Bulk and Nanoparticle Forms of Tea Catechins and Polyphenols - Lab Report Example

?Bulk and nanoparticle forms of tea catechins and polyphenols exhibit opposite effects on DNA damaged lymphocytes of colorectal cancer patients Abstract Tea catechin epigallocatechin-3-gallate (EGCG), and other polyphenols such as theaflavins (TF) are increasingly proving useful as chemopreventives in a number of human cancers, but without affecting the normal cells. The polyphenols in tea are known to have antioxidant properties which can quench free radical species, and also pro-oxidant activities that appear to be responsible for the induction of apoptosis in tumor cells. The bioavailability of these natural compounds is an important factor that determines their efficacy. Studies have shown a generally low bioavailability and stability of oral catechins in humans. Nanoparticle-mediated delivery techniques of EGCG and TF have been found to improve their bioavailability to a level that would benefit their effectiveness as chemopreventives. The present study was conducted to compare the effects of theaflavins and EGCG, when used in the bulk form and in the biopolymer (polylactide-co-glycolide)-based nanoparticle form, on oxaliplatin- and satraplatin-treated lymphocytes from colorectal cancer patients and healthy volunteers. The results of DNA damage measurements by comet assay revealed opposite trends in bulk and nanoparticle forms of TF as well as EGCG. Both the compounds in the bulk form produced stastically significant concentration-dependent reductions in DNA damage in oxaliplatin- or satraplatin-treated lymphocytes. In contrast to this, when used in the nanoparticle form both TF and EGCG caused a concentration-dependent increase in DNA damage in the lymphocytes. The maximum increase noted with TF was around 2.5-fold. The reverse activities exhibited by the two forms, namely bulk- and nanoparticle forms, of TF as well as EGCG support the notion that TF and EGCG act as both antioxidant and pro-oxidant, depending on the form in which they are administered. In the bulk form, the compounds likely act as antioxidants, which was observed as a decrease in the DNA damage measured as Olive Tail Moment in the comet assays. It is also our hypothesis that, changing their mode of action in the nanoparticle form, both TF and EGCG act as pro-oxidants, and cause an increase in the DNA damage. Introduction One of the major causes of cancer development is oxidative stress. Oxidative stress leads to the cellular redox imbalance that has been observed in various cancer cells as compared to normal cells (Valko et al., 2006). Tissue homeostasis is disrupted when the balance between cell growth and apoptosis (cell death) is lost provoking uncontrolled cell growth that results in cancer. Oxidative stress, which is prevalent in the tumour micro-environment, can affect the apoptotic potential of tumour cells. It can also affect many functions in cancer cells, including cell proliferation, promotion of mutations and genetic instability, modification of cellular sensitivity to anticancer compounds, invasion, and metastasis (Kumar et al., 2008). Reactive oxygen species (ROS) such as hydroxyl radicals, peroxides and superoxides, nitric oxide (NO') and peroxynitrite (ONOO-) that are generated in a normal cell both endogenously (by mitochondria, through metabolic processes, inflammation etc.) and via external sources, play a vital role in regulating several biological phenomena (Barzilai et al., 2002; Hussain et al, 2003). An excessive production of ROS or an inadequate anti-oxidant defense system, or both, in a normal cell can produce oxidative stress leading to DNA damage, and, further, induce an increased mutation rate and tumour development, possibly through a preferential selection of tumour cell mutations that confer a growth advantage (Sotgia et al., 2011). There is much evidence to show that oxidative stress plays an important role in the molecular mechanism of colorectal cancer (Keshavarzian et al., 1992; Bartsch et al., 2002). Free radicals formed during the metabolic activation of environmental genotoxic agents have been thought to cause genetic alterations that initiate colorectal neoplastic progression (Bartsch et al., 2002). A complete elimination or even reduction of exposure to such environmental factors may not be feasible. However, antioxidants, endogenously derived and primarily comprising of reduced glutathione and Phase II enzymes, as well as those obtained from the diet, are important for the protection of the integrity of DNA on exposure to genotoxic compounds. Dietary factors have been found to reduce colon cancer risk (Reddy et al., 1993). Natural anti-oxidants e.g., phytochemicals such as phenolics, flavonoids, isoflavonoids, and carotenoids have numerous biochemical activities, such as inhibition of ROS generation, direct or indirect scavenging of free radicals e.g., hydroxyl radicals, peroxyl radicals