New cancer treatment strategy using combination of green tea catechins and anticancer drugs. 3 To whom correspondence should be addressed.

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Review Article

New cancer treatment strategy using combination of

green tea catechins and anticancer drugs

Masami Suganuma,1,3Achinto Saha2and Hirota Fujiki2

1Research Institute for Clinical Oncology, Saitama Cancer Center, Saitama;2Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan

(Received October 4, 2010⁄Revised November 8, 2010⁄Accepted November 13, 2010⁄Accepted manuscript online November 19, 2010⁄Article first published online December 30, 2010)

Green tea is now recognized as the most effective cancer preven-tive beverage. In one study, 10 Japanese-size cups of green tea daily supplemented with tablets of green tea extract limited the recurrence of colorectal polyps in humans to 50%. Thus, cancer patients who consume green tea and take anticancer drugs will have double prevention. We studied the effects of combining ())-epigallocatechin gallate (EGCG) and anticancer drugs, focus-ing on inhibition of cell growth and induction of apoptosis. Numerous anticancer drugs, such as tamoxifen, COX-2 inhibitors, and retinoids were used for the experiments, and the combina-tion of EGCG and COX-2 inhibitors consistently induced the enhancement of apoptosis. To study the mechanism of the enhancement, we paid special attention to the enhanced expres-sions of DDIT3 (growth arrest and DNA damage-inducible 153, GADD153), GADD45A, and CDKN1A (p21⁄WAF1⁄CIP1) genes, based on our previous evidence that a combination of EGCG and sulindac specifically induced upregulated expression ofGADD153

andp21genes in PC-9 lung cancer cells. The synergistic enhance-ments of apoptosis and GADD153 gene expression in human non-small cell lung cancer cells by the combination of EGCG and celecoxib were mediated through the activation of the MAPK signaling pathway. This article reviews the synergistic enhance-ment of apoptosis, gene expression, and anticancer effects using various combinations of EGCG and anticancer drugs, including the combination of ())-epicatechin (EC) and curcumin. Based on the evidence, we present a new concept: green tea catechins as synergists with anticancer drugs. (Cancer Sci2011; 102: 317–323)

‘‘C

ancer chemoprevention’’ was defined in 1976,(1)and Michael Sporn(2) introduced the term ‘‘combination cancer chemoprevention’’ in the journal Nature in 1980. He defined the term as the combined use of several drugs with dif-ferent mechanisms of action exerting marked synergistic preven-tive effects. In 1983, we began to study the cancer prevenpreven-tive activity of green tea catechins.(3,4)Green tea extract chemically contains at least four tea catechins: EGCG, ECG, EGC, and EC. The first three catechins induce cancer preventive activities, whereas EC is inactive.(5)We first reported that combinations with the active catechins and inactive EC induced synergistic effects on induction of apoptosis and inhibition of cell growth of human lung cancer cell line PC-9, and inhibition of TNF-a release from BALB⁄3T3 cells treated with okadaic acid, a tumor promoter.(6) This suggests that whole green tea, which is a mixture of green tea catechins, is a more effective and practical cancer preventive than green tea catechins alone.(6) Using tritium (3H)-EGCG we showed that EC induced the enhanced incorporation of3H-EGCG and other active green tea catechins into cells.(6) In addition, we recently showed that drinking 10

cups of green tea supplemented with green tea tablets signifi-cantly (50%) prevented recurrence of colorectal adenomas in patients who had received polypectomy 1 year before, and it also reduced the size of adenomas.(7) These exciting results prompted us to think that green tea catechins together with anticancer agents are effective cancer treatments. Our study on cancer prevention with green tea thus moves to focus on a new strategy of cancer treatment based on a combination of green tea catechins and anticancer drugs.

