Hanyong Chen

Src kinase is a direct target of apigenin against UVB-induced skin inflammation

Apigenin, a flavonoid abundant in various vegetables and fruits, including parsley and onions, has been reported to possess anticarcinogenic effects. However, the direct molecular target of apigenin and its chemopreventive effect on ultraviolet (UV)-induced skin inflammation are not understood fully. Herein, we examined the anti-inflammatory effect of apigenin and its associated mechanisms in JB6 P+ cell line and SKH-1 hairless mouse model. Apigenin inhibited UVB-induced cyclooxygenase-2 (COX-2) expression, which is a well-known key mediator of inflammation and cancer, and restored the upstream stimulatory factor level in JB6 P+ cells. Immunoblot and kinase assay data demonstrate that Src activity was attenuated by apigenin, and this led to subsequent inhibition of UVB-induced phosphorylation of epidermal growth factor receptor, mitogen-activated protein kinases and Akt signaling. Inhibitory effects of apigenin on UVB-induced signaling were also confirmed in HaCaT human keratinocytes. In addition, in vitro pull-down assays revealed that apigenin binds Src in an adenosine triphosphate-competitive manner. Results using in vivo skin model indicate apigenin significantly inhibits UVB-induced ear edema development, COX-2 expression and Src kinase activity in SKH-1 hairless mice. Collectively, these findings suggest that apigenin exerts potent chemopreventive activity against UVB-induced skin inflammation primarily by targeting Src.

Curcumin Suppresses Proliferation of Colon Cancer Cells by Targeting CDK2

Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the Protein Data Bank against curcumin. Cyclin-dependent kinase 2 (CDK2), a major cell-cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell-cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of retinoblastoma (Rb), a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell-cycle arrest, we investigated the antiproliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine whether CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantially relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells. Cancer Prev Res; 7(4); 466–74. ©2014 AACR.

ERK1 phosphorylates Nanog to regulate protein stability and stem cell self-renewal

Nanog regulates human and mouse embryonic stem (ES) cell self-renewal activity. Activation of ERKs signaling negatively regulates ES cell self-renewal and induces differentiation, but the mechanisms are not understood. We found that ERK1 binds and phosphorylates Nanog. Activation of MEK/ERKs signaling and phosphorylation of Nanog inhibit Nanog transactivation, inducing ES cell differentiation. Conversely, suppression of MEK/ERKs signaling enhances Nanog transactivation to inhibit ES cell differentiation. We observed that phosphorylation of Nanog by ERK1 decreases Nanog stability through ubiquitination-mediated protein degradation. Further, we found that this phosphorylation induces binding of FBXW8 with Nanog to reduce Nanog protein stability. Overall, our results demonstrated that ERKs-mediated Nanog phosphorylation plays an important role in self-renewal of ES cells through FBXW8-mediated Nanog protein stability.

Isoliquiritigenin Induces Apoptosis and Inhibits Xenograft Tumor Growth of Human Lung Cancer Cells by Targeting Both Wild Type and L858R/T790M Mutant EGFR

Background: Non-small-cell lung cancer (NSCLC) exhibits EGFR mutation. Results: Treatment with isoliquiritigenin (ILQ) inhibited growth and induced apoptosis in tyrosine kinase inhibitor-sensitive and -resistant NSCLC cells. ILQ suppressed wild type and mutant (L858R/T790M) EGFR kinase activity and attenuated H1975 lung cancer cell xenograft tumor growth. Conclusion: ILQ directly targets wild type or mutant EGFR. Significance: ILQ could be a potential therapeutic agent against NSCLC. Non-small-cell lung cancer (NSCLC) is associated with diverse genetic alterations including mutation of epidermal growth factor receptor (EGFR). Isoliquiritigenin (ILQ), a chalcone derivative, possesses anticancer activities. In the present study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor (TKI)-sensitive and -resistant NSCLC cells and elucidated its underlying mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both TKI-sensitive and -resistant NSCLC cells. ILQ-induced apoptosis was associated with the cleavage of caspase-3 and poly-(ADP-ribose)-polymerase, increased expression of Bim, and reduced expression of Bcl-2. In vitro kinase assay results revealed that ILQ inhibited the catalytic activity of both wild type and double mutant (L858R/T790M) EGFR. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wild type or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells, and attenuated the kinase activity of Akt1 and ERK2 in vitro. ILQ directly interacted with both wild type and double-mutant EGFR in an ATP-competitive manner. A docking model study showed that ILQ formed two hydrogen bonds (Glu-762 and Met-793) with wild type EGFR and three hydrogen bonds (Lys-745, Met-793, and Asp-855) with mutant EGFR. ILQ attenuated the xenograft tumor growth of H1975 cells, which was associated with decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wild type or mutant EGFR.

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.

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.

