Isorhamnetin Suppresses Skin Cancer Through Direct Inhibition of MEK1 and PI3-K

Published OnlineFirst February 17, 2011; DOI: 10.1158/1940-6207.CAPR-11-0032

Cancer Prevention Research Article Research

Isorhamnetin Suppresses Skin Cancer through Direct Inhibition of MEK1 and PI3-K

Jong-Eun Kim1,2, Dong-Eun Lee1,2, Ki Won Lee1,3, Joe Eun Son1, Sang Kwon Seo1, Jixia Li2, Sung Keun Jung1,2, Yong-Seok Heo4, Madhusoodanan Mottamal2, Ann M. Bode2, Zigang Dong2, and Hyong Joo Lee1

Abstract 30-Methoxy-3,40,5,7-tetrahydroxyflavone (isorhamnetin) is a plant flavonoid that occurs in fruits and medicinal herbs. Isorhamnetin exerts anticancer effects, but the underlying molecular mechanism for the chemopreventive potential of isorhamnetin remains unknown. Here, we report anti–skin cancer effects of isorhamnetin, which inhibited epidermal growth factor (EGF)-induced neoplastic cell transformation. It also suppressed anchorage-dependent and -independent growth of A431 human epithelial carcinoma cells. Isorhamnetin attenuated EGF-induced COX-2 expression in JB6 and A431 cells. In an in vivo mouse xenograft using A431 cells, isorhamnetin reduced tumor growth and COX-2 expression. The EGF-induced phosphorylation of extracellular signal-regulated kinases, p90 and p70 ribosomal S6 kinases, and Akt was suppressed by isorhamnetin. In vitro and ex vivo kinase assay data showed that isorhamnetin inhibited the kinase activity of MAP (mitogen-activated protein)/ERK (extracellular signal regulated kinase) kinase (MEK) 1 and PI3-K (phosphoinositide 3-kinase) and the inhibition was due to direct binding with isorhamnetin. Notably, isorhamnetin bound directly to MEK1 in an ATP-noncompetitive manner and to PI3-K in an ATP-competitive manner. This report is the first mechanistic study identifying a clear molecular target for the anticancer activity of isorhamnetin. Overall, these results indicate that isorhamnetin has potent anticancer activity and it primarily targets MEK and PI3-K, which might contribute to the chemopreventive potential of certain foods. Cancer Prev Res; 4(4); 582–91. 2011 AACR.

Introduction not BCC, can metastasize to other areas of the body (2). Because NMSC is readily detectable and has limited malig- Nonmelanoma skin cancer (NMSC) is the most com- nancy, the mortality of NMSC is low compared with other monly diagnosed cancer in the United States. About 1.3 cancers (3). However, because of its high incidence, NMSC million individuals suffer from this disease (1). The most is the fifth most costly cancer in the United States. Medicare common types of NMSC are basal cell carcinoma (BCC) spends $13 billion each year on skin cancer treatment (4). and squamous cell carcinoma (SCC), which develop from Changing environmental conditions, such as the destruc- basal cells and keratinocytes, respectively. BCC comprises tion of the ozone layer and environmental pollution, and 75% of all NMSC cases and SCC makes up 20%. SCC, but alterations in eating habits and increased longevity enhance the incidence of NMSC (5). A suitable chemopreventive agent could prevent and cure NMSC, which would

Authors' Affiliations: 1WCU Major in Biomodulation, Department of help people avoid the pain of skin cancer and reduce Agricultural Biotechnology, Research Institute for Agriculture and Life Medicare costs (6). Sciences and Center for Agricultural Biomaterials, Seoul National Uni- UV radiation is the most important cause of skin cancer versity, Seoul, Republic of Korea; 2The Hormel Institute, University of Minnesota, Minnesota; and Departments of 3Bioscience and Biotechnol- and chronic illnesses, and carcinogenic chemical exposures ogy and 4Chemistry, Konkuk University, Seoul, Republic of Korea are also major factors (1). These causes of skin cancer Note: Supplementary data for this article are available at Cancer Preven- activate multiple cellular signaling pathways and convert tion Research Online (http://cancerprevres.aacrjournals.org/). normal cells to cancerous cells. An important mediator of J-E. Kim, D-E. Lee, and K.W. Lee contributed equally to this work. these signaling pathways is the epidermal growth factor Corresponding Author: Zigang Dong, The Hormel Institute, University of (EGF) receptor (EGFR), which belongs to the ErbB family Minnesota, 801 16th Avenue NE, Austin, MN 55912. Phone: 507-437- of receptor tyrosine kinases (RTK). This receptor is dimer- 9600; Fax: 507-437-9606; E-mail: [email protected] or Hyong Joo Lee, Department of Agricultural Biotechnology, Seoul National University, ized by binding a ligand, such as EGF (7), which activates Seoul 151-742, Republic of Korea. Tel.: +82-2-880-4860; Fax: +82-2- the intercellular tyrosine kinase domain. The activated 873-5095; E-mail: [email protected] EGFR tyrosine kinase domain activates the mitogen-acti- doi: 10.1158/1940-6207.CAPR-11-0032 vated protein (MAP) kinases and phosphoinositide 3- 2011 American Association for Cancer Research. kinase (PI3-K; ref. 8). Previous studies have shown that

