, generated ROS may reach elevated levels and eventually overwhelm cellular defenses and induce
16
oxidative stress. The FOX assay, performed during my internship and also by Lee et al. (2005), showed that gallic acid generated H2O2, confirming that gallic acid itself actually produced this ROS. Schuck et al. (2013) focused on the quantity of H2O2 produced and whether the level generated was sufficient to cause oxidative stress and cytotoxicity to HSC2 cells. While the addition of gallic acid resulted in generation of H2O2, studies indicated that levels of H2O2 were lowered by co-incubations with catalase, pyruvate, and divalent copper, all scavengers of H2O2. HSC-2 cells were treated with cytotoxic concentrations of gallic acid in the presence of catalase, pyruvate, and Co2+. Upon co-incubation of gallic acid with these
Percent of control (NR assay)
H2O2 scavengers, the toxicity of gallic acid was lessened (Figure 9).
100
HSC-2 cells 24-hr exposure
80
60
40
*
* * 75 µM gallic acid 100 µM gallic acid 125 µM gallic acid
20
0
No additions
Catalase Pyruvate Cobalt chloride (200 Units/ml) (110 mg/L) (250 µM)
Figure 9. Cytotoxicity of gallic acid to HSC-2 cells in the presence of scavengers of hydrogen peroxide as compared to control.
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These studies indicated that gallic acid-induced cytotoxicity occurred through the generation of H2O2 since cytotoxicity was lessened in the presence of scavengers of H2O2. Similar studies by Chen et al. (2009) found that catalase lessened the toxicity of gallic acid to human prostate DU145 cancer cells. Studies have also shown that exposure to gallic acid caused an increase in the production of ROS, which subsequently results in a decreased level of intracellular GSH and subsequent cytotoxicity due to a decrease in cellular defenses. HSC-2 cells, maintained in growth medium in tissue culture plates, were washed with phosphate buffered saline (PBS) and then treated with 25 to 150 µM of gallic acid. Thereafter, cells were then washed again with PBS, lysed with Triton X-100, and proteins were precipitated with sulfosalicylic acid. After cells were harvested by scraping and centrifugation, GSH levels were analyzed by determining the oxidation of GSH (in a 6 mM solution of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and prepared in a phosphate buffer/EDTA (pH 7.5) to GSSG, with a stoichiometric formation of 5-thio-2-nitrobenzoic acid, a yellow chromagen measured spectrophotometrically at 412 nm. My studies showed that a gradual increase in the concentration of gallic acid from 25 to 150 µM resulted in progressively reduced levels of intracellular GSH (figure 10). Similar studies showing a decrease in intracellular GSH with an increase in concentration of gallic acid were performed using human pulmonary adenocarcinoma Calu-6 cells (You et al., 2011) and human primary vein endothelial cells (Park et al., 2012).
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6
Intracellular glutathione (nmoles/10 cells)
HSC-2 cells 4-hr exposure in DMEM
35
30
25
* *
*
*
20
* 15
*
10 0
20
40
60
80
100
120
140
Gallic acid (µM) Figure 10. Effect of a 4-hr exposure to gallic acid on the intracellular levels of reduced glutathione in HSC-2 cells
Cells were also exposed to gallic acid in the presence of 250 µM Co2+, a scavenger of H2O2. Levels of intracellular GSH were unaffected by gallic acid in the presence of the H2O2 scavenger. HSC-2 cells were also treated with various substances that reduced the levels of GSH to confirm that the pro-oxidant nature of gallic acid was responsible for growth-inhibitory effects. With lower levels of GSH, ROS can more easily overwhelm the cell and cause oxidative stress. Each depleter of GSH interfered with a different aspect of GSH-GSSG cycling. The redox cycle of GSH/GSSG employs many enzymes to synthesize the production of GSH and subsequently aid in its oxidation GSSG and recycling to GSH. Intracellularly, GSH is produced in two steps: the first step controlled by ɣ-glutamylcysteine synthetase, which catalyzes the ATP-dependent condensation of cysteine and glutamate to form the dipeptide, ɣ-glutamylcysteine; the second step is controlled by glutathione
19
synthetase, which catalyzes the condensation of ɣ-glutamylcysteine and glycine, forming GSH (Njålsson, 2005). Glutathione-S-transferase can then aid in catalyzing the conjugation of electrophilic substrates to GSH (Sheehan et al., 2001). Subsequently, GSH is oxidized to GSSG via glutathione peroxidase. Finally, GSSG is reduced back to GSH using glutathione reductase. GSH-depleting reagents used included 1-chloro-2,4-dinitrobenzene (CDNB), DLbuthionine-[S,R]-sulfoximine (BSO), and bis-(2-chloroethyl)-N-nitrosourea (BCNU). CDNB inactivated glutathione S-transferase, BSO inhibited glutamylcysteine synthetase, and BCNU inhibited glutathione reducatse (Babich et al., 2011). Pretreatment with CDNB or BCNU and co-exposure with BSO potentiated the toxicity of gallic acid to the HSC-2 cells, as these treatments lessened the intracellular level of GSH (Figure 11).
