Jixia Li

Isorhamnetin suppresses skin cancer through direct inhibition of MEK1 and PI3-K

3′-Methoxy-3,4′,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.

Norathyriol Suppresses Skin Cancers Induced by Solar Ultraviolet Radiation by Targeting ERK Kinases

Ultraviolet (UV) irradiation is the leading factor in the development of skin cancer, prompting great interest in chemopreventive agents for this disease. In this study, we report the discovery of norathyriol, a plant-derived chemopreventive compound identified through an in silico virtual screening of the Chinese Medicine Library. Norathyriol is a metabolite of mangiferin found in mango, Hypericum elegans, and Tripterospermum lanceolatum and is known to have anticancer activity. Mechanistic investigations determined that norathyriol acted as an inhibitor of extracellular signal-regulated kinase (ERK)1/2 activity to attenuate UVB-induced phosphorylation in mitogen-activated protein kinases signaling cascades. We confirmed the direct and specific binding of norathyriol with ERK2 through a cocrystal structural analysis. The xanthone moiety in norathyriol acted as an adenine mimetic to anchor the compound by hydrogen bonds to the hinge region of the protein ATP-binding site on ERK2. Norathyriol inhibited in vitro cell growth in mouse skin epidermal JB6 P+ cells at the level of G(2)-M phase arrest. In mouse skin tumorigenesis assays, norathyriol significantly suppressed solar UV-induced skin carcinogenesis. Further analysis indicated that norathyriol mediates its chemopreventive activity by inhibiting the ERK-dependent activity of transcriptional factors AP-1 and NF-κB during UV-induced skin carcinogenesis. Taken together, our results identify norathyriol as a safe new chemopreventive agent that is highly effective against development of UV-induced skin cancer.

Sunlight UV-Induced Skin Cancer Relies upon Activation of the p38α Signaling Pathway

The activation of cellular signal transduction pathways by solar ultraviolet (SUV) irradiation plays a vital role in skin tumorigenesis. Although many pathways have been studied using pure ultraviolet A (UVA) or ultraviolet B (UVB) irradiation, the signaling pathways induced by SUV (i.e., sunlight) are not understood well enough to permit improvements for prevention, prognosis, and treatment. Here, we report parallel protein kinase array studies aimed at determining the dominant signaling pathway involved in SUV irradiation. Our results indicated that the p38-related signal transduction pathway was dramatically affected by SUV irradiation. SUV (60 kJ UVA/m(2)/3.6 kJ UVB/m(2)) irradiation stimulates phosphorylation of p38α (MAPK14) by 5.78-fold, MSK2 (RPS6KA4) by 6.38-fold, and HSP27 (HSPB1) by 34.56-fold compared with untreated controls. By investigating the tumorigenic role of SUV-induced signal transduction in wild-type and p38 dominant-negative (p38 DN) mice, we found that p38 blockade yielded fewer and smaller tumors. These results establish that p38 signaling is critical for SUV-induced skin carcinogenesis.

Quercetin-3-methyl ether suppresses proliferation of mouse epidermal JB6 P+ cells by targeting ERKs

Chemoprevention has been acknowledged as an important and practical strategy for the management of skin cancer. Quercetin-3-methyl ether, a naturally occurring compound present in various plants, has potent anticancer-promoting activity. We identified this compound by in silico virtual screening of the Traditional Chinese Medicine Database using extracellular signal-regulated kinase 2 (ERK2) as the target protein. Here, we showed that quercetin-3-methyl ether inhibited proliferation of mouse skin epidermal JB6 P+ cells in a dose- and time-dependent manner by inducing cell cycle G(2)-M phase accumulation. It also suppressed 12-O-tetradecanoylphorbol-13-acetate-induced neoplastic cell transformation in a dose-dependent manner. Its inhibitory effect was greater than quercetin. The activation of activator protein-1 was dose-dependently suppressed by quercetin-3-methyl ether treatment. Western blot and kinase assay data revealed that quercetin-3-methyl ether inhibited ERKs kinase activity and attenuated phosphorylation of ERKs. Pull-down assays revealed that quercetin-3-methyl ether directly binds with ERKs. Furthermore, a loss-of-function ERK2 mutation inhibited the effectiveness of the quercetin-3-methyl ether. Overall, these results indicated that quercetin-3-methyl ether exerts potent chemopreventive activity by targeting ERKs.