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Inhibition of Skin Cancer by Isorhamnetin

the EGFR is required for UV-induced NMSC development A431 cells (1106 cells in 50 mL of medium and 50 mLof (9). Chemical carcinogens, such as arsenite, also activate Matrigel). Cells were allowed to form tumors, and once the EGFR signal transduction. Both SCC and BCC highly over- tumors reached a size of 40 mm3, the mice were randomly express the EGFR (10). Thus, a compound that targets the assigned into groups (6 mice/group) and treated with (1 or EGFR signaling pathway might be useful in NMCS preven- 5 mg/kg body weight) or without isorhamnetin in 40% tion and treatment. DMSO/PBS buffer, administered intraperitoneally every COX-2 plays a crucial role in inflammation, tumorigen- other day for 28 days. Tumor size was measured every esis, and angiogenesis. It is overexpressed in many types of week with calipers, and the tumor volume was calculated cancer. The development of intestinal tumors and skin according to a standard formula: width length height/ papillomas were reportedly reduced in a COX-2 knockout 2. Mice were sacrificed after 28 days of treatment when the mouse model (11). The pharmacologic inhibition of COX- control tumors reached approximately 600 mm3. The 2 also impairs tumor formation in many animal models tumors were harvested, photographed, and weighed. (12), and COX-2 is apparently required for neoplastic Tumor tissues were used for Western blot analysis and transformation (13). Thus, the inhibition of COX-2 expres- immunohistochemical analysis. sion might also be a promising target for preventing carcinogenesis. Immunohistochemistry 30-Methoxy-3,40,5,7-tetrahydroxyflavone (i.e., isorham- For an immunohistochemical analysis of COX-2 protein netin) is an active compound in herbal medicinal plants, expression, excised tumors were fixed in 10% formalin for 1 such as Persicaria thunbergii H. and gross Hippophae rham- day, embedded in paraffin, and cut into 5-mm-thick sec- noides L., and it has been used to treat cardiovascular tions. Serial sections were mounted on Silane-coated slides, disease, rheumatism, and hemorrhage (14–16). Recent deparaffinized 3 with xylene, and dehydrated through a studies have shown that isorhamnetin exerts anticancer gradient alcohol series. The deparaffinized sections were effects. Isorhamnetin inhibits the proliferation of several boiled in 0.01 mol/L citrate buffer (pH 6.0) for 15 minutes cancer cell lines and suppresses the weight and size of for antigen retrieval. Sections were washed in PBST (PBS tumors of Lewis lung carcinoma cell allografts in mice with Tween 20) and placed in blocking buffer for 30 (15, 17, 18). However, the inhibitory effect of isorhamne- minutes, followed by incubation with a primary antibody tin on skin cancer and the underlying molecular mechan- against COX-2 (1:50 dilution) for 12 hours at 4C. Next, isms are not fully understood. In this study, we examined the endogenous peroxidase activity of paraffin-embedded the inhibitory effects of isorhamnetin against skin cancer sections was treated with 3% hydrogen peroxide for 10 and suggest that MAP/ERK (extracellular signal regulated minutes in blocking solution, consisting of PBS with 1% kinase) kinase (MEK) 1 and PI3-K as novel targets of bovine serum albumin (BSA). The slides were then incu- isorhamnetin. bated with anti-rabbit horseradish peroxidase–conjugated secondary antibody for 30 minutes. The immunoreactive Materials and Methods complexes were detected by staining with 3,30-diamino- benzidine tetrahydrochloride hydrate and counterstained Chemicals and reagents with Mayer’s hematoxylin. Isorhamnetin was purchased from Sigma Aldrich and its purity is more than 95%. EGF was purchased from R&D Luciferase assays for COX-2 promoter activity and Systems. Antibodies were from Cell Signal Biotechnology AP-1 transcription activity and Santa Cruz Biotechnology. The MEK1 and PI3-K assay The luciferase activity was determined as previously kits were obtained from Millipore Corporation. Other reported (19). chemicals were from Sigma-Aldrich. Kinase assays Western blot analysis Kinase assays were conducted as reported in our previous Western blot analyses were conducted as described in our studies (19, 20). previous studies (19). Direct and cell-based pull-down assays Anchorage-independent cell transformation assay Active MEK1 protein, PI3-K (200 ng), or a JB6 cellular This assay was conducted as reported in our previous supernatant fraction (500 mg) was incubated with isorham- studies (19). netin-Sepharose 4B or Sepharose 4B beads alone as a control (100 mL, 50% slurry) in reaction buffer Cell viability (50 mmol/L Tris, pH 7.5, 5 mmol/L EDTA, 150 mmol/L Cell viability was assessed as explained in our previous NaCl, 1 mmol/L DTT, 0.01% Nonidet P-40, 2 mg/mL BSA, studies (19). 0.02 mol/L phenylmethylsulfonylfluoride (PMSF), and 1 protease inhibitor mixture). After incubation with gentle Xenograft study rocking overnight at 4C, the beads were washed 5 with Female athymic nude mice were purchased from Orient. reaction buffer (50 mmol/L Tris, pH 7.5, 5 mmol/L EDTA, Each animal was injected subcutaneously in the flank with 150 mmol/L NaCl, 1 mmol/L DTT, 0.01% Nonidet P-40,