We studied the effects of anticancer drugs tamoxifen, sulin-dac, celecoxib, and retinoids in combination with EGCG or green tea extract. Although all anticancer drugs are structurally and functionally different, the combination with EGCG and an anticancer drug synergistically enhanced the induction of apop-tosis 10–15 times as strongly as any anticancer drug alone in PC-9 cells.(6,8) Moreover, cotreatment with green tea extract and sulindac showed enhanced prevention of intestinal tumor development in Min mice.(9) Using a human cancer cDNA expression array, we found that a combination of EGCG and sulindac induced upregulated expression ofDDIT3(GADD153) and CDKN1A (p21⁄WAF1⁄CIP1) genes, and downregulated expression of four genes,PLAT (T plasminogen activator), tis-sue inhibitors of metalloproteinase (TIMP3), IL1B(IL-1b), and ITGB4 (integrin b4).(3,10) These significant results led us to write a review article on a new strategy of cancer treatment based on the combination of EGCG and anticancer drugs.

Curcumin is a phenolic compound present in the plant Curcuma longa(L.) making up 2–5% of total spices in turmeric, a popular spice in India and neighboring countries.(11,12) We recently reported that the combination of EC, an inactive cate-chin, and curcumin induced cancer preventive effects associated with enhanced induction of GADD153 and GADD45 gene expression.(13)The results gave us a hypothesis indicating that numerous anticancer compounds that are present in vegetables and foods can induce cancer preventive activity in combination with green tea catechins. Weinstein and colleagues and our research group both reported enhanced anticancer effects by a combination of EGCG and anticancer drugs such as 5-FU, taxol, and gefitinib.(14–16)The combination of green tea catechins and anticancer drugs induces synergistic enhancement of expression of theGADD153gene, resulting in a new mechanism of antican-cer treatment that is different from that of green tea catechin or anticancer drugs alone. We anticipate that the combination can-cer treatment with green tea catechins and anticancan-cer drugs will be an effective method to enhance therapeutic effects, and that this strategy will reduce the adverse effects of anticancer drugs in cancer patients.

3To whom correspondence should be addressed.

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Combination of EGCG and tamoxifen

Tamoxifen is an anti-estrogenic compound used for prevention of breast cancer.(17) The combination of EGCG (75 and 100lM) and tamoxifen (5–200lM) induced apoptosis in PC-9 cells more strongly than EGCG alone or tamoxifen alone. Table 1 shows that apoptosis was induced in 27.3% of cells by cotreatment with 100lM EGCG and 10lM tamoxifen, a 1.3-fold additive enhancement.(6) The combination of EGCG and tamoxifen also induced the inhibition of TNF-arelease from BALB⁄3T3 cells treated with okadaic acid, a tumor promoter,(6) along with the inhibition of cell growth in human breast cancer cell line MCF-7 (twofold enhancement, data not shown).(6) Spe-cifically, we showed that TNF-a is an endogenous tumor pro-moter and cancer mediator, using TNF-a deficient mice.(18–20) Thus, our results significantly showed that the essential mecha-nism of tumor promotion was inhibited by the combination. Another research group found enhanced antitumor effects on the development of spontaneous mammary tumors in C3H⁄Ouj mice with a combination of green tea extract and tamoxifen.(21) This combination also inhibited proliferation of ER-positive (MCF-7, ZR-75–1, and T-47D) and ER-negative (MDA-MB-231) human breast cancer cells, and inhibited growth of xeno-grafts of MCF-7 and MDA-MB-231 in nude mice more strongly than tamoxifen alone.(22,23)The combination of EGCG or green tea extract with tamoxifen increases preventive effects on breast cancer cells regardless of their ER status, thus it may be an ideal tool for prevention of breast cancer in general.

Combination of EGCG and COX-2 inhibitors

EGCG and sulindac. Sulindac, a non-selective COX-2 inhibi-tor and non-steroidal anti-inflammainhibi-tory drug, suppresses colo-rectal tumorigenesis in patients with familial adenomatous polyposis, whose condition is caused by germline mutation of theadenomatous polyposis coli(APC) gene.(24)Although sulin-dac is a preventive drug for colon cancer in patients with famil-ial adenomatous polyposis, its chronic treatment is restricted

because it also causes bleeding and peptic ulceration in the gas-trointestinal tract. The combination with catechins may over-come this side-effect by decreasing the concentration of sulindac.

