N. R. Thimmegowda

Patulin induces colorectal cancer cells apoptosis through EGR-1 dependent ATF3 up-regulation

Patulin is a fungal mycotoxin of Aspergilus and Penicillium that is commonly found in rotting fruits and exerts its potential toxic effect mainly by reactive oxygen species (ROS) generation. However, the effect of patulin on cancer cells as well as its intracellular mechanism has been controversial and not clearly defined yet. In this study, patulin was found to induce G1/S accumulation and cell growth arrest accompanied by caspase-3 activation, PARP cleavage and ATF3 expression in human colon cancer cell line HCT116. Ser/Thr phosphorylation of a transcription factor, EGR-1, was increased while its expression did not change upon patulin treatment to the cells. Knockdown of ATF3 and EGR-1 using their respective siRNAs showed EGR-1 dependent ATF3 expression. Moreover, treatment of the cells with antioxidants N-acetylcysteine (NAC) and glutathione (GSH) revealed that patulin induced ATF3 expression and apoptosis were dependent on ROS generation. ATF3 expression was also increased by patulin in other colorectal cancer cell types, Caco2 and SW620. Collectively, our data present a new anti-cancer molecular mechanism of patulin, suggesting EGR-1 and ATF3 as critical targets for the development of anti-cancer chemotherapeutics. In this regard, patulin could be a candidate for the treatment of colorectal cancers.

A Derivative of Chrysin Suppresses Two-Stage Skin Carcinogenesis by Inhibiting Mitogen- and Stress-Activated Kinase 1

Mitogen- and stress-activated kinase 1 (MSK1) is a nuclear serine/threonine protein kinase that acts downstream of both extracellular signal-regulated kinases and p38 mitogen-activated protein kinase in response to stress or mitogenic extracellular stimuli. Increasing evidence has shown that MSK1 is closely associated with malignant transformation and cancer development. MSK1 should be an effective target for cancer chemoprevention and chemotherapy. However, very few MSK1 inhibitors, especially natural compounds, have been reported. We used virtual screening of a natural products database and the active conformation of the C-terminal kinase domain of MSK1 (PDB id 3KN) as the receptor structure to identify chrysin and its derivative, compound 69407, as inhibitors of MSK1. Compared with chrysin, compound 69407 more strongly inhibited proliferation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ cells with lower cytotoxicity. Western blot data demonstrated that compound 69407 suppressed phosphorylation of the MSK1 downstream effector histone H3 in intact cells. Knocking down the expression of MSK1 effectively reduced the sensitivity of JB6 P+ cells to compound 69407. Moreover, topical treatment with compound 69407 before TPA application significantly reduced papilloma development in terms of number and size in a two-stage mouse skin carcinogenesis model. The reduction in papilloma development was accompanied by the inhibition of histone H3 phosphorylation at Ser10 in tumors extracted from mouse skin. The results indicated that compound 69407 exerts inhibitory effects on skin tumorigenesis by directly binding with MSK1 and attenuates the MSK1/histone H3 signaling pathway, which makes it an ideal chemopreventive agent against skin cancer. Cancer Prev Res; 7(1); 74–85. ©2013 AACR.

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.

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.

STK295900, a Dual Inhibitor of Topoisomerase 1 and 2, Induces G2 Arrest in the Absence of DNA Damage

STK295900, a small synthetic molecule belonging to a class of symmetric bibenzimidazoles, exhibits antiproliferative activity against various human cancer cell lines from different origins. Examining the effect of STK295900 in HeLa cells indicates that it induces G2 phase arrest without invoking DNA damage. Further analysis shows that STK295900 inhibits DNA relaxation that is mediated by topoisomerase 1 (Top 1) and topoisomerase 2 (Top 2) in vitro. In addition, STK295900 also exhibits protective effect against DNA damage induced by camptothecin. However, STK295900 does not affect etoposide-induced DNA damage. Moreover, STK295900 preferentially exerts cytotoxic effect on cancer cell lines while camptothecin, etoposide, and Hoechst 33342 affected both cancer and normal cells. Therefore, STK295900 has a potential to be developed as an anticancer chemotherapeutic agent.

Synthesis and antitumor activity of natural compound aloe emodin derivatives.