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and 0.02 mmol/L PMSF) and proteins bound to the beads sons. A probability value of P < 0.05 was used as the were analyzed by Western blotting. criterion for significance.

ATP and isorhamnetin competition assay Results Recombinant MEK1 or PI3-K (0.2 mg) was incubated with 100 mL of isorhamnetin-Sepharose 4B or 100 mLof Isorhamnetin strongly inhibits EGF-induced Sepharose 4B beads in reaction buffer (see the previous neoplastic transformation of JB6 cells and anchorage- text) for 12 hours at 4C, and ATP was added at either 10 or dependent and -independent cell growth of A431 cells 100 mmol/L to a final volume of 500 mL, followed by We first examined the effects of isorhamnetin on EGF- incubation for 30 minutes. The samples were washed induced neoplastic transformation in JB6 cells. On the and proteins were detected by Western blotting. basis of the numbers of cell colonies, EGF-promoted neoplastic transformation was markedly inhibited in JB6 cells Molecular modeling treated with 10 mmol/L isorhamnetin (Fig. 1A and B). Insight II (Accelrys Inc.) was utilized for the docking Isorhamnetin concentrations between 10 and 40 mmol/L study and structural analysis by using the crystal coordi- had no effect on cell viability after 3 days (Supplementary nates for MEK1 (PDB code 1S9J) and PI3-K complexed with Fig. S1), suggesting that isorhamnetin inhibits EGF- ATP or quercetin (1E8X or 1E8W), available from the induced neoplastic transformation without affecting viabi- Protein Data Bank (http://www.rcsb.org/pdb/). lity of JB6 cells. Because A431 cells overexpress EGFR and can grow in athymic nude mice, this is an excellent model Statistical analysis for studying EGF-mediated skin cancer (21). We measured When necessary, data are expressed as means SD and a anchorage-dependent and -independent cell growth of one-way ANOVA was used for single statistical compari- A431 cells by MTS assay and a soft agar assay, respectively.