A combination of 100lM EGCG and 50lM sulindac increased the induction of apoptosis 10.5-fold in PC-9 cells (Table 1), whereas sulindac alone at concentrations up to 100lM did not induce any apoptosis.(6)We next looked at the synergistic effects of the combination with EGCG and metabo-lites of sulindac – sulindac sulfide and sulindac sulfone – on induction of apoptosis in PC-9 cells. The combination of 75lM EGCG and 10lM sulindac induced apoptosis more than 20 times stronger than sulindac alone. Combinations of 75lM EGCG and the same concentrations of sulindac sulfide, an inhib-itor of COX-1 and COX-2, and with EGCG and sulindac sulfone, an inactive metabolite, also synergistically induced apoptosis of the cells (data not shown).(25)We therefore believe that the synergistic effects on apoptosis by the combination are not necessarily related to inhibition of COX.(25)

Moreover, the combination synergistically inhibited cell growth of mouse colon adenocarcinoma cell line, Colon 26, more strongly than EGCG alone or sulindac alone (data not shown). To study the enhanced effects of the combination of EGCG and sulindac, Min mice, which have a germline mutation of the murine Apc gene, were given drinking water with 0.1% green tea extract and diet containing 0.03% sulindac for 10 weeks. Table 2 shows the additive inhibition of tumor forma-tion by the combinaforma-tion: control Min mice without any treat-ment developed 72.3 ± 28.3 tumors per mouse at 16 weeks of age, and the combination with green tea extract and sulindac reduced the number of tumors to 32.0 ± 18.7 tumors per mouse, a decrease of 44.3%.(9)The combination also resulted in signifi-cantly smaller tumor size than in any of the other groups.(9)In addition, Ohishiet al.(26)reported that a combination of 0.01% EGCG in drinking water and sulindac (10 mg⁄kg, 3 per week) synergistically suppressed the formation of aberrant crypt foci from 46.2 ± 4.9 to 10.0 ± 3.2 in F344 rats induced with azoxymethane.

Modulation of gene expression by combination of EGCG and sulindac. The cDNA expression array made it possible for us to monitor the expression levels of a multitude of genes Table 1. Synergistic induction of apoptosis and GADD153 gene

expression by combining ())-epigallocatechin gallate (EGCG) and anticancer drugs in PC-9 lung cancer cells

Anticancer drugs Induction of apoptosis† % of apoptotic cells (fold)‡ Expression of

GADD153gene fold expression (fold)§ Effects Anti-estrogen Tamoxifen– 27.3 (·1.3) n.d. Additive Cox-2 inhibitors Sulindac– 42.9 (·10.5) 11.2 (·10.2) Synergistic Celecoxib– 56.3 (·16.1) 16.8 (·12.9) Synergistic Retinoids ATRA– 80.2 (·26.7) 12.1 (·5.0) Synergistic 13-cis-RA– 45.5 (·12.6) 6.0 (·4.6) Synergistic 9-cis-RA– 25.3 (·11.0) 4.9 (·8.3) Synergistic 4-HPR– 63.8 (·0.9) 36.0 (·1.7) No effect

†Percent of apoptotic cells was determined by flow cytometry.‡Fold enhancement by combination with EGCG was calculated compared with that of anticancer drug alone. Percentages of apoptotic cells in non-treated and EGCG-treated cells were 5.0 and 6.1%, respectively. §Expression ofGADD153gene is the fold-expression compared with that of non-treated PC-9 cells as assessed by RT-PCR.–Cells were treated with combinations of 100lM EGCG and 10lM tamoxifen, 50lM sulindac, 10lM celecoxib, 10lM all-trans-retinoic acid (ATRA), 10lM 13-cis-retinoic acid (RA), 10lM 9-cis-RA, and 50lMN -(4-hydroxyphenyl)retinamide (4-HPR) for 24 h (for gene expression) or 40 h (for apoptosis). n.d., not determined.