hypochlorous acid and superoxide radical, O2?, and alteration of anti-oxidant potential (Finkel and Holbrook, 2000; Halliwell, 2003), besides inhibition of apoptosis. Therefore, they are potentially anti-carcinogenic (Johnson et al., 1994). Numerous studies have revealed a protective effect of dietary polyphenols present in fruits, vegetables and other plant products against colorectal cancer (Araujo et al., 2011). The antioxidant protective effects of polyphenols are particularly important to the gastrointestinal (GI) tract because it is constantly exposed to several reactive species, such as superoxide and H2O2 produced endogenously by NADPH oxidases and “dual oxidases” in the epithelial cells of GI tract (Kanner and Lapidot, 2001), or H2O2 in beverages and nitrite present in foods converted to HNO2 by gastric acid, forming nitrosating and DNA-deaminating species (Halliwell et al., 2005). Epidemiological studies have revealed the protective effects of tea against a number of human cancers (Yang and Wang, 1993; Stoner and Mukhtar, 1995; Fujiki et al., 1996). Tea is composed of polyphenols, alkaloids such as caffeine, theophylline and theobromine, and several other organic compounds (Cabrera et al., 2003). Fresh tea leaves of the plant Camellia sinensis are rich in polyphenolic compounds known as catechins. Soon after harvesting, catechins undergo oxidation due to the activity of polyphenol oxidase enzymes present in the tea leaves. The process of oxidation of catechins, described as fermentation, causes them to polymerise and form larger, more complex polyphenols known as theaflavins and thearubigins. Green tea made from unwilted leaves that are not oxidized, is rich in catechins. The biological activity of green tea is attributed to its four major catechins namely Epigallocatechin-3-gallate (EGCG), Epigallocatechin (EGC), Epicatechin-3-gallate (ECG) and Epicatechin (EC). The most active and abundant catechin in green tea is EGCG (Cabrera, 2006; Reto et al., 2007). On the other hand, black tea comprising of tea leaves that are rolled and allowed to oxidise or ferment fully, possesses high concentrations of the polyphenols, theaflavins and thearubigins, but comparatively less of catechins. Tea catechins and polyphenols such as theaflavins and thearubigins are effective scavengers of ROS and nitrogen species in vitro and possibly also function indirectly as antioxidants through their effects on redox-sensitive transcription factors and nuclear factor-kappaB and activator protein-1, inhibition of "pro-oxidant" enzymes, such as inducible nitric oxide synthase, lipoxygenases, cyclooxygenases and xanthine oxidase, and induction of phase II and antioxidant enzymes, such as glutathione S-transferases and superoxide dismutases (Frei and Higdon, 2003). EGCG has been shown to induce apoptosis in many types of neoplastic cells, without causing damage to normal cells (Chen et al., 1998). Similarly, theaflavins (TF) from black tea has been found to induce apoptogenic (Bhattacharyya et al., 2003) and antiproliferative (Liang et al., 1999) effects in various human cancer cells. EGCG was reported by Valcic et al. (1996) to be the most potent cancer preventive green tea catechin against HT-29 colon cancer line, acting through induction of apoptosis and promotion of cell growth arrest (Butt and Sultan, 2009). In addition to the amount consumed, the bioavailability of the tea catechins and other polyphenols is an important factor that dictates the benefits of the compounds as chemopreventives. The bioavailability varies depending on the chemical structure of the various catechins. Studies have shown generally low bioavailability of oral catechins in humans (Chow et al., 2001). Intestinal absorption of orally administered catechins was indicated in studies with rats (Okushio et al., 1996). Nanoparticle-mediated delivery of EGCG as also TF has been found to be advantageous in improving their systemic delivery and bioavailability (Singh et al., 2011). Nano-encapsulated EGCG has a significantly longer half-life compared to nonencapsulated EGCG which is completely degraded within 4 h (Siddiqui et al., 2010). Besides, the encapsulated formulations have 10-fold or more dose advantage. Nanoparticles have a mesoscopic size range of 5 to 200 nm (or microns), which facilitates their interaction with biological systems at the molecular level. Many types of nanoparticles-based drug delivery systems which include lipid-based and polymeric particle systems have been developed. The use of polymer-based drug delivery systems in oncology has grown rapidly, especially with the advent of biodegradable polymers. In the nanoparticle formulations, drugs are physically dissolved, entrapped, encapsulated, or covalently attached to the polymer matrix (Rawat et al., 2006). Poly-[D,L-lactide-co-glycolide] (PGLA) is a synthetic biodegradable polymer that is used as a nanocarrier. EGCG or TF when present is released by slow diffusion