Figure 1. Effects of isorhamnetin (IR) on neoplastic transformation of JB6 cells and growth of A431 cells. A and B, isorhamnetin inhibits EGF-induced neoplastic transformation of JB6 cells (A). The effect of isorhamnetin on EGF-induced cell transformation compared with untreated control cells (a); and cells treated with EGF alone (b); EGF and 10 mmol/L isorhamnetin (c); EGF and 20 mmol/L isorhamnetin (d); or EGF and 40 mmol/L isorhamnetin (d). The colonies were counted under a microscope (B). C and D, isorhamnetin inhibits anchorage- dependent (C) or -independent (D) cell proliferation. (C) A431 cells were treated with isorhamnetin at the concentrations indicated (0, 10, 20, 40 mmol/L) for 24, 48, or 72 hours. Cell viability was measured as described in Materials and Methods. D, the effect of isorhamnetin on anchorage-independent growth compared with untreated control cells. The colonies were counted under a microscope. For B, asterisks indicate a significant inhibition by isorhamnetin compared to the group treated with EGF alone (*, P < 0.05; **, P < 0.01). For D, asterisks indicate a significant inhibition by isorhamnetin compared to untreated control (*, P < 0.05; **, P < 0.01).

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Isorhamnetin suppressed both anchorage-dependent and expression in A431 cells and found that after 12 hours -independent growth of A431 cells (Fig. 1C and D). of treatment with 40 mmol/L isorhamnetin, COX-2 expression had completely disappeared (Fig. 2C). Isorhamnetin Isorhamnetin suppresses COX-2 protein expression in at 10 mmol/L also suppressed COX-2 expression completely JB6 and A431 cells in A431 cells (Fig. 2D). Because COX-2 is one of the most important inflammatory mediators of skin cancer (22), we examined the effects Isorhamnetin suppresses growth and COX-2 of isorhamnetin on EGF-induced COX-2 expression in JB6 expression in A431 xenograft tumors in nude mice cells. COX-2 expression was inhibited dose-dependently by Because isorhamnetin was observed to be effective in isorhamnetin treatment (Fig. 2A). To determine whether suppressing the proliferation of A431 cells, we studied the the inhibitory effects of isorhamnetin on COX-2 expression effects of isorhamnetin in an in vivo xenograft model to are mediated by transcriptional regulation, we investigated further confirm the antitumorigenic activity of isorhamne- the effects of isorhamnetin on EGF-induced cox-2 promoter tin. No significant body weight loss or appearance change activity. The luciferase assay revealed that exposure to EGF was observed in mice treated with isorhamnetin compared (10 ng/mL) significantly induced cox-2 promoter activity, with controls, indicating that the doses used were not toxic and it was suppressed by isorhamnetin treatment in a dose- to the animals (Supplementary Fig. S3). Photographic data dependent manner (Fig. 2B). JB6 cells stably transfected showed that isorhamnetin treatment suppressed tumor with an AP-1 luciferase reporter plasmid were used to development in mice (Fig. 3A). The average volume of measure AP-1 transactivation, which is a well-known tran- tumors in untreated mice increased over time and reached a scription factor regulating COX-2 expression. Isorhamnetin volume of 623 mm3 at 4 weeks postinoculation; however, inhibited EGF-induced AP-1 transactivation (Supplemen- at this time, in mice treated with 1 or 5 mg/kg isorhamne- tary Fig. S2) in a dose-dependent manner. Next, we exam- tin, the average tumor volume was only 280 or 198 mm3, ined the effect of isorhamnetin on COX-2 protein respectively (Fig. 3B). At the end of the study, we removed

Figure 2. Effects of isorhamnetin (IR) on COX-2 expression. A, EGF-induced COX-2 expression in JB6 cells is inhibited by isorhamnetin. Data are representative of 3 independent experiments. COX-2 and b-actin bands were quantified by densitometric analysis, using the ImageJ software program (NIH). B, isorhamnetin suppresses EGF-induced COX-2 promoter activity. Relative activities were determined using a luciferase assay as described in Materials and Methods. C and D, isorhamnetin inhibits COX-2 expression in A431 cells, which were treated with isorhamnetin (40 mmol/L) for 4, 8, 12, 18, or 24 hours (C). A431 cells were also treated with isorhamnetin at the concentrations indicated (0, 10, 20, 40 mmol/L) for 24 hours (D). Western blot analysis was conducted as described in Materials and Methods. b-Actin was used to verify equal protein loading. For A and B, asterisks indicate a significant inhibition by isorhamnetin compared with the group treated with EGF alone (*, P < 0.05; **, P < 0.01).

