Purpurogallin is a novel mitogen-activated protein kinase kinase 1/2 inhibitor that suppresses esophageal squamous cell carcinoma growth in vitro and in vivo.

Purpurogallin is a natural compound that is extracted from nutgalls and oak bark and it possesses antioxidant, anticancer, and anti-inflammatory properties. However, the anticancer capacity of purpurogallin and its molecular target have not been investigated in esophageal squamous cell carcinoma (ESCC). Herein, we report that purpurogallin suppresses ESCC cell growth by directly targeting the mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling pathway. We found that purpurogallin inhibits anchorage-dependent and -independent ESCC growth. The results of in vitro kinase assays and cell-based assays indicated that purpurogallin also strongly attenuates the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and also directly binds to and inhibits MEK1 and MEK2 activity. Furthermore, purpurogallin contributed to S and G2 phase cell cycle arrest by reducing cyclin A2 and cyclin B1 expression and also induced apoptosis by activating poly (ADP ribose) polymerase (PARP). Notably, purpurogallin suppressed patient-derived ESCC tumor growth in an in vivo mouse model. These findings indicated that purpurogallin is a novel MEK1/2 inhibitor that could be useful for treating ESCC.

Cell growth inhibition by 3-deoxysappanchalcone is mediated by directly targeting the TOPK signaling pathway in colon cancer.

BACKGROUND: Colorectal cancer is one of the most common causes of cancer death worldwide. Unfortunately, chemotherapies are limited due to many complications and development of resistance and recurrence. The T-lymphokine-activated killer cell-originated protein kinase (TOPK) is highly expressed and activated in colon cancer, and plays an important role in inflammation, proliferation, and survival of cancer cells. Therefore, suppressing TOPK activity and its downstream signaling cascades is considered to be a rational therapeutic/preventive strategy against colon cancers. PURPOSE: 3-Deoxysappanchalcone (3-DSC), a component of Caesalpinia sappan L., is a natural oriental medicine. In this study, we investigated the effects of 3-DSC on colon cancer cell growth and elucidated its underlying molecular mechanism of targeting TOPK. STUDY DESIGN AND METHODS: To evaluate the effects of 3-DSC against colon cancer, we performed cell proliferation assays, propidium iodide- and annexin V-staining analyses and Western blotting. Targeting TOPK by 3-DSC was identified by a kinase-binding assay and computational docking models. RESULTS: 3-DSC inhibited the kinase activity of TOPK, but not mitogen-activated protein kinase (MEK). The direct binding of 3-DSC with TOPK was explored using a computational docking model and binding assay in vitro and ex vivo. 3-DSC inhibited colon cancer cell proliferation and anchorage-independent cell growth, and induced G2/M cell cycle arrest and apoptosis. Treatment of colon cancer cells with 3-DSC induced expression of protein that are involved in cell cycle (cyclin B1) and apoptosis (cleaved-PARP, cleaved-caspase-3, and cleaved-caspase-7), and suppressed protein expressions of extracellular signal-regulated kinase (ERK)-1/2, ribosomal S6 kinase (RSK), and c-Jun, which are regulated by the upstream kinase, TOPK. CONCLUSION: 3-DSC suppresses colon cancer cell growth by directly targeting the TOPK- mediated signaling pathway.

Gossypin inhibits gastric cancer growth by direct targeting of AURKA and RSK2.

Gossypin is a flavone extracted from Hibiscus vitifolius, which has been reported to exhibit anti-inflammatory, antioxidant, and anticancer activities. However, the anticancer properties of gossypin and its molecular mechanism of action against gastric cancer have not been fully investigated. In the present study, we report that gossypin is an Aurora kinase A (AURKA) and RSK2 inhibitor that suppresses gastric cancer growth. Gossypin attenuated anchorage-dependent and anchorage-independent gastric cancer cell growth as well as cell migration. Based on the results of in vitro screening and cell-based assays, gossypin directly binds to and inhibits AURKA and RSK2 activities and their downstream signaling proteins. Gossypin decreased S phase and increased G2/M phase cell cycle arrest by reducing the expression of cyclin A2 and cyclin B1 and the phosphorylation of the CDC protein. Additionally, gossypin also induced intrinsic apoptosis by activating caspases and PARP and increasing the expression of cytochrome c. Our results demonstrate that gossypin is an AURKA and RSK2 inhibitor that could be useful for treating gastric cancer.

