Naomi Oi

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.

Prediction of molecular targets of cancer preventing flavonoid compounds using computational methods.

Plant-based polyphenols (i.e., phytochemicals) have been used as treatments for human ailments for centuries. The mechanisms of action of these plant-derived compounds are now a major area of investigation. Thousands of phytochemicals have been isolated, and a large number of them have shown protective activities or effects in different disease models. Using conventional approaches to select the best single or group of best chemicals for studying the effectiveness in treating or preventing disease is extremely challenging. We have developed and used computational-based methodologies that provide efficient and inexpensive tools to gain further understanding of the anticancer and therapeutic effects exerted by phytochemicals. Computational methods involving virtual screening, shape and pharmacophore analysis and molecular docking have been used to select chemicals that target a particular protein or enzyme and to determine potential protein targets for well-characterized as well as for novel phytochemicals.

[6]-Shogaol inhibits growth and induces apoptosis of non-small cell lung cancer cells by directly regulating Akt1/2

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. Despite progress in developing chemotherapeutics for the treatment of NSCLC, primary and secondary resistance limits therapeutic success. NSCLC cells exhibit multiple mutations in the epidermal growth factor receptor (EGFR), which cause aberrant activation of diverse cell signaling pathways. Therefore, suppression of the inappropriate amplification of EGFR downstream signaling cascades is considered to be a rational therapeutic and preventive strategy for the management of NSCLC. Our initial molecular target-oriented virtual screening revealed that the ginger components, including [6]-shogaol, [6]-paradol and [6]-gingerol, seem to be potential candidates for the prevention and treatment of NSCLC. Among the compounds, [6]-shogaol showed the greatest inhibitory effects on the NSCLC cell proliferation and anchorage-independent growth. [6]-Shogaol induced cell cycle arrest (G1 or G2/M) and apoptosis. Furthermore, [6]-shogaol inhibited Akt kinase activity, a downstream mediator of EGFR signaling, by binding with an allosteric site of Akt. In NCI-H1650 lung cancer cells, [6]-shogaol reduced the constitutive phosphorylation of signal transducer and activator of transcription-3 (STAT3) and decreased the expression of cyclin D1/3, which are target proteins in the Akt signaling pathway. The induction of apoptosis in NCI-H1650 cells by [6]-shogaol corresponded with the cleavage of caspase-3 and caspase-7. Moreover, intraperitoneal administration of [6]-shogaol inhibited the growth of NCI-H1650 cells as tumor xenografts in nude mice. [6]-Shogaol suppressed the expression of Ki-67, cyclin D1 and phosphorylated Akt and STAT3 and increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positivity in xenograft tumors. The current study clearly indicates that [6]-shogaol can be exploited for the prevention and/or treatment of NSCLC.

Taxifolin Suppresses UV-Induced Skin Carcinogenesis by Targeting EGFR and PI3K

Skin cancer is one of the most commonly diagnosed cancers in the United States. Taxifolin reportedly exerts multiple biologic effects, but the molecular mechanisms and direct target(s) of taxifolin in skin cancer chemoprevention are still unknown. In silico computer screening and kinase profiling results suggest that the EGF receptor (EGFR), phosphoinositide 3-kinase (PI3K), and Src are potential targets for taxifolin. Pull-down assay results showed that EGFR, PI3K, and Src directly interacted with taxifolin in vitro, whereas taxifolin bound to EGFR and PI3K, but not to Src in cells. ATP competition and in vitro kinase assay data revealed that taxifolin interacted with EGFR and PI3K at the ATP-binding pocket and inhibited their kinase activities. Western blot analysis showed that taxifolin suppressed UVB-induced phosphorylation of EGFR and Akt, and subsequently suppressed their signaling pathways in JB6 P+ mouse skin epidermal cells. Expression levels and promoter activity of COX-2 and prostaglandin E2 (PGE2) generation induced by UVB were also attenuated by taxifolin. The effect of taxifolin on UVB-induced signaling pathways and PGE2 generation was reduced in EGFR knockout murine embryonic fibroblasts (MEF) compared with EGFR wild-type MEFs. Taxifolin also inhibited EGF-induced cell transformation. Importantly, topical treatment of taxifolin to the dorsal skin significantly suppressed tumor incidence, volume, and multiplicity in a solar UV (SUV)-induced skin carcinogenesis mouse model. Further analysis showed that the taxifolin-treated group had a substantial reduction in SUV-induced phosphorylation of EGFR and Akt in mouse skin. These results suggest that taxifolin exerts chemopreventive activity against UV-induced skin carcinogenesis by targeting EGFR and PI3K. Cancer Prev Res; 5(9); 1103–14. ©2012 AACR.

