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* This work was supported by the Hormel Foundation and Grants CA81064, CA74916, and CA27502 from the National Cancer Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
In this study, we investigated the effect of tea polyphenols, (−)-epigallocatechin-3-gallate or theaflavins, on UVB-induced phosphatidylinositol 3-kinase (PI3K) activation in mouse epidermal JB6 Cl 41 cells. Pretreatment of cells with these polyphenols inhibited UVB-induced PI3K activation. Furthermore, UVB-induced activation of Akt and ribosomal p70 S6 kinase (p70 S6-K), PI3K downstream effectors, were also attenuated by the polyphenols. In addition to LY294002, a PI3K inhibitor, pretreatment with a specific mitogen-activated protein/extracellular signal-regulated protein kinases (Erks) kinase 1 inhibitor, U0126, or a specific p38 kinase inhibitor, SB202190, blocked UVB-induced activation of both Akt and p70 S6-K. Pretreatment with LY294002 restrained UVB-induced phosphorylation of Erks, suggesting that in UVB signaling, the Erk pathway is mediated by PI3K. Moreover, pretreatment with rapamycin, an inhibitor of p70 S6-K, inhibited UVB-induced activation of p70 S6-K, but UVB-induced activation of Akt did not change. Interestingly, UVB-induced p70 S6-K activation was directly blocked by the addition of (−)-epigallocatechin-3-gallate or theaflavins, whereas these polyphenols showed only a weak inhibition on UVB-induced Akt activation. Because PI3K is an important factor in carcinogenesis, the inhibitory effect of these polyphenols on activation of PI3K and its downstream effects may further explain the anti-tumor promotion action of these tea constituents.
atypical protein kinase C
epidermal growth factor
extracellular signal-regulated protein kinases
fetal bovine serum
c-Jun N-terminal kinases
minimal essential medium
mammalian target of rapamycin
ribosomal p70 S6 protein kinase
phosphatidylinositol 3,4,5-triphosphate-dependent protein kinase
4,5-P3, phosphatidylinositol 3,4,5-triphosphate
protein kinase B
protein kinase C
Nonmelanoma skin cancer is the most frequently diagnosed malignancy in the United States, and the most crucial risk factor in development of nonmelanoma skin cancer is UV radiation from sunlight (
). Although the UV portion of sunlight is divided into three components based on wavelength, the UV light that reaches the surface of the earth is comprised of UVA (320–400 nm) and UVB (280–320 nm). UVB is a major cause of skin cancer (
). The mitogen-activated protein (MAP)1 kinase family (e.g. extracellular signal-regulated protein kinases (Erks), c-Jun N-terminal kinases (JNKs), and p38 kinase) at least partially mediates the activation of transcription factors (
). In addition, UV radiation triggers activation of tyrosine receptors, such as the epidermal growth factor (EGF) receptor and the insulin receptor, Src family tyrosine kinases, and the small GTP-binding protein, Ras, and MAP kinases (
). PI3K plays a central role in a broad range of biological effects such as cell growth, apoptosis, intercellular vesicle trafficking/secretion, regulation of actin, cell migration, and integrin function (
). Those findings suggested that PI3K activation by UV exposure is a critical factor in UV-induced carcinogenesis. The serine/threonine proto-oncogene Akt (also known as protein kinase B or PKB) was first identified as the cellular homolog of the transforming oncogene product v-Akt (
). Activation of p70 S6-K regulates a variety of cellular functions involved in the mitogenic response including protein synthesis, translation of specific mRNA species, cell cycle regulation, and cell motility (
). The polyphenols from green tea and black tea, (−)-epigallocatechin-3-gallate (EGCG) and theaflavins, respectively, are generally considered to be the most effective components for inhibition of carcinogenesis (
). Thus, the inhibitory effect of the tea polyphenols in UVB-induced carcinogenesis may result from the blocking of these signal transduction pathways. In the present study, we investigated the effects of tea polyphenols on UVB-induced activation of the PI3K and its downstream effectors, which are critical factors in carcinogenesis.
Eagle's minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); l-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma. Dominant negative mutants of JNK1 were generous gifts from Dr. Roger J. Davis (
). EGCG (purity > 98%) was a gift from Dr. Yukihiro Hara of Mitsui Norin Co. (Fujieda, Japan). Theaflavins (a mixture of theaflavin, theaflavin-3-gallate, theaflavin-3′-gallate, theaflavin-3,3′-digallate, and unknowns, accounting for 21, 30, 15, 28, and 6%, respectively) were gifts from the Thomas J. Lipton Co. (Englewood Cliffs, NJ).