The Ashitaba (Angelica keiskei) Chalcones 4-hydroxyderricin and Xanthoangelol Suppress Melanomagenesis By Targeting BRAF and PI3K.

Malignant melanoma is an aggressive tumor of the skin and still lacks effective preventive and therapeutic treatments. In melanoma, both the BRAF/MEK/ERK and PI3-K/AKT signaling pathways are constitutively activated through multiple mechanisms, which result in cell-cycle progression and prevention of apoptosis. Therefore, the development of novel strategies for targeting BRAF and PI3K are of utmost importance. In this study, we found that Ashitaba (Angelica keiskei) chalcones, 4-hydroxyderricin (4HD) and xanthoangelol (XAG), suppressed melanoma development by directly targeting both BRAFV600E and PI3K, which blocked the activation of downstream signaling. This led to the induction of G1 phase cell-cycle arrest and apoptosis in melanoma cells. Importantly, 4HD or XAG dramatically attenuated tumor incidence and volume in the BRAF-activated Pten-deficient melanoma mouse model. Our findings suggest that 4HD and XAG are promising chemopreventive or potential therapeutic agents against melanomagenesis that act by targeting both BRAF and PI3K, providing hope for rapid clinical translation. Cancer Prev Res; 11(10); 607-20. ©2018 AACR.

Targeting AKT with Oridonin Inhibits Growth of Esophageal Squamous Cell Carcinoma In Vitro and Patient-Derived Xenografts In Vivo.

Overexpression or activation of AKT is very well known to control cell growth, survival, and gene expression in solid tumors. Oridonin, an inflammatory medical and diterpenoid compound isolated from Rabdosia rubescens, has exhibited various pharmacologic and physiologic properties, including antitumor, antibacterial, and anti-inflammatory effects. In this study, we demonstrated that oridonin is an inhibitor of AKT and suppresses proliferation of esophageal squamous cell carcinoma (ESCC) in vitro and in vivo The role of AKT in ESCC was studied using immuno-histochemical analysis of a tumor microarray, the effect of AKT knockdown on cell growth, and treatment of cells with MK-2206, an AKT inhibitor. Oridonin blocked AKT kinase activity and interacted with the ATP-binding pocket of AKT. It inhibited growth of KYSE70, KYSE410, and KYSE450 esophageal cancer cells in a time- and concentration-dependent manner. Oridonin induced arrest of cells in the G2-M cell-cycle phase, stimulated apoptosis, and increased expression of apoptotic biomarkers, including cleaved PARP, caspase-3, caspase-7, and Bims in ESCC cell lines. Mechanistically, we found that oridonin diminished the phosphorylation and activation of AKT signaling. Furthermore, a combination of oridonin and 5-fluorouracil or cisplatin (clinical chemotherapeutic agents) enhanced the inhibition of ESCC cell growth. The effects of oridonin were verified in patient-derived xenograft tumors expressing high levels of AKT. In summary, our results indicate that oridonin acts as an AKT inhibitor to suppress the growth of ESCC by attenuating AKT signaling. Mol Cancer Ther; 17(7); 1540-53. ©2018 AACR.

PKCι is a target of 7,8,4'-trihydroxyisoflavone for the suppression of UVB-induced MMP-1 expression.