Quercetin-3-methyl ether inhibits lapatinib-sensitive and -resistant breast cancer cell growth by inducing G2/M arrest and apoptosis

Lapatinib, an oral, small‐molecule, reversible inhibitor of both EGFR and HER2, is highly active in HER2 positive breast cancer as a single agent and in combination with other therapeutics. However, resistance against lapatinib is an unresolved problem in clinical oncology. Recently, interest in the use of natural compounds to prevent or treat cancers has gained increasing interest because of presumed low toxicity. Quercetin‐3‐methyl ether, a naturally occurring compound present in various plants, has potent anticancer activity. Here, we found that quercetin‐3‐methyl ether caused a significant growth inhibition of lapatinib‐sensitive and ‐resistant breast cancer cells. Western blot data showed that quercetin‐3‐methyl ether had no effect on Akt or ERKs signaling in resistant cells. However, quercetin‐3‐methyl ether caused a pronounced G2/M block mainly through the Chk1‐Cdc25c‐cyclin B1/Cdk1 pathway in lapatinib‐sensitive and ‐resistant cells. In contrast, lapatinib produced an accumulation of cells in the G1 phase mediated through cyclin D1, but only in lapatinib‐sensitive cells. Moreover, quercetin‐3‐methyl ether induced significant apoptosis, accompanied with increased levels of cleaved caspase 3, caspase 7, and poly(ADP‐ribose) polymerase (PARP) in both cell lines. Overall, these results suggested that quercetin‐3‐methyl ether might be a novel and promising therapeutic agent in lapatinib‐sensitive or ‐resistant breast cancer patients.

Lapatinib, a preventive/therapeutic agent against mammary cancer, suppresses RTK-mediated signaling through multiple signaling pathways.

Activation of receptor tyrosine kinases (RTK) plays a key role in the prognosis of mammary cancer. Lapatinib is a small molecule dual RTK inhibitor that targets epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2). Identifying the protein targets involved in the effects of lapatinib and other RTK inhibitors might help determine why preventive efficacy varies. In this study, female Sprague-Dawley rats were given methylnitrosourea (MNU) by intravenous injection resulting in the development of multiple estrogen receptor–positive tumors. Treatment with lapatinib beginning 5 days after MNU was highly effective in preventing cancer development. In addition, we treated rats with palpable mammary tumors with lapatinib daily. In these tumor-bearing animals, treatment continued for 42 days and therapeutic results were obtained. Some rats bearing cancers were treated for 5 days, and the resulting lesions were examined for biomarker modulation. Lapatinib effectively suppressed the abundance of HER2, phosphorylated HER2 (Tyr1221/1222), and phosphorylated EGFR (Tyr1173, Tyr1110) compared with tumors from untreated rats. Protein array analyses allowed parallel determination of the effect of lapatinib on the relative levels of protein phosphorylation and proteins associated with apoptosis. These results combined with immunoreactivity data indicated that, in addition to EGFR and HER2, lapatinib treatment was associated with changes in a number of other signaling molecules, including IGF-1R, Akt, and downstream targets such as GSK3, p27, p53, and cyclin D1 presumably leading to impaired proliferation, apoptosis, or cell-cycle arrest. Cancer Prev Res; 4(8); 1190–7. ©2011 AACR.

Abstract 1039: CREB (Ser133) phosphorylation is abnormally increased in tumors showing resistance to Iressa treatment