UVB irradiation was performed on serum-starved monolayer cultures utilizing a transluminator emitting UVB (
). The source of UVB is a bank of four Westinghouse F520 Lamps (National Biological, Twinsburg, OH) at 6 J/S/m light in the UVB range. Approximately 10% of the remaining radiation from the F520 Lamp was in the UVA region (320 nm). Although almost no UVC leakage occurs, the UVB irradiation was carried out in a UVB exposure chamber fitted with a Kodak Kodacel K6808 filter that eliminates all wavelengths below 290 nm. This lamp is one of the most frequently used UVB sources for the study of carcinogenesis. The International Agency for Research on Cancer refers to this lamp as a source emitting mainly UVB irradiation for the studies of cancer induction in animals. UVB irradiation was measured using the UVX radiometer from UVP (UVX-31).
The JB6 mouse epidermal cell line Cl 41 and its stable transfectants, Cl 41 CMV-neo and the dominant negative mutant of JNK1 were grown at 37 °C in MEM supplemented with 5% heat-inactivated FBS, 2 mm l-glutamine, and 25 μg/ml gentamicin.
Generation of Stable Cotransfectants
The dominant negative mutant of JNK1 subcloned into a mammalian expression vector plasmid, CMV-neo, was transfected into JB6 Cl41 cells by using LipofectAMINE (Life Technologies Inc.) following the manufacturer's instructions. The stable transfectants were obtained by selection for G418 resistance (300 μg/ml) and further confirmed by activity assays.
Immunoblotting was carried out as described previously (
). In brief, JB6 Cl 41 cells and its stable transfectants were cultured to 80% confluence. The cells were starved in 0.1% FBS MEM for 48 h at 37 °C. Then the media were changed to fresh 0.1% FBS MEM, and the cells were incubated for another 2–4 h at 37 °C. Before the cells were irradiated with UVB, they were incubated with or without LY294002, PD98059, SB202190, rapamycin, EGCG, or theaflavins for 1 h. Then the cells were exposed to UVB (4 kJ/m2) and subsequently incubated for an additional 30 min at 37 °C in the presence of inhibitors or tea polyphenols. The cells were then lysed, and immunoblot analysis was performed by using antibodies against total Akt, p70 S6-K, Erks, JNKs, or p38 kinase proteins or the phospho-specific antibodies against their phosphorylated proteins. Antibody-bound proteins were detected by chemiluminescence (ECF Western blotting kit; Amersham Pharmacia Biotech) and analyzed using the Storm 840 Scanner (Molecular Dynamics, Sunnyvale, CA).
PI3K activity was assayed as described previously (
). In brief, the cells were treated with UVB (4 kJ/m2) as described above. The cells were lysed in 400 μl of lysis buffer (20 mm Tris-HCl, pH 7.4, 137 mmNaCl, 1 mm MgCl2, 10% glycerol, 1% Nonidet P-40, 1 mm DTT, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 μmaprotinin, 10 μm leupeptin). The lysate was sonicated and centrifuged, and the supernatant fraction was incubated with 20 μl of agarose conjugated with a monoclonal antiphosphotyrosine antibody (PY20) with gentle rocking overnight at 4 °C. The agarose beads were washed twice with each of the following buffers: 1) PBS with 1% Nonidet P-40, 1 mm DTT, 0.1 mm sodium orthovanadate; 2) 100 mm Tris-HCl, pH 7.6, 0.5m LiCl, 1 mm DTT, 0.1 mm sodium orthovanadate; and 3) 10 mm Tris-HCl, pH 7.6, 0.1m NaCl, 1 mm DTT, 0.1 mm sodium orthovanadate. The beads were incubated for 5 min on ice in 20 μl of buffer 3, and then 20 μl of 0.5 mg/ml phosphatidylinositol (previously sonicated in 50 mm HEPES, pH 7.6, 1 mm EGTA, 1 mm NaH2PO4) was added. After 5 min at room temperature, 10 μl of the reaction buffer were added (50 mm MgCl2, 100 mm HEPES, pH 7.6, 250 μm ATP containing 10 μCi of [γ-32P]ATP), and the beads were incubated for an additional 15 min. The reaction was stopped by the addition of 15 μl of 4 n HCl and 130 μl of chloroform:methanol (1:1). After vortexing for 30 s, 30 μl of the chloroform phase was spotted onto 1% potassium oxalate-coated silica gel H plates (previously baked at 110 °C for 1 h). The plates were developed in choloroform/methanol/NH4OH/H2O (60:47:2:11.3) and dried at room temperature. Radiolabeled spots were quantified using the Storm 840 scanner (Molecular Dynamics).
