A Novel Cinnamon-Related Natural Product with Pim-1 Inhibitory Activity Inhibits Leukemia and Skin Cancer.

The Pim-1 kinase regulates cell survival, proliferation, and differentiation and is overexpressed frequently in many malignancies, including leukemia and skin cancer. In this study, we used kinase profiling analysis to demonstrate that 2'-hydroxycinnamicaldehyde (2'-HCA), a compound found in cinnamon, specifically inhibits Pim-1 activity. Cocrystallography studies determined the hydrogen bonding pattern between 2'-HCA and Pim-1. Notably, 2'-HCA binding altered the apo kinase structure in a manner that shielded the ligand from solvent, thereby acting as a gatekeeper loop. Biologically, 2'-HCA inhibited the growth of human erythroleukemia or squamous epidermoid carcinoma cells by inducing apoptosis. The compound was also effective as a chemopreventive agent against EGF-mediated neoplastic transformation. Finally, 2'-HCA potently suppressed the growth of mouse xenografts representing human leukemia or skin cancer. Overall, our results offered preclinical proof of concept for 2'-HCA as a potent anticancer principle arising from direct targeting of the Pim-1 kinase.

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.

Phosphatidylinositol 3-kinase, a novel target molecule for the inhibitory effects of kaempferol on neoplastic cell transformation.

Kaempferol (KF), which is a natural dietary flavonoid, has potential beneficial effects as a chemopreventive agent for critical health conditions, such as cancer. However, the molecular mechanisms underlying the activity of KF remain unknown. We report on the inhibition of neoplastic cell transformation by KF through the suppression of phosphatidylinositol 3-kinase (PI3K) activity. Epidermal growth factor (EGF)-induced neoplastic transformation of mouse epidermal JB6 P+ cells was inhibited by 40 microM KF. The activation of activator protein-1 and nuclear factor-kappaB induced by EGF was also attenuated by KF. The EGF-induced phosphorylation of Akt (protein kinase B) was completely suppressed by KF, although extracellular signal-regulated kinase, p38, c-Jun N-terminal kinase and p90 ribosomal S6 kinase were unaffected by KF. Kinase assay data revealed that KF bound directly to PI3K, which is upstream of Akt, and suppressed its activity. Furthermore, KF inhibited ultraviolet B (UVB)-induced PI3K activity and attenuated UVB-induced phosphorylation of Akt. Our results suggest that KF docks at the adenosine triphosphate-binding site of PI3K, which is located between the N-lobe and C-lobe of the kinase domain. Inhibition by KF of PI3K, which is an important factor in carcinogenesis, and its downstream effects may explain the chemopreventive action of KF.

7,3',4'-Trihydroxyisoflavone inhibits epidermal growth factor-induced proliferation and transformation of JB6 P+ mouse epidermal cells by suppressing cyclin-dependent kinases and phosphatidylinositol 3-kinase.

Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3',4'-Trihydroxyisoflavone (7,3',4'-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3',4'-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G(1) phase. As shown by Western blot, 7,3',4'-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G(1) phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3',4'-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3beta/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3',4'-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3',4'-THIF provide a molecular basis for the possible development of new chemoprotective agents.

5-deoxykaempferol plays a potential therapeutic role by targeting multiple signaling pathways in skin cancer.

Nontoxic small molecules with multitargeting effects are believed to have potential in cancer prevention. Dietary phytochemicals were shown to exhibit cancer-preventive effects attributed to their antioxidant capacities. In this report, we show that the natural compound 5-deoxykaempferol (5-DK) exerts a chemopreventive effect on UVB-induced skin carcinogenesis by targeting multiple signaling molecules. 5-DK suppressed the UVB-induced expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor in mouse skin epidermal JB6 P+ cells. Moreover, 5-DK inhibited phosphorylation of MKK3/6, MKK4, and Akt, but had no effect on phosphorylation of Src, extracellular signal-regulated kinases, or ribosomal S6 kinase (RSK). However, 5-DK affected multiple targets by reducing Src, phosphoinositide 3-kinase (PI3K), and RSK2 activities. In particular, pull-down assays revealed that 5-DK specifically bound to and competed with ATP for binding with Src, PI3K, and RSK2. Exposure to 5-DK significantly suppressed UVB-induced tumorigenesis in mouse skin in a dose-dependent manner, and it inhibited the UVB-induced expression of COX-2, proliferating cell nuclear antigen, vascular endothelial growth factor, and matrix metalloproteinase-9. Our data suggest that 5-DK docks at the ATP-binding site of Src, PI3K, and RSK2. For RSK2, the ATP-binding site is located between the N- and C-lobes of the kinase domain. Taken together, our results indicate that 5-DK holds promise for the treatment of UVB-induced skin cancer by targeting Src, PI3K, and RSK2 signaling.

Cyanidin suppresses ultraviolet B-induced COX-2 expression in epidermal cells by targeting MKK4, MEK1, and Raf-1.

Skin cancer is the most frequently diagnosed cancer in the United States. Ultraviolet B (UVB) rays (wavelength: 280-320nm) play a pivotal role in the development of skin cancer by inducing the expression of inflammatory proteins such as cyclooxygenase-2 (COX-2). Cyanidin, the most plentiful of the plant pigments known as anthocyanidins, is a potent chemopreventive agent. In the present study, we examined the molecular mechanisms underlying the chemopreventive activity of cyanidin and identified its molecular targets. Cyanidin inhibited UVB-induced COX-2 expression and prostaglandin E(2) secretion in the epidermal skin cell line JB6 P+ by suppressing the transactivation of nuclear factor-kappaB and activator protein-1 which are well-known transcription factors regulated by mitogen-activated protein kinase. Cyanidin markedly inhibited the phosphorylation of JNK1/2, ERK1/2, and MEK1/2 than the of MKK4 and Raf-1, two upstream kinases of JNK1/2, ERK1/2, and MEK1/2. Cyanidin significantly suppressed the activities of MKK4, MEK1, and Raf-1 through direct binding. Transient transfection of a small interfering RNA specific for MKK4 inhibited the UVB-induced expression of COX-2 in JB6 P+ cells, as did the expression of a dominant-negative ERK2 mutant. We conclude that MKK4, MEK1, and Raf-1 are targets of cyanidin for the suppression of UVB-induced COX-2 expression.