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL EGFR is a validated therapeutic target; but unfortunately, only a small percentage of patients with EGFR-overexpressing tumors respond to therapy, and resistance develops even in responsive patients. Gefitinib (Iressa) is a small molecule tyrosine kinase inhibitor that suppresses the activation of EGFR. Understanding the molecular mechanisms and protein targets involved in the effects of Iressa requires the simultaneous identification of specific molecular markers in the complex network of signaling pathways that are secondarily modulated by Iressa in mammary cancer. In this study, female Sprague-Dawley rats (50 days old) were given methylnitrosourea (MNU) by IV injection through the jugular vein (75 mg/kg BW). The rats were palpated for mammary cancers 2 times per week. When a palpable mammary cancer of approximately 200-250 mm2 was present, the rat was administered Iressa (6 mg/kg BW/day) by gavage for 2 days. The tumor was then biopsied. The rats were treated with Iressa at the same dose for an additional 40 days and tumors were harvested. At sacrifice, the mammary cancer was rapidly removed and frozen for protein array analysis. The tumors were sorted into two groups based on sensitivity to treatment with Iressa and analyzed by a phospho-protein Proteome Profiler™ Array (R&D, Minneapolis, MN). Three tumors that regressed 27, 39, and 59% were compared to 4 tumors that continued to grow, increasing in size by 56, 106, 210, and 236%. Most notably, tumors that continued to grow in spite of Iressa treatment consistently showed a marked increased phosphorylation of CREB (Ser133) and Src (Tyr419) compared to tumors that regressed. CREB phosphorylation at Ser133 enhances its transactivation and transcriptional activities, resulting in increased expression of many downstream target genes involved in cell proliferation and cancer development. In addition, tumors that continued to grow also showed increased phosphorylation of p27 (Thr157) compared to regressed tumors. Phosphorylation of p27 at Thr157 is known to impair its nuclear localization, suggesting an acceleration of the G1/S cell cycle transition, resulting in enhanced cell proliferation. In contrast, tumors that regressed showed marked increased phosphorylation of Hck (Tyr411) compared to tumors that continued to grow. The hck gene is located at 20q11-q12, which is a region affect by interstitial deletions in some acute myeloid leukemias and myeloproliferative disorders and damage to hck might contribute to the pathogenesis of these conditions, suggesting that autophosphorylation of Hck (Tyr411) might be involved in tumor suppression. These results indicated that Iressa resistance in mammary cancer induced by MNU is closely related with activation of the signaling axis of the cytosolic tyrosine kinase, CREB, which could contribute to continuous proliferation. Supported by NCI # HHSN-261200433009C – NO1-CN-55006-72. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1039. doi:10.1158/1538-7445.AM2011-1039

Abstract 1589: Isorhamnetin suppresses skin cancer through direct inhibition of MEK1 and PI3-K

Isorhamnetin is a plant flavonoid that is found in fruits and medicinal herbs and exerts anticancer effects. However, the underlying molecular mechanism of the chemopreventive potential of isorhamnetin remains unknown. Here, we report anti-skin cancer effects of isorhamnetin. In an in vivo mouse xenograft model injected with A431 human skin epithelial carcinoma cells, isorhamnetin reduced tumor growth and COX-2 expression. Isorhamnetin inhibited epidermal growth factor (EGF)-induced neoplastic cell transformation of JB6 mouse epidermis cells and anchorage-dependent and -independent growth of A431 cells. Isorhamnetin attenuated EGF-induced cyclooxygenase (COX)-2 expression in JB6 cells and A431 cells. 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 demonstrated that isorhamnetin inhibited the kinase activity of MEK1 and PI3-K 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. Overall, these results indicate that isorhamnetin has potent anti-cancer activity and it primarily targets MEK and PI3-K, which might contribute to the chemopreventive potential of certain foods. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1589. doi:1538-7445.AM2012-1589