Assay for JNK Activity
The cells were treated with UVB (4 kJ/m2) as described above. The cells were lysed in 400 μl of lysis buffer (20 mm Tris, pH 7.4, 150 mmNaCl, 1 mm Na2EDTA, 1 mm EGTA, 1 mm Na3VO4, 1 mm β-glycerophosphate, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, and 1 μm microcystin). The lysates were sonicated and centrifuged, and the supernatant fractions were incubated with a phospho-specific JNKs antibody with gentle rocking overnight at 4 °C. Then, protein A/G plus agarose was added, and the incubation continued for another 4 h at 4 °C. The beads were washed twice with 500 μl of lysis buffer and twice with 500 μl of kinase buffer (25 mm Tris, pH 7.5, 5 mmβ-glycerolphosphate, 2 mm DTT, 0.1 mmNa3VO4, 10 mm MgCl2). The kinase reactions were carried out in the presence of 200 μm ATP at 30 °C for 30 min using 2 μg of c-Jun as substrate for JNKs. The phosphorylated protein was detected by immunoblotting using a phospho-specific antibody.
Akt and p70 S6-K Immunoprecipitation Kinase Assay
The cells were treated with the inhibitors or tea polyphenols before irradiation with UVB (4 kJ/m2), lysates were prepared from the cells, and the immunoprecipitation was carried out using 4 μg of Akt1/PKBα PH domain antibody or 3 μg of anti-p70 S6-K antibody as described above. The enzyme-immune complex was washed three times with 0.5 ml of lysis buffer and once with 100 μl of assay dilution buffer (20 mm MOPS, pH 7.2, 25 mmβ-glycerophosphate, pH 7.0, 1 mm sodium orthovanadate, 1 mm DTT). For the Akt kinase assay, the enzyme immune complex was added to 10 μl of assay dilution buffer, 10 μm protein kinase A inhibitor peptide, 0.1 mmAkt substrate peptide, and 10 μCi of [γ-32P]ATP, and for the p70 S6-K assay, it was added to 20 μl of assay dilution buffer, 10 μl of inhibitor mixture, 50 μm S6 kinase substrate peptide, and 10 μCi of [γ-32P]ATP. The reaction was incubated for 10 min at 30 °C and centrifuged, and then 30 μl of the supernatant fraction was transferred onto p81 phosphocellulose paper and allowed to bind for 30 s. The p81 papers were washed three times in 0.75% phosphoric acid and then washed once in acetone, and γ-32P incorporation was measured by scintillation counting.
Direct Inhibition of Akt and p70 S6-K Activities by Tea Polyphenols
The cells were treated with UVB (4 kJ/m2) and lysed, and the immunoprecipitation was carried out using 4 μg of anti-Akt1/PKBα PH domain or 3 μg of anti-p70 S6-K antibody, and the enzyme-immune complex was washed with 0.5 ml of lysis buffer and with 100 μl of assay dilution buffer as described above. For the Akt kinase assay, the enzyme immune complex was added to 10 μl of different concentrations of tea polyphenols in assay dilution buffer, 10 μm protein kinase A inhibitor peptide, 0.1 mm Akt substrate peptide, and 10 μCi of [γ-32P]ATP, and for the p70 S6-K assay, it was added to 20 μl of different concentration of tea polyphenols in assay dilution buffer, 10 μl of inhibitor mixture, 50 μm S6 kinase substrate peptide, and 10 μCi of [γ-32P]ATP. The reaction and measurement were performed as described under “Akt and p70 S6-K Immunoprecipitation Kinase Assay.”
Significant differences in the kinase activities were determined by using both Student's t test and Welch's t test.