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Figure 3. Effects of isorhamnetin (IR) on tumor growth and COX-2 expression in the A431 xenograft model. A, photographs of tumors excised from each group. B, the average tumor volume of control and isorhamnetin-treated mice plotted over 28 days after tumor cell inoculation. The P values indicate statistical significance of the inhibition of tumor growth by isorhamnetin (**, P < 0.01). C, at the end of the study, the tumors excised from each group were weighed (n ¼ 6). The asterisks indicate a significant decrease in tumor weight between the control group and the group treated with isorhamnetin (*, P < 0.05; **, P < 0.01). D, effects of isorhamnetin on COX-2 expression in xenograft tumors in athymic nude mice. The immunostaining data are representative of 3 tissue samples from each group and COX-2 appears brown. Arrows indicate the regions exhibiting immunoreactivity for COX-2 proteins.

and weighed the tumors for each group. Clearly, isorham- rhamnetin on the ERKs and PI3-K signaling pathways, netin treatment (1 or 5 mg/kg) reduced tumor weight which play key roles in EGFR-mediated cell growth and compared with the untreated control group (Fig. 3C). COX-2 expression (19, 23). Exposure of JB6 cells to EGF (10 Collectively, these results suggest that isorhamnetin might ng/mL) markedly induced the phosphorylation of ERKs and serve as an effective anticancer treatment with the potential Akt. Isorhamnetin suppressed ERK/p90RSK but not MEK1/2 to suppress or delay the tumorigenicity of A431 cells in an (Fig. 4A) and also inhibited the phosphorylation of Akt/ in vivo system. To further confirm the inhibitory effect of p70S6K (Fig. 4B). Next, we investigated the change in phos- isorhamnetin on COX-2 protein expression in an in vivo phorylation status in A431 cells by isorhamnetin treatment model, we examined the level of COX-2 expression in in a time- and concentration-dependent manner. As for JB6 xenograft tumors of athymic nude mice by immunohisto- cells, isorhamnetin suppressed the phosphorylation of chemical analysis. Consistent with the results using A431 ERKs and p90RSK but not MEK and also inhibited AKT/ cells, the levels of COX-2 expression in the isorhamnetin- p70S6K (Fig. 4C and D, Supplemental Fig. S4). treated groups were lower than those in the control group (Fig. 3D). Isorhamnetin binds to and directly inhibits MEK1 and PI3-K activities Isorhamnetin inhibits the phosphorylation of ERKs To examine the mechanism of isorhamnetin inhibition and Akt in JB6 and A431 cells of the EGF-induced signaling pathway, we searched for the To examine the mechanism of the inhibitory effects of molecular target of isorhamnetin. Because isorhamnetin isorhamnetin on EGF-induced cell transformation and suppressed EGF-induced phosphorylation of ERKs, but COX-2 protein expression, we examined the effects of iso- increased MEK1/2 phosphorylation, we measured the

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Figure 4. Effect of isorhamnetin (IR) on ERKs and Akt signaling pathways. A and B, JB6 cells were treated with isorhamnetin at the concentrations indicated (0, 10, 20, 40 mmol/L) for 1 hour before being treated with EGF (10 ng/mL) and harvested after 15 minutes. B and D, isorhamnetin inhibits phosphorylation of ERKs and Akt in A431 cells, which were treated with isorhamnetin at the concentrations indicated (0, 10, 20, 40 mmol/L) for 24 hours (C). A431 cells were also treated with isorhamnetin (40 mmol/L) for 4, 8, 12, 18, or 24 hours (D). Western blot analysis was conducted as described in Materials and Methods.