Resveratrol induces apoptosis by directly targeting Ras-GTPase-activating protein SH3 domain-binding protein 1.

Resveratrol (trans-3,5,4′-truhydroxystilbene) possesses a strong anticancer activity exhibited as the induction of apoptosis through p53 activation. However, the molecular mechanism and direct target(s) of resveratrol-induced p53 activation remain elusive. Here, the Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) was identified as a potential target of resveratrol, and in vitro binding assay results using resveratrol-conjugated Sepharose 4B beads confirmed their direct binding. Depletion of G3BP1 significantly diminishes resveratrol-induced p53 expression and apoptosis. We also found that G3BP1 negatively regulates p53 expression by interacting with ubiquitin-specific protease 10 (USP10), a deubiquitinating enzyme of p53. Disruption of the interaction of p53 with USP10 by G3BP1 interference leads to the suppression of p53 deubiquitination. Resveratrol, on the other hand, directly binds to G3BP1 and prevents the G3BP1/USP10 interaction, resulting in enhanced USP10-mediated deubiquitination of p53, and consequently increased p53 expression. These findings disclose a novel mechanism of resveratrol-induced p53 activation and resveratrol-induced apoptosis by direct targeting of G3BP1.

6-C-(E-phenylethenyl)-Naringenin Suppresses Colorectal Cancer Growth by Inhibiting Cyclooxygenase-1

Recent clinical trials raised concerns regarding the cardiovascular toxicity of selective cyclooxygenase-2 (COX-2) inhibitors and cyclooxygenase-1 (COX-1) is now being reconsidered as a target for chemoprevention. Our aims were to determine whether selective COX-1 inhibition could delay or prevent cancer development and also clarify the underlying mechanisms. Data clearly showed that COX-1 was required for maintenance of malignant characteristics of colon cancer cells or tumor promoter-induced transformation of preneoplastic cells. We also successfully applied a ligand-docking computational method to identify a novel selective COX-1 inhibitor, 6-C-(E-phenylethenyl)-naringenin (designated herein as 6CEPN). 6CEPN could bind to COX-1 and specifically inhibited its activity both in vitro and ex vivo. In colorectal cancer cells, it potently suppressed anchorage-independent growth by inhibiting COX-1 activity. 6CEPN also effectively suppressed tumor growth in a 28-day colon cancer xenograft model without any obvious systemic toxicity. Taken together, COX-1 plays a critical role in human colorectal carcinogenesis, and this specific COX-1 inhibitor merits further investigation as a potential preventive agent against colorectal cancer.

Embryonic stem‐cell‐preconditioned microenvironment induces loss of cancer cell properties in human melanoma cells

The cancer microenvironment affects cancer cell proliferation and growth. Embryonic stem (ES)‐preconditioned 3‐dimensional (3‐D) culture of cancer cells induces cancer cell reprogramming and results in a change in cancer cell properties such as differentiation and migration in skin melanoma. However, the mechanism has not yet been clarified. Using the ES‐preconditioned 3‐D microenvironment model, we provide evidence showing that the ES microenvironment inhibits proliferation and anchorage‐independent growth of SK‐MEL‐28 melanoma cells. We also found that the ES microenvironment suppresses telomerase activity and thereby induces senescence in SK‐MEL‐28 cells. Furthermore, we observed that gremlin, an antagonist of BMP4, is secreted from ES cells and plays an important role in cellular senescence. Knocking down gremlin in the ES microenvironment increases proliferation and anchorage‐independent growth of SK‐MEL‐28 melanoma cells. Taken together, these results demonstrated that gremlin is a crucial factor responsible for abrogating melanoma properties in the ES‐preconditioned 3‐D microenvironment.