Table 2. Synergistic inhibition of tumor formation by combining green tea extract and COX-2 inhibitors

Tumor incidence (%)

Average no.

tumors⁄mouse (%) References Intestine (Min mice)†

Non-treated 100.0 72.3 ± 28.3 (100.0) 9 Green tea extract 100.0 56.7 ± 3.5 (78.4)

Sulindac 100.0 49.0 ± 12.7 (67.7) Green tea extract + sulindac 100.0 32.0 ± 18.7 (44.3)§ Lung (A⁄J mice treated with NNK)‡ NNK 100.0 3.2 (100.0) 32 NNK + green tea extract 100.0 2.2 (100.0) NNK + celecoxib 90.0 1.5 (46.9) NNK + green tea extract + celecoxib 73.3§ 1.1 (34.4)§

†Multiple intestinal neoplasia (Min) mice were treated with 0.1% green tea extract in drinking water and 0.03% sulindac in diet.‡A⁄J mice were injected i.p. with 100 mg⁄kg 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), then treated with 0.3% green tea extract in drinking water and 0.05% celecoxib in diet. §P< 0.05.

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simultaneously.(27) Using Clontech’s Atlas cDNA expression array, which deals with 588 known cancer-related genes, we found that treatment with EGCG in PC-9 cells induced upregu-lation of one gene, retinoic acid receptora1, and downregulation of four genes, MAP3KI4 (NF-jB inducing kinase), DAPK1 (death-associated protein kinase I), rho B, and tyrosine–protein kinase (SKY).(10,28)The downregulation ofNIKgene expression resulted in less activation of the NF-jB pathway, becauseNIK is an activator of IKKa, and related to constitutive activation of the NF-jB pathway, one of the essential signals for cancer development.

Using the same experimental procedure as with the human cancer cDNA expression array, the levels of gene expression in PC-9 cells treated with 200lM EGCG and 10lM sulindac, with 200lM EGCG alone, or with 10lM sulindac alone, using non-treated cells as a control, were determined. The combina-tion induced upregulated expression of GADD153 and p21 genes dramatically, approximately 10.2-fold and threefold, respectively, whereas those genes were not affected by treat-ments with either EGCG or sulindac alone.(3,10) The upregula-tion of theGADD153gene induces apoptosis in the cells,(29)and upregulation of thep21gene is related to inhibition of cell pro-liferation by blocking the cell cycle.(30)Furthermore, the combi-nation induced downregulated expression of T plasminogen activator, TIMP3, IL-1b, and integrin b4 genes, all <0.3-fold.(3,10) These altered genes were completely different from the previously mentioned genes found in EGCG treated cells, and this finding is our first evidence that the combination with EGCG and sulindac induces some new mechanism of cancer treatment associated with expression patterns in genes, patterns not observed with EGCG alone or sulindac alone.(3,10) These results were well supported by evidence that the combination induces synergistic effects on apoptosis of the cells (Table 1). Upregulation of the GADD153 gene was much stronger than that of thep21gene, as mentioned above, and we think that the enhanced expression of theGADD153gene is a new mechanism of combination cancer prevention.

EGCG and celecoxib. Celecoxib is a COX-2 selective inhibi-tor, and both sulindac and EGCG are non-selective COX-2 inhibitors. Celecoxib is a promising preventive drug but its long-term use causes adverse cardiovascular effects.(31)A com-bination of EGCG and celecoxib was used on three human non-small cell lung cancer cell lines, PC-9, A549, and ChaGo K-1, to determine the induction of apoptosis and upregulation of GADD153gene expression. The combination of 100lM EGCG

and 10lM celecoxib induced the apoptosis of PC-9 cells approximately 16.1-fold, along with synergistic expression of the GADD153 gene (12.9-fold) (Table 1). As a result of the strong gene expression, a large amount of GADD153 protein was confirmed in the cells treated with the combination. Syner-gistic induction of apoptosis by the combination was also observed in both A549 and ChaGo K-1 cells, along with enhanced GADD153 gene expression.(8)However, the expres-sion of other apoptosis related genes, such asp21andGADD45, was not enhanced by the combination.