effect of isorhamnetin on MEK1 kinase activity. Data from experimental observation that isorhamnetin binds to our in vitro kinase assays indicated that isorhamnetin MEK1 noncompetitively with ATP, we docked this com- strongly inhibited MEK1 kinase activity [Fig. 5A (a)] and pound into the pocket separate from but adjacent to the also suppressed EGF-induced MEK1 activity [Fig. 5A (b)]. ATP-binding site, similar to PD318088 in the crystal struc- Because isorhamnetin attenuated the EGF-induced Akt/ ture of the MEK1–PD318088 complex (Fig. 6C; ref. 24). p70S6K pathway, and PI3-K is an upstream kinase of The putative binding mode of isorhamnetin was also Akt, we investigated whether PI3-K was another molecular similar to that of PD318088. The hydroxyl groups at the target of isorhamnetin. We found that isorhamnetin inhib- 7-position can form a hydrogen bond with the backbone ited PI3-K in vitro [Fig. 5B (a)] and ex vivo [Fig. 5B (b)]. carbonyl group of Val127 in the ATP-noncompetitive bind- Next, we investigated whether isorhamnetin interacted ing site. In addition, isorhamnetin can form hydrophobic directly with MEK1 or PI3-K. The direct binding of iso- interactions with the side chains of Ile99, Ile141, Phe209, rhamnetin to MEK1 or PI3-K was shown by an in vitro pull- and Leu118. The methoxy group at the 30-position and the down assay [Fig. 5C (a) and D (a)]. In addition, we hydroxyl group at the 40-position can form hydrogen bonds observed ex vivo binding between isorhamnetin and with the backbone atoms of Ser212 in the activation loop MEK1 or PI3-K in JB6 cell lysates [Fig. 5C (b) and D (b)]. of the nonphosphorylated MEK1 and the benzene ring moiety can form van der Waals interactions with Val211 Isorhamnetin binds to MEK1 in an ATP- and Leu215 in the activation loop. These interactions noncompetitive manner and to PI3-K in an ATP- between isorhamnetin and the activation loop would ren- competitive manner der MEK1 catalytically inactive by stabilizing the inactive We determined whether the direct binding between conformation of the activation loop. isorhamnetin and MEK1 or PI3-K occurred in an ATP- To further investigate the binding mode of isorhamnetin competitive manner. Results indicated that the amount to PI3-K, we also conducted a modeling study by using the of MEK1 bound to isorhamnetin-Sepharose beads crystal structure of PI3-K in complex with ATP or quercetin remained constant with increasing concentrations of ATP (25, 26). PI3-K consists of 4 domains: a Ras-binding (Fig. 6A) whereas the amount of PI3-K bound to isorham- domain, C2 domain, helical domain, and a catalytic netin-Sepharose beads decreased with increasing concen- domain. The catalytic domain of the enzyme consists of trations of ATP (Fig. 6B). Thus, isorhamnetin bound to a C-lobe and N-lobe, with a fold similar to that of most MEK1 in an ATP-noncompetitive manner and to PI3-K in protein kinases, and this structural similarity is also con- an ATP-competitive manner. Thus, to predict the binding served in the ATP-binding site that is flanked by these 2 mode between isorhamnetin and MEK1 or PI3-K, we con- lobes. Consequently, ATP binds between these lobes in a ducted a molecular modeling study. Considering the manner similar to ATP binding in protein kinases. The N-

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Figure 5. Effects of isorhamnetin (IR) on MEK1 and PI3-K activity and direct binding with MEK1 and PI3-K. A and B, MEK1 activity assays (A) and PI3-K activity assays (B) in vitro (a) and ex vivo (b) were conducted as described in Materials and Methods. C and D, isorhamnetin binds directly to the MEK1 (C) or PI3-K (D) protein in vitro (a) and ex vivo (b). MEK1- or PI3-K–isorhamnetin binding was confirmed by immunoblotting using an antibody against MEK1/2 (C) or p110 for PI3-K (D): lane 1 (input control), MEK1 (C, a), PI3-K (D, a) protein standard, or JB6 cell lysate (C, b and D, b); lane 2 (control), Sepharose 4B was used for a pull-down assay as described in Materials and Methods; lanes 3; MEK1 (C, a), PI3-K (D, a) protein standard, or JB6 cell lysate (C, b and D, b) was pulled down using isorhamnetin- Sepharose 4B beads as described in Materials and Methods.