out of the biodegradable polymeric matrix when the water diffusing in causes the polymer chains to lose their structure and degrade due to hydrolysis (Hrkach, 2008). (This describes the actual mechanism of the technique by which EGCG or TF or any nanoparticle associated compound, for that matter, is released in a sustained manner over a period of time from the biodegradable nanocarrier. The water from the tissue/cells enters the nanoparticle matrix and causes the breakdown of the structure of the nanocarrier polymer, thereby setting the EGCG/TF/or any other bound material free) The specific objective of the present study was to compare the effects of theaflavins and EGCG on oxaliplatin- and satraplatin-treated lymphocytes from colorectal cancer patients and healthy volunteers when used in the bulk form and poly-[D,L-lactide-co-glycolide]-based nanoparticle form. (the term D,L describes the exact chemical structure of the nanocarrier. You can omit it, if you want) Results Lymphocytes from normal volunteers and patients of colon cancer treated with either oxaliplatin or satraplatin at 2?M, were subsequently incubated with different concentrations of theaflavin (TF) and epigallocatechin-3-gallate (EGCG). This dose of TF and EGCG to be used in the study was determined from pilot experiments conducted with TF (bulk: 100?M-800?M; nano: 1?g/ml-6?g/ml) and EGCG (bulk: 1?M -1000?M; nano: 1?g/ml-6?g/ml), to choose the best concentration that does not cause genotoxicity as determined by comet assay (Figs. 1 to 4). In the next set of experiments, the lymphocytes from both normal volunteers and colon cancer patients treated previously with 2?M of either oxaliplatin or satraplatin were subjected to 30-min incubation with TF (bulk concentration used: 100?M, 300?M and 600?M; nano concentration used: 0.5?g/ml; 1.0?g/ml and 1.5?g/ml), and EGCG (bulk concentration: 1?M, 10?M and 100?M; nano concentration: 1?g/ml, 2?g/ml and 4 ?g/ml). The cells were subsequently submitted to comet assay and the results are shown in Figs. 5 to 8. For comparison, lymphocytes without any treatment (negative control), and H2O2-treated positive controls were used. Strong induction of DNA damage was evident through comet assay in oxaliplatin- and satraplatin- treated lymphocytes and the relative DNA breakage is expressed as the olive tail moment in Figs. 5 to 8. DNA damage induced by oxaliplatin in normal cells was the highest and was nearly 4.5-fold higher than the corresponding control value, and double that observed in CRC patients. In satraplatin-treated normal lymphocytes the DNA damage was again higher (~2-fold) than in the untreated control cells, but not very different from that observed in the lymphocytes from CRC patients. Exposure to bulk TF in the range of 100?M-600?M caused an overall significant reduction in DNA damage induced by oxaliplatin or satraplatin (2?M concentration), both in normal and patient-derived lymphocytes (Fig. 5). In healthy oxaliplatin-exposed cells the OTM value observed with 600?M TF used in the bulk form was about 2.5 times lower than the value seen with 100?M TF. The corresponding value obtained with satraplatin was half that observed with 100?M TF. In contrast, when TF was used in the nanoparticle form, it showed a significant enhancement of 2?M oxaliplatin or satraplatin induced DNA damage, particularly in lymphocytes from CRC patients (Fig. 6). The DNA damage was found to increase with the concentration of nanoparticle form of TF used. As observed in Fig. 6, the increase was about 2.5-fold in the highest concentration i.e., 1.5?g/ml. Similarly, in the case of satraplatin-treated lymphocytes from CRC patients, a statistically-significant rise in OTM i.e., DNA damage was noted on exposure to TF in the nanoparticle form. EGCG in bulk form caused a suppression of DNA damage in lymphocytes obtained from normal volunteers as well as CRC patients resulting from treatment with either oxaliplatin or satraplatin, 2?M concentration (Fig. 7). The maximum reduction in OTM observed in normal cells treated with oxaliplatin was around 3.75-fold, in the case of 100?M EGCG, while in satraplatin-treated normal cells the corresponding reduction was 2.2-fold. In the case of lymphocytes from CRC-afflicted persons, the maximum reduction was around 2-fold, observed in both oxaliplatin and satraplatin exposed lymphocytes. The reduction in OTM was correlated to the concentration of EGCG in bulk form. In contrast to the above observations, EGCG used in the form of nanoparticles led to an increase in DNA damage induced by oxaliplatin and satraplatin in normal as well as CRC patient-derived lymphocytes. As seen in Fig. 8, an increase in DNA damage, as indicated by the rise in OTM values, occurred when oxaliplatin- or satraplatin-treated normal and patient lymphocyte samples were exposed to nanoparticle form of EGCG. The extent of DNA damage increased proportionately with increasing doses of the green tea