The synergistic inhibition of tumor formation by the combina-tion of green tea extract and celecoxib was also confirmed in NNK-induced lung tumorigenesis in A⁄J mice. Female A⁄J mice were given an i.p. injection of 100 mg⁄kg body weight of NNK, and 3 days later mice began treatment with 0.3% green tea extract in drinking water and 0.05% celecoxib in diet, continuing for 16 weeks. The combination significantly reduced tumor incidence (from 100% to 73.3%) and the average number of tumors⁄mouse (from 3.2 to 1.1), a 34.4% inhibition (Table 2).(32)The results clearly indicated effective cancer pre-vention in the lungs by a combination of EGCG and celecoxib.

Synergistic anticancer effects in vitro andin vivoon human prostate cancer cells were also achieved by the combination of EGCG and 2 selective inhibitors. Specifically, new COX-2 selective inhibitor NS398, in combination with EGCG, enhanced induction of apoptosis and inhibited growth of LNCaP, PC-3, and CWR22Rm1 cells, and the combination of green tea catechin and celecoxib resulted in enhanced inhibition of tumor growth in athymic nude mice implanted with CWR22Rm1 cells.(33)

Enhancement ofGADD153gene expression and MAPK signaling pathway

GADD153, also known as CHOP (C⁄EBP homology protein), is a transcription factor belonging to the CCAAT⁄enhancer bind-ing protein (C⁄EBP) family.(34) GADD153 gene expression becomes highly upregulated in cells as a result of various stress conditions, and some reports show that GADD153 is directly involved in the regulation of apoptosis, mediated through activa-tion of the MAPK signaling pathway.(35) The combination of 100lM EGCG and celecoxib at various concentrations (1, 10, and 50lM) dose-dependently induced phosphorylation of ERK1⁄2 and p38 MAPK in PC-9 cells 1 h after treatment, indi-cating the significant activation of ERK1⁄2 and p38 (Fig. 1A).

Fig. 1. Significance of ERK1⁄2 activation in enhanced expression of theGADD153gene by the combination of ())-epigallocatechin gallate (EGCG) and celecoxib. (A) Combination of EGCG and celecoxib significantly increased phophorylated ERK 1⁄2 and p38MAPK in PC-9 cells 1 h after treatment. (B) A specific inhibitor of ERK1⁄2, PD98059 (PD), and a selective inhibitor of MEK, UO126 (UO), both inhibited the enhanced expression of theGADD153

gene induced by the combination of EGCG and celecoxib, whereas a specific inhibitor of p38MAPK, SB203580 (SB), and a specific inhibitor of protein kinase C, calphostin C (Cal), did not. C, celecoxib; E, EGCG.

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Using specific inhibitors of the protein kinases PD98059 (a specific ERK1⁄2 inhibitor), UO126 (a selective MEK inhibitor), SB203580 (a specific p38 MAPK inhibitor), and calphostin C (a specific protein kinase C inhibitor), we studied the involvement of ERK1⁄2 activity in both expression of the GADD153 gene and synergistic induction of apoptosis by the combination. Pre-treatments with PD98059 and UO126 dose-dependently reduced the expression ofGADD153gene upregulated by the combina-tion, but other inhibitors, SB203580 and calphostin C, were not effective (Fig. 1B). Moreover, two inhibitors, PD98059 and UO126, dose-dependently inhibited the synergistic induction of apoptosis by the combination (data not shown).(8)Similar results were also obtained in other lung cancer cell lines, A549 and ChaGo K-1. All the results indicate that the combination of EGCG and celecoxib induced the activation of the ERK signal-ing pathway, followed by enhanced expression of theGADD153 gene, and then induction of apoptosis.

