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Green tea catechins are considered as possible cancer preventive agents for several cancer types but little is known regarding their effects on pancreatic cancer cells. The best studied catechin and the major polyphenol present in green tea is epigallocatechin gallate (EGCG). In the present study, we investigated the in vitro anti-tumoral properties of EGCG on human pancreatic ductal adenocarcinoma (PDAC) cells PancTu-I, Panc1, Panc89 and BxPC3 in comparison with the effects of two minor components of green tea catechins, catechin gallate (CG) and epicatechin gallate (ECG). We found that all three catechins inhibited proliferation of PDAC cells in a dose- and time-dependent manner. Interestingly, CG and ECG exerted much stronger anti-proliferative effects than EGCG. Western blot analyses performed with PancTu-I cells revealed catechin-mediated modulation of cell cycle regulatory proteins (cyclins, cyclin-dependent kinases [CDK], CDK inhibitors). Again, these effects were clearly more pronounced in CG or ECG than in EGCG-treated cells. Importantly, catechins, in particular ECG, inhibited TNFα-induced activation of NF-κB and consequently secretion of pro-inflammatory and invasion promoting proteins like IL-8 and uPA. Overall, our data show that green tea catechins ECG and CG exhibit potent and much stronger anti-proliferative and anti-inflammatory activities on PDAC cells than the most studied catechin EGCG. (Cancer Sci 2011; 102: 728–734)
Pancreatic ductal adenocarcinoma (PDAC) is the 10th most commonly diagnosed cancer, but it has the fourth highest mortality rate among all cancer-related deaths in the US. Since 1975 long-term survival rates have improved only marginally and are currently around 5%.(1) The poor prognosis of PDAC is attributed to an aggressive cancer progression with a high invasive and metastatic potential. Conventional chemotherapy and radiotherapy are still largely ineffective in improving pancreatic cancer and surgical resection has only limited benefits because of a high rate of recurrent disease.(2) Recently, we have shown in a murine orthotopic xenotransplantation model that tumor recurrence and metastasis after surgical resection of PDAC is substantially driven by tumor cell-derived, tumor necrosis factor α (TNFα).(3) Furthermore, we showed a significant contribution of endogenous TNFα to the growth and invasiveness of primary PDAC tumors. We proposed that TNF-inhibiting drugs, infliximab and etanercept, which are broadly used for the treatment of chronic inflammatory diseases, will also be beneficial in PDAC treatment, especially after subtotal pancreaticoduodenectomy.
Plant phytochemicals like quercetin, resveratrol and catechins are some examples of alternative chemotherapeutics being extensively tested for their anti-tumoral and anti-inflammatory potential.(4,5)
Catechins are polyphenols present in green and black tea, red wine and chocolate. Catechins, which mainly occur in green tea, comprise epigallocatechin gallate (EGCG), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (CG), epicatechin gallate (ECG), gallocatechin gallate (GCG) and catechin (C).(6) One gram of dried green tea leaves contains more than 200 mg catechins.(7) The best studied catechin is EGCG, the major polyphenol in green tea.(8)
Epigallocatechin gallate has been shown to inhibit the proliferation of many cancer cells by inducing apoptosis and cell cycle arrest.(9–14) Beyond the suppression of tumor cell proliferation, EGCG may inhibit inflammation, invasion and metastasis of carcinoma cells.(15–17) There is some evidence that other catechins, including CG and ECG, may also act as anti-proliferative agents.(18–21) Babich et al.(22) reported that CG and its epimer ECG, more potently than EGCG, inhibit proliferation of human cancer cells of the oral cavity. The effect of green tea catechins other than EGCG on pancreatic cancer cells is currently unknown.
Thus, in the present study we analyzed the potential anticancer properties of CG and ECG in comparison with EGCG on pancreatic cancer cell line PancTu-I, which has been extensively characterized by us in vitro as well as in an appropriate PDAC model system.(3,23) We examined the effects of catechins on proliferation and the TNFα-mediated inflammatory response, and the uncovered mechanisms underlying this activity.
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Epidemiological studies have failed to identify a definite and consistent association between the consumption of green tea and the risk of pancreatic cancer.(29–31) On the other hand, some cell culture(32–34) and animal studies(35,36) demonstrated beneficial effects of green tea catechins on pancreatic cancer cells in vitro and in vivo. Several studies on cancer cells of other origin strengthen the evidence for potential anticancer activity of both EGCG and catechin mixtures.(37,38) To extend these findings, we investigated in vitro the potential anticancer action of three different catechins in PancTu-I cells. We chose this human PDAC cell line due to its aggressive phenotype characterised by a resistance to death receptor-induced apoptosis, as well as its high potential for invasion and metastasis.(3,39) Cancer preventive agents that modulate this phenotype are in demand for therapies of pancreatic cancer.