Percent of control (NR assay)
HSC-2 cells 24-hr exposure
25 µM gallic acid 50 µM gallic acid 75 µM gallic acid 100 µM gallic acid
100
80
*
60
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*
40
*
20
*
* *
* *
* *
0
No treatment
BCNU (0.5 mM)
CDNB (20 µM)
BSO (5 mM)
Figure 11. Cytotoxicity of gallic acid toward HSC-2 cells in the presence of depleters of intracellular reduced glutathione.
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As noted earlier, gallic acid also induced lipid peroxidation in HSC-2 cells, another indicator of cytotoxicity by ROS. This was verified, as a three-hour exposure of HSC-2 cells to gallic acid (100 µM) induced lipid peroxidation, which was moderately potentiated in the presence of ferrous iron (Fe2+) (Schuck et al., 2013). Gallic Acid: an Inducer of Apoptosis Apoptosis, the process of programmed cell death, is generally regulated in an organism as a whole, working to balance cellular proliferation with cellular destruction, while maintaining constant cell numbers in somatic tissues. It also functions to rid the body of genetically damaged and potentially malignant cells by their removal before they multiply. In the process of apoptosis, chromosomal DNA is fragmented, chromatin condenses, the nucleus dissociates into small pieces, and the cytoskeleton is disrupted. Eventually, the cell shrinks and is broken into various membrane-enclosed fragments. Apoptotic cells and their cellular fragments are recognized and phagocytosed by macrophages, thereby removing them from the somatic tissues. Apoptosis occurs through a series of cellular pathways. One such method includes mitochondria sending out a signal to induce apoptosis, triggering the inactivation of a molecule termed the inhibitor of apoptosis proteins (IAP). Once inactivated, apoptosis occurs through caspases, which are proteases containing a cysteine residue at their active sites and that cleave substrate proteins after aspartic acid residues. A cascade chain of initiator and effector caspases cleave various cellular target proteins, ultimately leading to cell death. Apoptotic protease activating factor (Apaf-1) and cytochrome c, released by mitochondria, bind to an apoptosome, a multi-subunit complex, which activates caspase-9 to cleave and thereby activate the effector caspase-3. The effector caspase then cleaves
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different cellular proteins, including nuclear lamins, cytoskeletal proteins, and an inhibitor of deoxyribonuclease (DNase), ultimately leading to cell death (Cooper et al., 2007). Genes, occurring either as antiapoptotic or proapoptotic activators, regulate apoptosis in cells. The triggering of apoptosis is based on the ratio of activity between the proapoptotic and antiapoptotic gene products. Proapoptotic genes include Bax, Bak, and BH3 and antiapoptotic genes include Bcl-2. When activated, Bax and Bak form oligomers in the mitochondrial outer membrane, leading to the release of cytochrome c and to caspase activation. Apoptosis can also be induced directly by activating certain receptors on the target cell. One such cell surface receptor is the Fas receptor. The Fas receptor activates an initiator caspase, caspase-8, which can then initiate downstream effector caspases. Studies have shown that gallic acid induced apoptosis by activating the Fas protein receptor and initiating the caspase cascade (Hsu et al., 2006). Studies with HeLa cells determined that apoptosis induction with gallic acid included the upregulation of Bax and the downregulation of Bcl-2 genes (You et al., 2010). Apoptosis induced by gallic acid was correlated with elevated levels of ROS, leading to increased oxidative stress (Chen et al., 2009). Increased oxidative stress may lead to decreased levels of GSH and thereby reduce cellular antioxidant defenses against ROS damage. Poot et al. (1995) observed that the induction of apoptosis was inversely related to the cellular GSH content. Using flow cytometry, my research was directed to correlating the prooxidant property of gallic acid with its subsequent induction of apoptosis in carcinoma HSC-2 cells. In addition, Western blotting was employed to visualize poly(ADP-ribose)-polymerase