Aspirin Prevents Colorectal Cancer by Normalizing EGFR Expression.

Background Aspirin intake reduces the risk of colorectal cancer (CRC), but the molecular underpinnings remain elusive. Epidermal growth factor receptor (EGFR), which is overexpressed in about 80% of CRC cases, is implicated in the etiology of CRC. Here, we investigated whether aspirin can prevent CRC by normalizing EGFR expression. Methods Immunohistochemistry staining was performed on paraffin-embedded tissue sections from normal colon mucosa, adenomatous polyps from FAP patients who were classified as regular aspirin users or nonusers. The interplay between cyclooxygenase-2 (COX-2) and EGFR was studied in primary intestinal epithelial cells isolated from ApcMin mice, immortalized normal human colon epithelial cells (HCECs) as well as murine embryonic fibroblasts (MEFs). Results Immunohistochemistry staining results established that EGFR overexpression is an early event in colorectal tumorigenesis, which can be greatly attenuated by regular use of aspirin. Importantly, EGFR and COX-2 were co-overexpressed and co-localized with each other in FAP patients. Further mechanistic studies revealed that COX-2 overexpression triggers the activation of the c-Jun-dependent transcription factor, activator protein-1 (AP-1), which binds to the Egfr promoter. Binding facilitates the cellular accumulation of EGFR and lowers the threshold required for pre-neoplastic cells to undergo transformation. Conclusion Aspirin might exert its chemopreventive activity against CRC, at least partially, by normalizing EGFR expression in gastrointestinal precancerous lesions.

A Chrysin Derivative Suppresses Skin Cancer Growth by Inhibiting Cyclin-dependent Kinases

Background: Binding to the ATP site results in poor selectivity; therefore, development of ATP-noncompetitive inhibitors is needed. Results: A modified chrysin with anticancer activity targets Cdks and binds to a Cdk2 allosteric site, not the ATP pocket. Conclusion: Modified chrysin is a novel ATP-noncompetitive inhibitor. Significance: This pharmacophore model might provide insights for the development of new ATP-noncompetitive agents. Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P+ cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P+ cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency.

Circulating Prostaglandin Biosynthesis in Colorectal Cancer and Potential Clinical Significance

Background Colorectal cancer (CRC) represents the third leading cause of cancer-related death in the United States. Lack of reliable biomarkers remains a critical issue for early detection of CRC. In this study, we investigated the potential predictive values of circulating prostaglandin (PG) biosynthesis in CRC risk. Methods Profiles of circulating PG biosynthesis and platelet counts were determined in healthy subjects (n = 16), familial adenomatous polyposis (FAP) patients who were classified as regular aspirin users (n = 14) or nonusers (n = 24), and CRC patients with (n = 18) or without FAP history (n = 20). Immunohistochemistry staining was performed on biopsy samples. Results Analysis of circulating PG biosynthesis unexpectedly revealed that CRC progression is accompanied by a pronounced elevation of circulating thromboxane A2 (TXA2) levels. When a circulating TXA2 level of 1000 pg/mL was selected as a practical cutoff point, 95% of CRC patients were successfully identified. Further study suggested that the TXA2 pathway is constitutively activated during colorectal tumorigenesis and required for anchorage-independent growth of colon cancer cells. Conclusions This study established the importance of the TXA2 pathway in CRC pathophysiology, and laid the groundwork for introducing a TXA2-targeting strategy to CRC prevention, early detection and management.