lobe and C-lobe are linked through a loop, called the Discussion "hinge region." The backbone of this loop interacts with the adenine moiety of ATP through hydrogen bonding. EGFR is a RTK that mediates multiple signaling pathways Considering the experimental result showing that isorham- including the MEK/ERKs and PI3-K pathways that regulate netin is an ATP-competitive inhibitor of PI3-K, we docked cell proliferation (27). It is an important drug target in the compound to the ATP-binding site of PI3-K (Fig. 6D). cancer and acts as a strong prognostic factor (8). This The carbonyl group at the 4-position and the hydroxyl receptor is expressed abundantly both in the basal layer groups at the 5-position of isorhamnetin can form hydro- of the epidermis and in the outer sheath of hair follicles and gen bonds with the backbone atoms of Val887 in the hinge is highly overexpressed in NMSC (28). It mediates the region of PI3-K. The hydroxyl groups at the 7- and 40- signaling pathway induced by major etiologic factors of positions can also form hydrogen bonds with the side skin cancer including UV light and heavy metal exposure. chains of Lys890 and Asp841, respectively. In addition, Activated EGFR promotes AP-1 transactivation by mediat- isorhamnetin would be sandwiched among the side chains ing signaling pathways such as PI3-K and MEK/ERKs (7). of the hydrophobic residues in the ATP-binding site, These pathways induce many inflammatory genes, such as including Met804, Trp812, Ile831, Ile879, and Ile881 from cox-2, which play pivotal roles in skin carcinogenesis (29). the N-lobe and Ala885, Phe961, Met953, and Ile963 from Here, we studied the inhibitory effects of a natural com- the C-lobe. The strong inhibitory activity of isorhamnetin pound, isorhamnetin, on EGFR-mediated skin cancer. for PI3-K may be attributable to this hydrogen bonding and To verify the inhibitory effects of isorhamnetin hydrophobic interactions. in EGFR-mediated skin cancer, we used 2 types of

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Figure 6. Isorhamnetin (IR) binds to MEK1 in an ATP-noncompetitive manner and to PI3-K in an ATP-competitive manner. Active MEK1 (A) or PI3-K (B): lane 1 (input control), MEK1 (A) and PI3-K (B) protein standard; lane 2 is the negative control, indicating that neither MEK1 (A) nor PI3-K (B) binds to Sepharose 4B; and lane 3 is the positive control, showing that MEK1 (A) or PI3-K (B) binds with isorhamnetin-Sepharose 4B. C and D, hypothetical model of MEK1 and PI3-K in complex with isorhamnetin. C, hypothetical model of the MEK1–isorhamnetin complex and its close-up view; isorhamnetin (atomic color) binds to the pocket adjacent to the ATP (orange)-binding site. PD318088 (white) is overlaid on the model structure of the MEK1–isorhamnetin complex for comparison. The partially disordered activation loop is colored yellow. The residues in gray ellipses are involved in hydrophobic interactions with isorhamnetin. The hydrogen bonds are depicted as white lines. D, hypothetical model of PI3-K in complex with isorhamnetin and its close-up view. The Ras-binding domain, C2 domain, and helical domain of PI3-K are colored in gray. The N-lobe, C-lobe, and hinge region of the catalytic domain are colored in yellow, purple, and cyan, respectively. Isorhamnetin (atomic color) binds to the ATP-binding site in the catalytic domain of PI3-K. The residues shown in gray ellipses represent the hydrophobic residues interacting with isorhamnetin. The hydrogen bonds are depicted as white lines. experimental models, including an EGF-induced JB6 cell such as EGF. This process represents the preneoplastic to transformation assay and a mouse xenograft model of neoplastic progression. In contrast, the A431 cell line was A431 cells. JB6 mouse epidermal cells are a well-devel- derivedfroman85-year-oldwomanwithskinepidermal oped cell culture system for studying tumor promotion ex carcinoma. Because this cell line highly overexpresses vivo. This model comprises 2 phenotypes, promotion- EGFR, forms colonies when cultivated in soft agar, sensitive (Pþ) and promotion-resistant (P)JB6mouse and develops tumors in nude mice, it is an excellent epidermal cell lines (30, 31). JB6 Pþ cells, but not P model for studying EGFR-mediated cellular signaling cells, are irreversibly transformed by a tumor promoter (32–34).