Combination of EGCG and retinoids

To extend our concept of the combination cancer treatment, we studied the effects of combining EGCG and retinoids. Numerous retinoids have been developed, and their mechanisms of action are varied.(36)Four retinoids, ATRA, 13-cis-RA, 9-cis-RA, and 4-HPR, were used for our experiments. The combination of 100lM EGCG with 10lM ATRA, or 10lM 13-cis-RA, or 10lM 9-cis-RA, induced dramatically upregulated expression of the GADD153 gene in PC-9 cells 24 h after treatment (Table 1). However, 4-HPR alone induced the enhancement, and did not show any further enhancement with EGCG (Table 1). Thus we think that 4-HPR acts differently from other retinoids, mediated through retinoic acid receptor-independent mechanisms. The synergistically enhanced expression of the GADD153gene by the combination of EGCG with the retinoids ATRA, 13-cis-RA, and 9-cis-RA showed patterns similar to those of apoptosis induction (Table 1).(37)Although the mecha-nisms of these three retinoids are different from those of the two COX-2 inhibitors, the combination of retinoids with EGCG induced similar, strong inductions ofGADD153gene expression and synergistic induction of apoptosis in human cancer cells. Combination of EC and curcumin

Curcumin is traditionally well known to have therapeutic effects on various types of diseases.(13)The combination of EGCG and curcumin showed synergistic interactions in growth inhibition and increased sigmoidicity of the dose-effect curves in human oral epithelial cells,(38)and the combination also provided higher efficacy in inhibiting ERabreast cancer cell growthin vitroand

in vivo.(39)Although the cancer preventive activity of curcumin has been intensively studied in animal experiments, human studies on cancer prevention with curcumin have not yet been reported, due to the low bioavailability, which means the poor absorption of curcumin into the cells and tissues, in contrast to catechins.

To overcome this low bioavailability, we used a combination of curcumin with catechin in experiments, by examining growth inhibition of PC-9 cells and induction of apoptosis. As EGCG and EC showed very similar synergistic effects with curcumin on these tests, we chose EC for further experiments, because EC is a relatively inexpensive compound compared with EGCG. The combination of 200lM EC and 20lM curcumin signifi-cantly induced much higher inhibition of human lung cancer cell lines PC-9 and A549 than either EC alone or curcumin alone (data not shown). The combination significantly increased induction of apoptosis to 59.0% of PC-9 cells after 72 h, whereas treatment with EC alone showed a marginal effect, and that with curcumin alone induced 42.3% apoptosis of the cells.(13) The results suggest that the enhancement of growth inhibition by the combination of EC and curcumin is also asso-ciated, in part, with the induction of apoptosis.

The combining of EC (100 and 200lM) and 20lM curcu-min induced 1.7- and 2.1-fold enhancement ofGADD153gene expression in PC-9 cells after 24 h, whereas treatment with EC alone or with curcumin alone was marginal.(13)In addition, the synergistic enhancement of GADD45 gene expression was observed by the combination in PC-9 cells. However, the combi-nation induced synergistic expression of the GADD153 gene, but not the GADD45 gene, in A549 cells, and synergistic enhancement of p21 gene expression was observed in PC-9 cells, but not in A549 cells.(13)

To characterize the molecular nature of the induction of apop-tosis and the enhanced expressions ofGADD153andGADD45 genes, siRNA duplexes were used to knock down their mRNA levels in PC-9 cells. The treatment of PC-9 cells with siRNAs for GADD153 and GADD45 significantly reduced apoptosis induced by the combination of EC and curcumin as compared with cells with control siRNA, along with the reduction of both mRNA and protein levels ofGADD153andGADD45genes.(13) Enhanced incorporation of curcumin with EC

We first found that EC enhanced the incorporation of3H-EGCG into PC-9 cells by 1.5-fold, and unlabeled EGCG inhibited that of 3H-EGCG dose-dependently (Fig. 2A), suggesting that the presence of EC facilitates 3H-EGCG incorporation into the cells. Therefore, using the spectrophotometric method, we investigated whether EC would enhance the incorporation of

(A) (B)

Fig. 2. Enhancement of ())-epigallocatechin gallate (EGCG) and curcumin incorporation with () )-epic-atechin (EC). (A) 3H-EGCG incorporation into PC-9

cells was enhanced with EC (

), but was inhibited with non-radioactive EGCG (s). (B) Intracellular curcumin levels determined by the spectrophoto-metric method. Curcumin levels in the cells were increased by combining with EC.