Inhibition of UVB-induced PI3K Activity by EGCG or Theaflavins
Exciting developments have recently implicated PI3K as an important factor in carcinogenesis (
). Therefore, PI3K was also suggested to play a critical role in carcinogenesis induced by UVB. We first investigated the effect of the tea polyphenols, EGCG or theaflavins, on UVB-induced PI3K activity in JB6 Cl 41 cells. The results showed that pretreatment of cells with these polyphenols inhibits UVB-induced PI3K activity (Fig.1). Although inhibition of UVB-induced PI3K activity by these polyphenols is suggested to be due to UVB absorption, our previous study showed that the attenuation of UVB-induced signaling by EGCG or theaflavins does not appear to result from UVB absorption (
). As shown in Fig. 2, pretreatment of cells with EGCG or theaflavins inhibited UVB-induced phosphorylation of Akt at both Thr308 and Ser473, which are prerequisites for the catalytic activation of Akt (
). UVB-induced phosphorylation of p70 S6-K at Thr389 and Thr421/Ser424 was also blocked by pretreatment with these polyphenols (Fig. 2). In addition, pretreatment with these polyphenols attenuated UVB-induced activation of both Akt and p70 S6-K (Fig. 3). Phosphoinositide-dependent kinase (PDK-1) is known to phosphorylate Akt at Thr308, and it is also a downstream target of PI3K (
). Therefore, the inhibition of UVB-induced activation of Akt and p70 S6-K by these polyphenols may also be implicated in blocking Erks or JNKs. To determine the mechanism by which EGCG or theaflavins inhibit UVB-induced activation of Akt and p70 S6-K, we investigated the signaling pathways involved in UVB-induced activation of kinases by using specific chemical inhibitors. Pretreatment of cells with a PI3K inhibitor, LY294002, blocked UVB-induced phosphorylation of Akt (at Thr308 and Ser473) and p70 S6-K (at Thr389 and Thr421/Ser424) (Fig.4). LY294002 also inhibited UVB-induced activation of these kinases (Fig. 5), showing that in UVB signaling the activation of both Akt and p70 S6-K is mediated through PI3K. In addition, the phosphorylation and activation of Akt and p70 S6-K were attenuated by pretreatment of cells with a specific MEK inhibitor, U0126, or a specific p38 kinase inhibitor, SB202190 (Figs. 4 and 5), whereas the inhibition of phosphorylation and activation of these kinases by UVB was not observed in cells expressing a dominant negative mutant of JNK1 (Fig.6) (
). Thus, Erks and p38 kinase, but not JNKs, mediate UVB-induced activation of Akt and p70 S6-K. Pretreatment with LY294002 restrained UVB-induced phosphorylation of Erks, suggesting that in UVB signaling the Erk pathway is mediated by PI3K. In agreement with this result, our previous study demonstrated that expression of a dominant negative mutant of the PI3K p85 subunit also inhibits UVB-induced phosphorylation of Erks, but not JNKs or p38 kinase (
). On the other hand, pretreatment with rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) an upstream effector of p70 S6-K, inhibited UVB-induced phosphorylation and activation of P70 S6-K, whereas UVB-induced phosphorylation and activation of Akt was not affected by rapamycin (Figs. 4 and 5).
Direct Inhibition of UVB-induced p70 S6-K Activation by EGCG or Theaflavins
To test whether EGCG and theaflavins directly inhibit kinase activity of Akt and p70 S6-K induced by UVB, we performed kinase activity assays for Akt and p70 S6-K in the presence of these polyphenols. The enzymes were immunoprecipitated from cell lysate treated with UVB (4 kJ/m2) using an antibody against p70 S6-K or Akt. UVB-induced p70 S6-K activation was significantly blocked by the addition of EGCG or theaflavins, whereas these polyphenols only weakly inhibited UVB-induced Akt activation (Fig.7). Thus, the inhibition of UVB-induced activation of p70 S6-K, but not Akt, by these polyphenols may be, in part, achieved by the direct blocking of kinase activity of p70 S6-K.
Green tea is widely used as a beverage in China, Japan, and other Asian countries, whereas black tea is more popular in Western countries (
). Thus, PI3K and its downstream effectors are suggested to be important regulatory proteins in the tumor promotion effects of UVB.
In this study, we found that EGCG and theaflavins inhibit UVB-induced activation of PI3K and its downstream effectors, Akt and p70 S6-K. Furthermore, we demonstrated that the Erks and p38 kinase pathways are critical for UVB-induced activation of Akt and p70 S6-K. Fig.8 shows a schematic of the proposed signaling model for Akt and p70 S6-K induced by UVB. Generally, Akt is believed to be regulated by a pathway mediated by PI3K and PDK-1 (
). In contrast, we recently demonstrated that UVB-induced Akt activation is mediated through Erks and p38 kinase-dependent mitogen- and stress-activated protein kinase-1 rather than the PI3K/PDK-1 pathway (
) and the present study also suggested that PI3K is located upstream of Erks, because pretreatment of cells with a PI3K inhibitor, LY294002, or the expression of a dominant negative mutant of the PI3K p85 subunit inhibited UVB-induced phosphorylation of Erks, but not JNKs or p38 kinase.