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Isorhamnetin is a dietary flavonoid, found in apples, liferation. Several studies have shown that PI3-K/Akt blackberries, and pears (35). It is also a major plasma pathway deregulation is associated with various types of metabolite of quercetin, which is a highly abundant cancer through subunit mutations or the regulation of flavonoid found in many dietary plants (36). Recent apoptosis (26). PI3-K is also involved in neoplastic cell studies showed that isorhamnetin exerts chemopreventive transformation and COX-2 expression (48). Thus, MEK1 effects against cancer, cardiovascular disease, obesity, and and PI3-K are attractive targets for pharmacologic inter- Alzheimer’s disease (15, 17, 18). These studies have attrib- ventions in proliferative and inflammatory diseases. uted the major chemopreventive mechanism of isorham- In this study, isorhamnetin inhibited the phosphoryla- netin to its antioxidant effects. Indeed, many reports have tion of ERKs, p90RSK, Akt, and p70S6K. We suggest that the suggested that the health benefits of other flavonoids and direct molecular targets of isorhamnetin are MEK1 and PI3- isorhamnetin come from antioxidant effects because they K. Isorhamnetin suppresses MEK1 kinase activity through are strong antioxidants (37). Nonetheless, the proposed direct binding, and because ERKs are substrates of MEK1, antioxidant effects cannot explain all of the effects of most the inhibition of MEK1 by isorhamnetin leads to the flavonoids, such as the low effective dose and specific inhibition of EGF-induced phosphorylation of ERKs. Sub- inhibition of oncogenic signal transduction pathways. sequently, EGF-induced phosphorylation of p90RSK, which Our findings and other previous studies suggest that fla- is the substrate of ERKs, is also inhibited by isorhamnetin. vonoids can also act as specific kinase inhibitors (38). The inhibition of PI3-K activity by isorhamnetin might A strong link has been reported between inflammation explain the manner in which isorhamnetin inhibits the and carcinogenesis (39). COX-2, a major mediator of phosphorylation of Akt and p70S6K. inflammation, and its product, prostaglandin E2, enhance In summary, isorhamnetin inhibits EGF-induced neo- carcinogenesis (22), increasing both the number of cells plastic transformation and COX-2 protein expression in during the exponential growth phase and the number of JB6 cells and also reduces tumor growth and COX-2 expres- colonies formed in soft agar (40). Knockout of cox-2 results sion in the A431 mouse xenograft model. This inhibition is in reduced tumor formation and progression, and epide- mediated primarily through the blocking of the MEK/ERK/ miologic studies and animal experiments have shown that p90RSK and PI3-K/Akt/p70S6K signaling pathways and the nonsteroidal anti-inflammatory drugs targeting COX-2 can subsequent suppression of AP-1 activity. Isorhamnetin reduce the incidence of colorectal carcinoma (41). Thus, strongly suppresses MEK1 and PI3-K activities. Overall, COX-2 has been recognized as a molecular target of many these results suggest that MEK1 and PI3-K are potent chemopreventive and anti-inflammatory agents. In the molecular targets for the suppression of neoplastic trans- present study, isorhamnetin inhibited EGF-induced formation by isorhamnetin. COX-2 protein expression both in JB6 cells and in A431 cells. These results indicate that COX-2 is the target of Disclosure of Potential Conflicts of Interest isorhamnetin and that isorhamnetin has chemopreventive potential. No potential conflicts of interest were disclosed. MEK1 is a dual-specificity protein kinase that phosphor- ylates ERK1/2 at specific tyrosine and threonine residues. Grant Support MEK is activated by the phosphorylation of key serine residues in its catalytic domain by an upstream serine This work was supported in part by The Hormel Foundation; NIH grants kinase, Raf. Many tumors express mutated Raf or MEK CA027502, CA120388, CA111536, CA077646, and CA081064; grants from World Class University Program (R31-2008-00-10056-0), World Class (42). A small-molecule inhibitor of MEK1 is capable of Institute Program (WCM 0100911), and National Leap Research Program inhibiting up to 80% of the growth of human and murine (No. 2010-0029233) through the National Research Foundation of Korea colon carcinomas in mice (43). The inhibition of MEK1 funded by the Ministry of Education, Science and Technology, Republic of Korea. attenuates neoplastic cell transformation (44). The expres- The costs of publication of this article were defrayed in part by the sion of dominant-negative ERK2 shows a similar result (45, payment of page charges. This article must therefore be hereby marked RSK advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate 46). p90 is also required for neoplastic cell transforma- this fact. tion (47). PI3-K, a family of heterodimeric kinases, plays a pivotal role in the control of multiple cell signaling path- Received August 2, 2010; revised January 17, 2011; accepted January 21, ways leading to cell survival, motility, apoptosis, and pro- 2011; published OnlineFirst February 17, 2011.

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www.aacrjournals.org Cancer Prev Res; 4(4) April 2011 591

Isorhamnetin Suppresses Skin Cancer through Direct Inhibition of MEK1 and PI3-K

Jong-Eun Kim, Dong-Eun Lee, Ki Won Lee, et al.

Cancer Prev Res 2011;4:582-591. Published OnlineFirst February 17, 2011.

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