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(PARP) fragments, an indicator of caspase activities, and microscopy to visualize morphological cellular alterations characteristic of apoptosis. Flow cytometry is a technique that can directly differentiate and measure the number of cells that are alive, dead, or undergoing apoptosis. In this technique, the cells were suspended in fluid and passed through an electronic detection apparatus. The cells, passed single file through a light beam and detected by a flow cytometer, had been treated with a DNA binding dye with differential permeability for viable, apoptotic, and nonviable cells. Data collection and analysis was performed using a computer. In my studies, HSC-2 cells were untreated (control) or treated with varying concentrations of gallic acid for 24 hours. The cells were then washed and diluted to a concentration slightly less than 5 x 105 cells/mL. A 20 L sample of cells and 380 L of Guava ViaCount Reagent were placed on ice in the dark for 5 minutes. A Guava Easycyte Miniflow Cytometer was used to determine cell viability, apoptosis, and cell death. As seen in Figure 12, gallic acid induced apoptosis in a concentration-dependent manner.
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(A)
(1) control
(2) 75 µM gallic acid
(3) 100 µM gallic acid
(4) 125 µM gallic acid
(5) 150 µM gallic acid
24
(B)
100
HSC-2 carcinoma cells 20-hr exposure
Percentage of total
80
Apoptotic cells ** ** **
60
Viable cells
Dead cells
40
*
** **
*
20
** 0
0 75 100 125 150
0 75 100 125150
0 75 100 125 150
Gallic acid (µM)
Figure 12. A) Representative FACS profiles of the viability and apoptosis of HSC-2 cells after a 20-hr treatment with varying concentrations of gallic acid. Viable cells are located on the left side of each panel; apoptotic cells between the two lines; and dead cells on the right side of the panel. Panel 1 is untreated cells, panel 2 is cells treated with 75 µM gallic acid; panel 3 is cells treated with 100 µM gallic acid; panel 4 is cells treated with 125 µM gallic acid; and panel 5 is cells treated with 150 µM gallic acid. (B) Cytometric analysis, using Guava ViaCount Reagent, of a 20-hr exposure of HSC-2 cells to increasing concentrations of gallic acid. Percentage of viable, apoptotic, and dead cells were quantified by the flow cytometer.
Gallic acid-induced apoptosis was apparently mediated by oxidative stress, as the number of apoptotic and dead cells were reduced in the presence of Co2+, a scavenger of H2O2 (Figure 13).
25
(A)
(1)
(2)
(3)
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(B)
100
Viable cells
HSC-2 carcinoma cells 20-hr exposure
Percentage of total
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Apoptotic cells
Dead cells
0
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75 125 0 75 125 0 75 Gallic acid (µM) + 250 µM CoCl 2
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Figure 13. (A) Representative FACS profiles of viabile and apoptotic HSC-2 cells after a 20-hr treatment with 250 µM CoCl2, alone (control) and in the presence of 75 and 125 µM gallic acid. Viable cells are located on the left side of each panel; apoptotic cells between the two lines; and dead cells on the right side of the panel. Panel 1 is control cells, panel 2 is cells treated with CoCl2 and 75 µM gallic acid; and panel 3 is cells treated with CoCl2 and 125 µM gallic acid. (B) Cytometric analysis, using Guava ViaCount Reagent, of a 20-hr exposure of HSC-2 cells to 250 µM CoCl2, alone (control) and in the presence of 75 and 125 µM gallic acid. Percentage of viable, apoptotic, and dead cells were quantified by the flow cytometer.