Our results demonstrate differences in anticancer activity between CG, ECG and EGCG. All catechins tested in this study possess a polyphenolic structure including a gallate group at position three of the C-ring, two hydroxyl groups at the A-ring and two or three hydroxyl groups at the B-ring. In contrast to CG and ECG, EGCG contains an additional 5′-OH group in the B-ring. Epicatechin gallate represents the epimer of CG showing a differential steric configuration of the B-ring. The typical chemical structure of CG, ECG and EGCG is responsible for their free radical scavenging activity.(40) Among green tea catechins, EGCG exhibits the most potent free radical scavenging activity in vitro.(41,42) Thus, differences in the anticancer potential of catechins in pancreatic cancer cells seem to be not mainly related to free radical scavenging activity.
It is known that catechins are susceptible to autoxidation under cell culture conditions. Auto-oxidized catechins accumulate in catechin dimers and H2O2 and may act as pro-oxidants, thereby influencing apoptosis, cell growth and several other cellular signalling.(43,44) Recent experimental data demonstrate that the extent of autoxidation differs between the different catechins as determined by the production of H2O2. Catechin gallate and ECG generated much less H2O2 in tissue culture medium than EGCG.(45) The clearly weaker effects of EGCG compared with CG and ECG regarding the inhibition of cell proliferation and inflammation may therefore be caused by increased degradation of EGCG due to autoxidation.
Interestingly, although catechins markedly inhibited cell proliferation we did not observe any specific cell cycle arrest. However, we found strong modulation of cell cycle regulators of the G1/S phase (cyclin D1 and D3, CDK4, p21) and the G2/M phase (cdc2, cyclin B1). These results are in line with previous studies in which catechins modulated G1-regulatory proteins like cyclin D1, cyclin E, p21, CDK4 and CDK6,(9,11,12,14,46) but in contrast to our results the authors observed clear G1-arrest under catechin treatment. It is known that catechins, generally EGCG, are incorporated into the phospholipid bilayer of membranes and inhibit the binding of ligands to the cell membrane-surface receptors.(47) Consequently, multiple signaling pathways, including membrane-associated receptor tyrosine kinases (EGFR, Her2),(48,49) MAP kinases, Akt(48) as well as AP-1 and NF-κB(48,50,51) have been determined as putative targets for catechins. Due to the inhibition of these and other still unknown pathways, many signaling molecules, for example the above mentioned G1-S- and G2-phase driving proteins and survivin, are modulated in parallel. Consequently, catechins most likely induce cell cycle arrest in all cell cycle phases and therefore FACS analyses of unsynchronized growing tumor cells revealed no specific cell cycle arrest.
Interestingly, we found that catechins, especially ECG, almost completely blocked TNFα-induced NF-κB activity and consequently strongly diminished the secretion of IL-8 and uPA following TNFα treatment. Both IL-8 and uPA are proteins overexpressed in pancreatic cancer cells and linked to invasion, angiogenesis and metastasis.(24,52–54) Thus, we selected IL-8 and uPA as molecular biomarkers of inflammation. It is known that tumor cells are exposed to TNFα by infiltrated immune cells or tumor cells themselves.(3,55) Blocking TNFα with TNF-neutralising drugs (infliximab, etanercept) inhibited IL-8 and uPA secretion and NF-κB activity and resulted in decreased invasion. Inhibition of TNFα has an even stronger effect in vivo where it reduced tumor growth and metastasis.(3) These data indicate that catechins, especially ECG, are also able to inhibit TNFα-mediated effects in vitro. Therefore, it is possible that catechin treatment could diminish the aggressive phenotype of pancreatic cancer supporting its potential use in anticancer therapy.
One possible mechanism for the catechin-mediated inhibition of TNFα-induced NF-κB activity could be the downregulation of TRAF2 expression, which correlated with the extent of inhibitory power for the particular catechin. However, because catechins can alter the plasma membrane structure,(47) it is also possible that they change the localization/function of TNFR1 and thereby diminish the TNF-mediated response. Further studies will be needed to clarify this issue.
In this study we used catechin concentrations comparable with other studies in pancreatic cancer cells in the range of 25–200 μM.(32–34) However, all three catechins showed anticancer activity at concentrations (≥40 μM) that are much higher than physiologically achievable in blood after green tea catechin consumption.(56) Peak plasma levels in healthy volunteers receiving 687.5 mg EGCG or 663.5 mg ECG reached 1.3 μM/L and 3.1 μM/L, respectively.(57) As CG is only a minor constituent in green tea catechins, there is no information about its level in blood plasma. It is therefore most likely that an anticancer therapy of pancreatic cancer with catechins in general and ECG in particular may be only effective when green tea catechins are administered at pharmacological concentrations.
In summary, our results provide evidence that catechins show an anti-proliferative and, especially ECG, an anti-inflammatory potential that could possibly increase the efficacy of classical anticancer therapies. The presented in vitro mechanisms of catechins need to be evaluated in animal models of pancreatic cancer in future in vivo studies.