catechin. Discussion Oxaliplatin has been proved an effective drug in clinical treatment of colorectal cancer. Its therapeutic effect occurs on account of covalent binding to DNA and forming cross-links (Misset et al., 2000). These are repaired by the cell process of the NER pathway. DNA breaks that are produced as intermediates during the process are visualized as DNA migration by the comet assay. Satraplatin, an orally bioavailable platinum drug, and its major metabolite, JM118, have shown in vitro as well as in vivo antineoplastic activity in clinical settings (Choy et al., 2008). Compared to oxaliplatin, satraplatin exposure results in a higher rate of cell death in vitro. Reportedly, satraplatin causes apoptosis via multiple death pathways including the caspase 8 cleavages and Fas protein expression (Kalimutho et al., 2011). In the present study, the effects of two tea polyphenols, TF (theaflavin) and EGCG (epigallocatechin gallate) on previously oxaliplatin- and satraplatin-treated lymphocytes from healthy and CRC patients were investigated by comet assay. The objective was to compare the effects of the compounds when used in the bulk and nanoparticle forms. DNA damage due to environmental, medical or lifestyle factors is an important factor in the initiation of carcinogenesis. Although several cellular mechanisms exist to counteract the hazardous events leading to DNA damage, they may not be adequate because the extent of possible encounters with potentially carcinogenic factors and the resultant oxidative stress is too large. Polyphenolic compounds including flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins are ubiquitous in nature. They are known to possess significant antioxidative potency. Oxidative stress has been linked to cancer, besides many other diseases. Chemoprevention using naturally occurring nontoxic agents, especially phytochemicals is deemed a promising strategy for cancer management. Chemopreventive agents could delay the process of carcinogenesis that is, the process by which normal cells are transformed into cancer cells. The major catechin component of green tea, epigallocatechin-3-gallate (EGCG), has been shown to inhibit carcinogenesis (Lambert et al., 2005). The proposed mechanisms of action of EGCG are inhibition of growth factor signalling, inhibition of kinases, and inhibition of DNA methyltransferase (Higdon and Frei, 2003). Sustained long-term treatment with green tea polyphenols have been reported to show a preventive effect against colorectal cancer recurrence (Hoensch et al., 2008). Inflammation is an important factor in the cascade of events leading to colorectal cancer. Green tea solids (this refers to the components present in green tea extract) have been shown to drastically bring down the levels of an inflammatory mediator, prostaglandin PGE2, in the rectal lining cells in human volunteers (August et al., 1999). The major black tea polyphenols such as theaflavin (TF), theaflavin-3-monogallate and theaflavin-3’-monogallate mixture, and theaflavin-3, 3’-digallate are basically oxidation products of green tea polyphenols. Of these various theaflavins, only theaflavin-3-monogallate and theaflavin-3’-monogallate mixture has been found to exhibit a definite differential growth-inhibitory and apoptotic effect against two colon cancer cell lines (Lu et al., 2000). The antiproliferative and cytotoxic effects of the theaflavin monomers were found to be higher in the carcinoma, than in the normal, tongue cells (gingival fibroblasts) (Babich et al., 2008). Tea polyphenols normally possess antioxidative properties (Yoshino et al., 1994; Shiraki et al., 1994). Besides differentially inducing apoptosis in transformed cells, the theaflavin monogallates also specifically inhibit Cox-2 gene expression, which is controlled predominantly at the transcription level (Kim and Fischer, 1998). Thus, green and black tea polyphenols, in addition to exhibiting antioxidant properties, act at several points to control cancer cell growth, survival, and metastasis, with effects at the DNA, RNA, and protein levels. Furthermore, theaflavin monogallates also act as prooxidants and induce oxidative stress, to which, compared to normal cells, carcinoma cells are more sensitive (Babich et al., 2008). The pro-oxidant activity of tea extracts has been shown to increase with the level of fermentation (Tan et al., 2010). To characterise tea based on the degree of fermentation, green tea is non-fermented, oolong tea is semi-fermented, while black tea is fermented post harvest. Thus, the pro-oxidant activity increases in the order of green tea < oolong tea < black tea. Also, according to Tan et al., theaflavins are stronger pro-oxidants than tea catechins (2010). The antioxidant activity of the green tea polyphenols has been implicated as a potential mechanism for the cancer preventive effect of green tea polyphenols. That they are antioxidants with the ability to quench free radical