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intracellular curcumin levels in the cells. As Figure 2(B) shows, the combination of 100lM EC and 20lM curcumin signifi-cantly increased the amounts of intracellular curcumin approxi-mately 1.3-fold over that with curcumin alone.(13) Thus, EC enhanced the uptake of curcumin into PC-9 cells.

The average daily individual intake of turmeric is approxi-mately 2.0–2.5 g in the Indian subcontinent, or up to 100 mg of curcumin ingestion on a regular basis without any adverse effects.(40) Based on the above-mentioned results, we assume that the combination of 1 g EC and 100 mg curcumin will result in much greater preventive activity than that produced by curcu-min alone in humans.

Combinations with EGCG and anticancer drugs

In 2001, I. B. Weinstein’s group reported that EGCG at 0.1lg⁄mL markedly enhanced the growth inhibitory effects of 5-FU on human head and neck squamous cell carcinoma lines, YCU-N861 (3.6-fold) and YCU-H891 (45-fold) (Table 3).(14) Interestingly, YCU-H891 cells that are resistant to 5-FU, became sensitive to the drug when combined with EGCG.(14) They also reported that treatment with EGCG inhibited the growth of both YCU-H891 cells and breast cancer cell line BT-474 more strongly than that with taxol alone did.(15)Lianget al. reported that treatment with EGCG significantly reduced the IC50value for doxorubicin from 36 to 1.9lg⁄mL, and that for

ECG to 2.3lg⁄mL in BEL-7404⁄DOX cells, and the combina-tion with EGCG and doxorubicin clearly enhanced the reduccombina-tion of tumor volumes in anin vivoxenograft model inoculated with BEL-7404⁄DOX cells (Table 3).(41)

We previously reported that EGCG has a sealing effect as its mechanism of cancer prevention, that is, the treatment of cells with EGCG interrupts the interaction of cellular factors in mem-brane receptors by covering the cell surface and intracellular organella.(42)In the experiments, we showed that treatment with EGCG inhibited both the activation of EGFR and the EGFR downstream signaling pathway.(42,43)The combination of EGCG and the EGFR tyrosine kinase inhibitor, gefitinib, additively inhibited the growth of human lung cancer cell lines PC-9 and A549, compared with EGCG alone or gefitinib alone.(16) The combination of EGCG and gefitinib more strongly inhibited phosphorylation of EGFR than gefitinib alone.(16)It is important to note that the combination induced expression of the GADD153 gene and apoptosis, whereas gefitinib alone did not induce expression of the GADD153 gene.(16) The synergistic inhibition by the combination of EGCG and another EGFR tyro-sine kinase inhibitor, erlotinib, was also observed in five head and neck squamous cell carcinoma cell lines, and the phospho-rylations of EGFR and AKT were strongly inhibited, followed by induction of apoptosis (Table 3). Furthermore, antitumor effects of the EGCG and erlotinib combination were shown on xenograft mice bearing Tu212 cells.(44) All the results showed that EGCG probably enhances the sensitivity of cancer cells to various anticancer drugs.

Discussion

Green tea is recognized as a cancer preventive beverage in Japan, and it is now developing as a cancer preventive drug in the USA and Europe. Two clinical phase II trials carried out Table 3. Enhancements of growth inhibitory effects by combining ())-epigallocatechin gallate and anticancer drugs

Anticancer drugs Cancer (cell lines) Effects References 5-Fluorouracil Head and neck squamous cell carcinoma

(YCU-N861, YCU-H891)

Synergistic growth inhibition 14 Taxol Head and neck squamous cell carcinoma

(YCU-H891) Breast carcinoma (BT-474)

Synergistic growth inhibition 15 Doxorubicin Hepatocellular carcinoma

(BEL-7404⁄DOX)