Abstract 2325: The crystal structure of the ERK2/norathyriol complex

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Norathyriol is an active metabolite of mangiferin, one of the mango polyphenolic compounds that have been demonstrated to have anti-proliferative and pro-apoptotic activities against human leukemia, as well as human lung, breast, colon and prostate cancer cells. Norathyriol has been reported to inhibit the PMA-induced respiratory burst and aggregation of rat neutrophils by suppressing protein kinase C activity. We show that norathyriol targets extracellular signal-regulated kinase 2 (ERK2), which is activated in response to growth factors and cytokines, and is a key kinase in the Ras/Raf/MAPK signaling pathway, which is deregulated in many cancers. We determined the crystal structure of ERK2 complexed with norathyriol at 1.6 A resolution. Human recombinant ERK2 was expressed, purified from E.coli and successfully crystallized. The protein crystals were soaked with norathyriol solution prior to the data collection. The norathyriol molecule is clearly visible in the electron density map inside the ATP-binding site below the phosphate-binding loop. Norathyriol is anchored to the ERK2 hinge region that connects the kinase N- and C-lobes by several hydrogen bonds. The side chain carboxyl of the gatekeeper residue Gln105 hydrogen bonded with the 7-OH group of norathyriol. The backbone of Asp106 and Met108 form hydrogen bonds with 6-OH group of norathyriol. We mutated Gln105 to alanine to weaken the norathyriol interaction with ERK2, and showed that the recombinant Q105A mutant exhibited impaired in vitro binding affinity to norathyriol compared with wild type. The Ile31, Val39, and Ala52 residues from the N-lobe contribute to the hydrophobic interactions with the xanthone moiety of norathyriol. These results indicate that norathyriol directly binds to the ATP-binding site of ERK2. The Ras/Raf mutation in many cancers causes the hyperactivation of ERK2 and unregulated cell proliferation. Recent reports indicate that knockdown of ERK2 inhibits growth of tumor cells, suggesting that ERK2 inhibition is a potential approach for the treatment of cancer. Norathyriol thus might be a potential therapeutic and chemopreventive lead compound targeting ERK2 in cancer cells where activation of the ERK2 pathway mediates the anti-apoptotic response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2325. doi:10.1158/1538-7445.AM2011-2325

Abstract 1050: Lapatinib suppresses RTK-mediated signaling through multiple signaling pathways in the chemically-induced ER+ model of mammary cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Activation of tyrosine kinase receptors, including EGFR, HER2, HER3 and HER4, plays a key role in the prognosis of mammary cancer. EGFR and HER2 are validated therapeutic targets; but unfortunately, only a small percentage of patients with EGFR or HER2-overexpressing tumors respond to therapy, and resistance develops even in responsive patients. Lapatinib is a small molecule dual tyrosine kinase inhibitor that suppresses the activation of EGFR and HER2. Completely understanding the molecular mechanisms and protein targets involved in the effects of Lapatinib and other RTK inhibitors can help determine why efficacy varies as well as identify biomarkers which may be indicative of effective therapy. This requires the simultaneous identification of specific molecular markers in the complex network of signaling pathways that are modulated by Lapatinib in mammary cancer. The purpose of this study was to identify potential direct and indirect protein targets modified by Lapatinib treatment. Female Sprague-Dawley rats (50 d) were given methylnitroso-urea (MNU) by IV injection via jugular vein (75 mg/kg BW). Rats developed multiple ER+ tumors. Two different approaches were taken to evaluate the efficacy of Lapatinib as either a chemopreventive or chemotherapeutic drug. First we demonstrated that treatment with Lapatinib beginning 5 days after MNU was highly effective in preventing tumor development. In addition, we treated rats with palpable mammary tumors with Lapatinib (75 mg/kg BW/day). In some of the tumor bearing animals, treatment proceeded for 42 days, and therapeutic results were obtained. In a separate group, rats bearing tumors were treated for 5 days and the resulting lesions examined for biomarker modulation. Female Sprague-Dawley rats (50 d) were given methylnitroso-urea (MNU) by IV injection via jugular vein (75 mg/kg BW). Rats developed multiple ER+ tumors. Two different approaches were taken to evaluate the efficacy of Lapatinib as either a chemopreventive or chemotherapeutic drug. First we demonstrated that treatment with Lapatinib beginning 5 days after MNU was highly effective in preventing tumor development. In addition, we treated rats with palpable mammary tumors with Lapatinib (75 mg/kg BW/day). In some of the tumor bearing animals, treatment proceeded for 42 days, and therapeutic results were obtained. In a separate group, rats bearing tumors were treated for 5 days and the resulting lesions examined for biomarker modulation. These results suggest that RTK inhibitors have a wide range of potential targets. More work is in progress to continue the dissection of the action of Lapatinib and other RTK inhibitors. Supported by The Hormel Foundation and NCI Contract Number HHSN-261200433009C – NO1-CN-55006-72. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1050. doi:10.1158/1538-7445.AM2011-1050