species and chelate chelate or bind by forming chemical bonds with transition metals has been demonstrated in vitro, while in vivo there are indications that they may act indirectly by upregulating phase II antioxidant enzymes (Forester and Lambert, 2011). On the other hand, Lambert and Elias (2010), found evidence for the fact that the effects of green tea catechins were also related to induction of oxidative stress. The pro-oxidant effects observed both in vitro and in vivo, appeared to be the cause of the induction of apoptosis in tumour cells. Elbling et al. (2005) while working in vitro with human promyelocytic leukemic HL60 cell lines found that EGCG does not directly scavenge H2O2 nor does it mediate other antioxidant activities, but actually increases H2O2-induced oxidative stress and DNA damage. The authors also hypothesised that EGCG at nanomolar concentrations may exhibit useful activities as second messenger through the generation of nanomolar H2O2 which, as shown by several studies, performs physiological functions (Forman et al., 2004). EGCG has also been reported to mediate the generation of H2O2 in pancreatic beta cells where it did not scavenge exogenous H2O2 but, on the contrary, it synergistically increased H2O2-induced oxidative cell damage through triggering Fe(II)-dependent formation of a highly toxic radical (Suh et al., 2010). However, in human bronchial epithelial cell lines, treatment with both black and green tea polyphenols have shown comparable inhibitory effects on the growth of cells (Yang et al., 2000). The same study also revealed that H2O2 formation was a key mechanism for growth inhibition and apoptosis induction by EGCG, but not in theaflavin-induced cell death. Furthermore, the results suggested that the degree of growth inhibition observed with the theaflavins was dependent on their chemical structure having gallate (i.e., gallic acid) groups. gallate The experiments done in the present study using bulk and nanoparticle forms of TF as well as EGCG revealed opposite trends in DNA damage measurements by comet assay. For instance, while TF in the bulk form produced a concentration-dependent reduction in DNA damage in oxaliplatin- or satraplatin-treated lymphocytes (Fig. 5), TF when used in the nanoparticle form caused a concentration-dependent increase in DNA damage in the lymphocytes, the maximum increase noted being nearly 2.5-fold more than that observed in 2?M oxaliplatin-treated colorectal cancer patient lymphocytes (Fig. 6). Similarly, a dose-dependent increase in DNA damage occurred with EGCG used in the nanoparticle form (Fig. 8). As opposed to this, EGCG in bulk form led to a significant (P< 0.01) reduction in the DNA damage caused by exposure to oxaliplatin or satraplatin in both normal and CRC patient-derived lymphocytes (Fig. 7), supporting the notion that EGCG acts as both an antioxidant and a pro-oxidant. The greatest protection was afforded by EGCG in bulk form to normal lymphocytes against DNA damage due to oxaliplatin (Fig. 7). This suggests a definite chemopreventive action by EGCG against cytotoxic effects of oxaliplatin. Also, as noted from the increasing trend in OTM values in the presence of nanoparticle forms of TF and EGCG (Figs. 6 and 8, respectively), it could be inferred that TF was a stronger pro-oxidant than EGCG. This is in agreement with the observation of Tan et al. (2010) that the pro-oxidant activity of tea extracts increases with the level of fermentation. what is his observation??. Our results also support the contention of Babich et al. (2008) that carcinoma cells are more sensitive to pro-oxidation by theaflavins as compared to normal cells. The maximum increase (2.7-fold) in OTM in our experiments occurred in (oxaliplatin-treated) colorectal cancer patient lymphoma in the presence of nano-theaflavin (Fig. 6). In conclusion, theaflavins and epigallocatechin-3-gallate (EGCG) administered in the nanoparticle form may change their mode of action and act as pro-oxidants in their nanochemoprevention role whereas, when used in the bulk form, they act as antioxidants in all the concentrations. References Araujo JR, Goncalves P & Martel F., 2011. Chemopreventive effect of dietary polyphenols in colorectal cancer cell lines. Nutr Res., 31(2):77-87. August DA, Landau J, Caputo D, et al., 1999. 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Results of pilot experiment to determine the ‘safe’ values of theaflavin in bulk form for use in the present study Fig. 2. Results of pilot experiment to determine the ‘safe’ values of theaflavin in nanoparticle form for use in the present study Fig. 3. Results of pilot experiment to determine the ‘safe’ values of epigallocatechin-3-gallate (EGCG) in bulk form for use in the present study. Fig. 4. Results of pilot experiment to determine the ‘safe’ values of epigallocatechin-3-gallate (EGCG) in nanoparticle form for use in the present study Fig. 5. Fig. 6. Fig. 7. Fig. 8. Read More