Synergistic growth inhibition Decreased tumor volume

41 Gefitinib Lung cancer (PC-9, A549) Synergistic growth inhibition 16 Erlotinib Head and neck squamous cell carcinoma

(Tu177, Tu212, 886LN, SQCCY1, SQCCY38)

Synergistic growth inhibition Decreased tumor volume

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Fig. 3. Green tea catechins as synergists with anticancer drugs. Combination treatment with () )-epigallocatechin gallate (EGCG) and cancer preventive agents (COX-2 inhibitors and retinoids), with EGCG and gefitinib, and with ())-epicatechin (EC) and curcumin synergistically enhanced

GADD153 gene expression and high induction of apoptosis in cancer cells.

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in Italy and the USA showed the preventive effects of green tea on prostate cancer in patients with prostate intraepithelial neoplasias and on the high-risk oral premalignant lesion.(45,46)

Considering the significant cancer preventive activity of green tea catechins in humans, we began to study an additional feature of green tea catechins: if patients consume sufficient amounts of green tea and also take anticancer agents, they get double prevention. Several experiments showed that the combi-nation of EGCG and COX-2 inhibitors sulindac and celecoxib, along with retinoids and curcumin, synergistically or additively induced apoptosis and enhanced the expression of GADD153 and GADD45 genes in PC-9 cells. The mechanisms of the enhanced gene expressions were shown to be mediated through activation of the MAPK signaling pathway. It is important to note that these modulations in gene expression are newly induced by the combination, but not by a single compound alone. It may subsequently be found that numerous anticancer compounds that are present in vegetables and foods can induce such synergistic cancer preventive effects with green tea cate-chins. As green tea catechins increase the anticancer activity of various anticancer drugs, this review presents our new concept of green tea as a synergist with anticancer drugs (Fig. 3). Acknowledgments

This work was supported by the Japan Society for the Promotion of Sci-ence: Scientific Research on Priority Areas for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan; Comprehensive Research on Aging and Health; and Cancer Research from the Ministry of Health, Labor, and Welfare, Japan; Selec-tive Applied and Developed Research, and Green Tea Extracts Research Development for Cancer Prevention by the Department of Agriculture and Forests and the Department of Health and Human Services of Saitama Prefecture, Japan; and the Smoking Research Fund. We thank Dr. Takashi Sugimura, President Emeritus of the National Cancer Center, for his encouragement, and Professor Emeritus Takuo Okuda at

the Faculty of Pharmaceutical Sciences of Okayama University for his collaboration. We also thank Mr. Yoshiaki Kitaoka, Mr. Kenta Nakajima, and Mr. Atsushi Takahashi at the Department of Agriculture and Forests of Saitama Prefecture for their fruitful collaborations, and Drs. Kei Nakachi, Kazue Imai, and Sachiko Okabe-Kidokoro, and Mrs. Kaori Suzuki, Miki Kurusu, and Ikuko Shiotani, who worked with us at the Saitama Cancer Center Research Institute. The author (A. S.) is supported by the Japanese Government Monbukagakusho Scholarship Program for doctoral study from the Ministry of Education, Culture, Sports, Science and Technology, Japan and expresses his special thanks to the American Association for Cancer Research and ITO-EN, Ltd for the 2009 AACR-ITO EN, Ltd. Scholar-in-Training Award at the AACR 100th Annual Meeting, Denver, CO, USA.

Disclosure Statement

All authors have no conflict of interest.

Abbreviations

4-HPR N-(4-hydroxyphenyl)retinamide 5-FU 5-fluorouracil

APC adenomatous polyposis coli ATRA all-trans-retinoic acid EC ())-epicatechin ECG ())-epicatechin gallate EGC ())-epigallocatechin EGCG ())-epigallocatechin gallate EGFR epidermal growth factor receptor ER estrogen receptor

GADD153 growth arrest and DNA damage-inducible 153 Min multiple intestinal neoplasia

NF-jB nuclear factorjB NIK NF-jB inducing kinase

NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone RA retinoic acid

TNF-a tumor necrosis factor-a

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