Naproxen induces cell-cycle arrest and apoptosis in human urinary bladder cancer cell lines and chemically induced cancers by targeting PI3K

Naproxen [(S)-6-methoxy-α-methyl-2-naphthaleneacetic acid] is a potent nonsteroidal anti-inflammatory drug that inhibits both COX-1 and COX-2 and is widely used as an over-the-counter medication. Naproxen exhibits analgesic, antipyretic, and anti-inflammatory activities. Naproxen, as well as other nonsteroidal anti-inflammatory drug, has been reported to be effective in the prevention of urinary bladder cancer in rodents. However, potential targets other than the COX isozymes have not been reported. We examined potential additional targets in urinary bladder cancer cells and in rat bladder cancers. Computer kinase profiling results suggested that phosphoinositide 3-kinase (PI3K) is a potential target for naproxen. In vitro kinase assay data revealed that naproxen interacts with PI3K and inhibits its kinase activity. Pull-down binding assay data confirmed that PI3K directly binds with naproxen in vitro and ex vivo. Western blot data showed that naproxen decreased phosphorylation of Akt, and subsequently decreased Akt signaling in UM-UC-5 and UM-UC-14 urinary bladder cancer cells. Furthermore, naproxen suppressed anchorage-independent cell growth and decreased cell viability by targeting PI3K in both cell lines. Naproxen caused an accumulation of cells at the G1 phase mediated through cyclin-dependent kinase 4, cyclin D1, and p21. Moreover, naproxen induced significant apoptosis, accompanied with increased levels of cleaved caspase-3, caspase-7, and PARP in both cell types. Naproxen-induced cell death was mainly because of apoptosis in which a prominent downregulation of Bcl-2 and upregulation of Bax were involved. Naproxen also caused apoptosis and inhibited Akt phosphorylation in rat urinary bladder cancers induced by N-butyl-N-(4-hydroxybutyl)-nitrosamine. Cancer Prev Res; 7(2); 236–45. ©2013 AACR.

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.

Kaempferol Targets RSK2 and MSK1 to Suppress UV Radiation-Induced Skin Cancer

Solar UV (SUV) irradiation is a major factor in skin carcinogenesis, the most common form of cancer in the United States. The MAPK cascades are activated by SUV irradiation. The 90 kDa ribosomal S6 kinase (RSK) and mitogen and stress-activated protein kinase (MSK) proteins constitute a family of protein kinases that mediate signal transduction downstream of the MAPK cascades. In this study, phosphorylation of RSK and MSK1 was upregulated in human squamous cell carcinoma (SCC) and SUV-treated mouse skin. Kaempferol, a natural flavonol, found in tea, broccoli, grapes, apples, and other plant sources, is known to have anticancer activity, but its mechanisms and direct target(s) in cancer chemoprevention are unclear. Kinase array results revealed that kaempferol inhibited RSK2 and MSK1. Pull-down assay results, ATP competition, and in vitro kinase assay data revealed that kaempferol interacts with RSK2 and MSK1 at the ATP-binding pocket and inhibits their respective kinase activities. Mechanistic investigations showed that kaempferol suppresses RSK2 and MSK1 kinase activities to attenuate SUV-induced phosphorylation of cAMP-responsive element binding protein (CREB) and histone H3 in mouse skin cells. Kaempferol was a potent inhibitor of SUV-induced mouse skin carcinogenesis. Further analysis showed that skin from the kaempferol-treated group exhibited a substantial reduction in SUV-induced phosphorylation of CREB, c-Fos, and histone H3. Overall, our results identify kaempferol as a safe and novel chemopreventive agent against SUV-induced skin carcinogenesis that acts by targeting RSK2 and MSK1. Cancer Prev Res; 7(9); 958–67. ©2014 AACR.

Licochalcone A, a Natural Inhibitor of c-Jun N-Terminal Kinase 1

The c-Jun N-terminal kinases (JNK) play an important role in many physiologic processes induced by numerous stress signals. Each JNK protein appears to have a distinct function in cancer, diabetes, or Parkinsons disease. Herein, we found that licochalcone A, a major phenolic constituent isolated from licorice root, suppressed JNK1 activity but had little effect on JNK2 in vitro activity. Although licochalcone A binds with JIP1 competitively with either JNK1 or JNK2, a computer simulation model showed that after licochalcone A binding, the ATP-binding cleft of JNK1 was distorted more substantially than that of JNK2. This could reduce the affinity of JNK1 more than JNK2 for ATP binding. Furthermore, licochalcone A inhibited JNK1-mediated, but not JNK2-mediated, c-Jun phosphorylation in both ex vivo and in vitro systems. We also observed that in colon and pancreatic cancer cell lines, JNK1 is highly expressed compared with normal cell lines. In cancer cell lines, treatment with licochalcone A or knocking down JNK1 expression suppressed colon and pancreatic cancer cell proliferation and colony formation. The inhibition resulted in G1 phase arrest and apoptosis. Moreover, an in vivo xenograft mouse study showed that licochalcone A treatment effectively suppressed the growth of HCT116 xenografts, without affecting the body weight of mice. These results show that licochalcone A is a selective JNK1 inhibitor. Therefore, we suggest that because of the critical role of JNK1 in colon cancer and pancreatic carcinogenesis, licochalcone A might have preventive or therapeutic potential against these devastating diseases. Cancer Prev Res; 7(1); 139–49. ©2013 AACR.