In addition, we previously showed that overexpression of a dominant negative mutant of PKCλ/τ (
), and the maximal phosphorylation and activation of aPKCs by PDK-1 require PI-3,4,5-P3. These findings suggested that in UVB signaling Erk activation is mediated by pathways involving PI3K/aPKCs or PI3K/PDK-1/aPKCs. On the other hand, PDK-1 is also suggested to serve as a Thr229 kinase of p70 S6-K because the sequence surrounding the activation loop including Thr229 of p70 S6-K is highly homologous to that of Akt, which is activated by PDK-1. Pullen et al. (
) reported that PDK-1 activates a p70 S6-K mutant with acidic residues at Thr389 and four phosphorylation sites in the C terminus and that the catalytically inactive PDK-1 blocked insulin-induced p70 S6-K activation. However, the activation of p70 S6-K by PDK-1 was found to be independent of PI-3,4,5-P3 or pretreatment with wortmannin or rapamycin (
) showed that expression of a dominant negative mutant of PKCλ antagonized p70 S6-K activation by EGF and PDK-1. They also demonstrated that p70 S6-K forms complexesin vivo with PDK-1 and PKCλ. In addition, the interaction of the N-terminal region of p70 S6-K with the kinase domain of PKCλ and the requirement for interaction with the regulatory domain of PKCλ of the C-terminal region of p70 S6-K have been reported by Akimoto et al. (
). Therefore, PDK-1 may be able to colocalize with aPKCs at the membrane. In this study, we showed that U0126, an inhibitor of MAP kinase/Erk kinase, repressed UVB-induced p70 S6-K phosphorylation at Thr389 and Thr421/Ser424. Thus, the activation of p70 S6-K by PI3K is suggested to be mediated through interaction with aPKCs or phosphorylation at Thr389 and Thr421/Ser424 by PDK-1/aPKCs/Erk pathways. Furthermore, although whether Akt phosphorylates mTOR is not clear (
). We also showed that rapamycin, an inhibitor of mTOR, not only represses phosphorylation of p70 S6-K at Thr389 but blocks phosphorylation at Thr421/Ser424. In addition, SB202190, a specific p38 kinase inhibitor, inhibited UVB-induced p70 S6-K phosphorylation at Thr389 and Thr421/Ser424 and p70 S6-K has also shown to be phosphorylate by MAP kinases (
). These findings suggested that p38 kinase and Erks activate p70 S6-K directly by themselves or indirectly through other kinases including mTOR mediated by the mitogen- and stress-activated protein kinase 1/Akt pathway.
Our previous study showed that EGCG or theaflavins inhibit UVB-induced Erks phosphorylation (
), and the present study demonstrated that these polyphenols attenuated UVB-induced PI3K activation. Although the inhibition of Erks phosphorylation may be due to suppressed PI3K activation, the blocking of the Erks-mediated pathway was suggested to be involved in the inhibitory effects of Akt and p70 S6-K by tea polyphenols. On the other hand, EGCG and theaflavins also directly inhibited the activation of p70 S6-K but not Akt activation. Polyphenols are known to bind to proteins, as exemplified by their binding to the proline-rich salivary proteins (
). These proteins have the general characteristics of a large size, a loose open structure, and a high proportion of hydrophobic amino acids and proline. Among the regulatory phosphorylation sites that are implicated in the activation of p70 S6-K, Ser404 is surrounded by large bulky hydrophobic amino acids in the −1 and +1 positions, similar to Thr229 and Thr389, and the remaining phosphorylation sites in C-terminal are followed by proline at the +1 position, similar to Ser371 (
). Therefore, EGCG and theaflavins may modulate the conformation of p70 S6-K by binding to the hydrophobic amino acids described above (Fig. 7B). In contrast, the binding of these polyphenols to Akt was suggested to be unnecessary or insufficient for inhibition of its activation (Fig.7B).
In summary, the present results show that EGCG and theaflavins inhibited the activation of PI3K and also attenuated the activation of Akt and p70 S6-K, downstream effectors of PI3K, by inhibiting PI3K and Erk activation in UVB signaling. Furthermore, these polyphenols directly blocked UVB-induced p70 S6-K activation. Because PI3K and its downstream effectors are considered to play a critical role in carcinogenesis, these results provide insight into the biological actions of tea polyphenols on UV-induced carcinogenesis and on the molecular basis for the development of new chemopreventive agents.
We thank Dr. Ann M. Bode for critical reading and Andria Hansen for secretarial assistance.