Another study to determine if gallic acid induced apoptosis in the HSC-2 cells was the observation of poly(ADP-ribose) polymerase (PARP) fragments. PARP catalyzes the formation of poly(ADP-ribose), a post-translational modification involved in various cellular functions, including surveillance of genome integrity (Isabelle et al., 2010). Proteolytic inactivation and cleavage of PARP by caspases, in particular by caspase-3, is a useful monitor of the induction of apoptosis. PARP fragmentation in untreated cells and in cells treated with gallic acid for 24 hours was determined. After the 24-hour incubation, the cells
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were lysed with RIPA buffer, centrifuged to remove cellular debris, and protein concentration of the cell lysates was quantified with a BCA Protein Assay Kit. Equivalent levels of total protein from each sample were separated by SDS-PAGE electrophoresis, electroblotted to nitrocellulose membranes, and blocked with 5% dry milk in Tris-buffered saline containing 0.1% Tween 20. Membranes, probed with polyclonal anti-PARP to detect full-length and cleaved PARP, were incubated with the appropriate peroxidase-conjugated secondary antibodies and were developed using an ECL detection kit. PARP cleavage products were detected via Western blot analysis only in those protein lysates of HSC-2 cells exposed to gallic acid. Again, oxidative stress induced by gallic acid was shown, as in the presence of CoCl2, gallic acid failed to trigger apoptosis (Figure 14).
Figure 14. Immunoblot analysis of PARP cleavage in HSC-2 cells untreated and treated with increasing concentrations of gallic acid (GA) after a 20-hr exposure in exposure medium without and with 250 µM ClCl 2. Cellular proteins, separated by 10% SDS-PAGE, were transferred to nitrocellulose and probed with an antibody specific for full length PARP and one specific for the ~85 kD C-terminal cleaved fragment of PARP, which were identified by comparison to a standard molecular weight marker. An anti-actin antibody was the loading control.
Induction of apoptosis by gallic acid, as mediated by oxidative stress, was also shown microscopically. Cells treated with gallic acid and in the presence of pyruvate and Co2+, both scavengers of ROS, were visualized via microscopy. Control HSC-2 cells had a large
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cytoplasm and nucleus, whereas for cells treated with gallic acid, the cytoplasmic mass was reduced, the nucleus was hypercondensed, and cell blebbing was evident - all morphological signs of apoptosis. In cells cotreated with gallic acid and either pyruvate or Co2+, however, these cellular indications of apoptosis were not observed (Figure 15).
A
B
C
D
Figure 15. Brightfield microscopy of HSC-2 cells untreated and treated with gallic acid for 24-hr, in the absence and presence of scavengers of hydrogen peroxide. (A) Control, untreated cells; (B) treated with 125 µM gallic acid, alone (C) treated with 125 µM gallic acid in the presence of 110 mg/L pyruvate; and (D) treated with 125 µM gallic acid in the presence of 250 µM CoCl2.
Conclusion Research has indicated gallic acid’s role as an antioxidant and/or as a prooxidant. The FOX assay, used to determine and quantify the generation of ROS (primarily
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H2O2) in cell culture medium, displayed an increase in the production of H2O2 in cell culture medium amended with increasing concentrations of gallic acid. Studies conducted in our lab have shown that gallic acid is more cytotoxic to HSC-2 cells than to normal cells. Similar studies have determined this to be a characteristic of other cancerous cell lines as well. Gallic acid, therefore, has the potential to be used as a less harmful chemotherapeutic agent than past cancer drugs. Exposure to gallic acid caused a decrease in levels of intracellular glutathione, increased lipophilicity, and an increase in intracellular ROS levels. The induction of apoptosis was noted in a concentration-dependent manner in cells exposed to gallic acid. In the presence of scavengers of ROS such as Co2+ or reagents that deplete intracellular glutathione levels, however, the previously described effects were not observed. These studies demonstrated a correlation between the autooxidation of gallic acid, which generates H2O2 to induce oxidative stress, and the subsequent initiation of apoptosis in cancer cells. Acknowledgments I would like to express my gratitude toward Dr. Harvey Babich for his constant help and motivation in researching and writing this paper. I would also like to thank Dr. Jeffrey Weisburg for his support and guidance in the laboratory when conducting the research presented. Lastly, I would like the express my appreciation to the S. Daniel Abraham Honors Program at Stern College for Women, Yeshiva University for support, in part, of this research.
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