The soy isoflavone daidzein is bioconverted to 7,8,4'-trihydroxyisoflavone (7,8,4'-THIF) by microorganisms. Here, we investigated the matrix metalloproteinase (MMP)-1 inhibitory properties of 7,8,4'-THIF that arise through the suppression of UVB-induced MMP-1 expression. 7,8,4'-THIF reduced UVB-induced MMP-1 expression at the transcriptional level in primary human dermal fibroblasts and inhibited UVB-induced transcriptional activity of AP-1, a major activator of MMP-1 expression. Additionally, it was observed that the mitogen-activated protein kinase (MAPK) pathway, a crucial signalling cascade for MMP-1 expression, was suppressed by 7,8,4'-THIF. Protein kinase C iota (PKCι) was suspected to be a direct target of 7,8,4'-THIF. The direct interaction between 7,8,4'-THIF and PKCι was confirmed using pull-down assays and immobilized metal ion affinity-based fluorescence polarization assays. Finally, we observed that 7,8,4'-THIF inhibited UVB-induced MMP-1 expression in a human skin equivalent model. Taken together, these results suggest that 7,8,4'-THIF, a bioconversion product of daidzein, suppresses UVB-induced MMP-1 expression.

Grifolin directly targets ERK1/2 to epigenetically suppress cancer cell metastasis.

Grifolin, a secondary metabolite isolated from the fresh fruiting bodies of the mushroom Albatrellus confluens, has been reported by us and others to display potent antitumor effects. However, the molecular target of grifolin has not been identified and the underlying mechanism of action is not fully understood. Here, we report that the ERK1/2 protein kinases are direct molecular targets of grifolin. Molecular modeling, affinity chromatography and fluorescence quenching analyses showed that grifolin directly binds to ERK1/2. And in vitro and ex vivo kinase assay data further demonstrated that grifolin inhibited the kinase activities of ERK1/2. We found that grifolin suppressed adhesion, migration and invasion of high-metastatic cancer cells. The inhibitory effect of grifolin against tumor metastasis was further confirmed in a metastatic mouse model. We found that grifolin decreased phosphorylation of Elk1 at Ser383, and the protein as well as the mRNA level of DNMT1 was also down-regulated. By luciferase reporter and ChIP assay analyses, we confirmed that grifolin inhibited the transcription activity of Elk1 as well as its binding to the dnmt1 promoter region. Moreover, we report that significant increases in the mRNA levels of Timp2 and pten were induced by grifolin. Thus, our data suggest that grifolin exerts its anti-tumor activity by epigenetic reactivation of metastasis inhibitory-related genes through ERK1/2-Elk1-DNMT1 signaling. Grifolin may represent a promising therapeutic lead compound for intervention of cancer metastasis, and it may also be useful as an ERK1/2 kinase inhibitor as well as an epigenetic agent to further our understanding of DNMT1 function.

Computational and Biochemical Discovery of RSK2 as a Novel Target for Epigallocatechin Gallate (EGCG).

The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2), as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals. The computer prediction was confirmed by an in vitro kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains in vitro and ex vivo. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2+/+ and RSK2-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting phosphorylation of histone H3 at Ser10. Overall, these results indicate that RSK2 is a novel molecular target of EGCG.

Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling.

Phosphatase and tensin homolog (PTEN) loss or mutation consistently activates the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway, which contributes to the progression and invasiveness of prostate cancer. Furthermore, the PTEN/PI3-K/Akt and Ras/MAPK pathways cooperate to promote the epithelial-mesenchymal transition (EMT) and metastasis initiated from prostate stem/progenitor cells. For these reasons, the PTEN/PI3-K/Akt pathway is considered as an attractive target for both chemoprevention and chemotherapy. Herein we report that eupafolin, a natural compound found in common sage, inhibited proliferation of prostate cancer cells. Protein content analysis indicated that phosphorylation of Akt and its downstream kinases was inhibited by eupafolin treatment. Pull-down assay and in vitro kinase assay results indicated that eupafolin could bind with PI3-K and attenuate its kinase activity. Eupafolin also exhibited tumor suppressive effects in vivo in an athymic nude mouse model. Overall, these results suggested that eupafolin exerts antitumor effects by targeting PI3-K.

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 Parkinson's 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.