In this study, we have synthesized novel water soluble derivatives of natural compound aloe emodin 4(a–j) by coupling with various amino acid esters and substituted aromatic amines, in an attempt to improve the anticancer activity and to explore the structure–activity relationships. The structures of the compounds were determined by 1H NMR and mass spectroscopy. Cell growth inhibition assays revealed that the aloe emodin derivatives 4d, 4f, and 4i effectively decreased the growth of HepG2 (human liver cancer cells) and NCI‐H460 (human lung cancer cells) and some of the derivatives exhibited comparable antitumor activity against HeLa (Human epithelial carcinoma cells) and PC3 (prostate cancer cells) cell lines compared to that of the parent aloe emodin at low micromolar concentrations.

Anticancer activity of a novel small molecule tubulin inhibitor STK899704

We have identified the small molecule STK899704 as a structurally novel tubulin inhibitor. STK899704 suppressed the proliferation of cancer cell lines from various origins with IC50 values ranging from 0.2 to 1.0 μM. STK899704 prevented the polymerization of purified tubulin in vitro and also depolymerized microtubule in cultured cells leading to mitotic arrest, associated with increased Cdc25C phosphorylation and the accumulation of both cyclin B1 and polo-like kinase 1 (Plk1), and apoptosis. Unlike many anticancer drugs such as Taxol and doxorubicin, STK899704 effectively displayed antiproliferative activity against multidrug-resistant cancer cell lines. The proposed binding mode of STK899704 is at the interface between αβ-tubulin heterodimer overlapping with the colchicine-binding site. Our in vivo carcinogenesis model further showed that STK 899704 is potent in both the prevention and regression of tumors, remarkably reducing the number and volume of skin tumor by STK899704 treatment. Moreover, it was significant to note that the efficacy of STK899704 was surprisingly comparable to 5-fluorouracil, a widely used anticancer therapeutic. Thus, our results demonstrate the potential of STK899704 to be developed as an anticancer chemotherapeutic and an alternative candidate for existing therapies.

Hirsutenone in Alnus extract inhibits akt activity and suppresses prostate cancer cell proliferation.

Although specific compounds found in some East Asian traditional medicines have been shown to exhibit bioactive properties, their molecular mechanisms of action remain elusive. The bark of the Alnus species has been used for the treatment of various pathological conditions including hemorrhage, alcoholism, fever, diarrhea, skin diseases, inflammation, and cancer in East Asia for centuries. In this study, we show that hirsutenone, a bioactive compound in Alnus japonica, exhibits anti‐cancer effects against prostate cancer through a direct physical inhibition of Akt1/2. Hirsutenone suppressed anchorage‐dependent and independent cell growth of PC3 and LNCaP human prostate cancer cells. Annexin V and Propidium iodide (PI) staining results demonstrated that hirsutenone strongly induces apoptotic cell death in both PC3 and LNCaP cells. Furthermore, treatment of hirsutenone attenuated phosphorylation of mammalian target of rapamycin (mTOR), a downstream substrate of Akt, without affecting Akt phosphorylation. Kinase and pull‐down assay results clearly show that hirsutenone inhibits Akt1 and 2 by direct binding in an adenosine triphosphate (ATP)‐noncompetitive manner in vitro and ex vivo. Our results show that hirsutenone suppresses human prostate cancer by targeting Akt1 and 2 as a key component to explain for anti‐cancer activity of Alnus species.

Hirsutenone Directly Targets PI3K and ERK to Inhibit Adipogenesis in 3T3-L1 Preadipocytes

Adipogenesis is a key driver of the expansion of adipose tissue mass that causes obesity. Hirsutenone (HST) is an active botanical diarylheptanoid present in Alnus species. In this study, we evaluated the effects of HST on adipogenesis, its mechanisms of action and the molecular targets involved. Using Oil Red O staining, we observed that HST dose‐dependently suppresses lipid accumulation during adipogenesis in 3T3‐L1 preadipocytes, concomitant with a decrease in peroxisome proliferator‐activated receptor‐γ (PPARγ), CCAAT/enhancer‐binding protein α (C/EBPα) and fatty acid synthase (FAS) protein expression. This inhibitory effect was largely limited to the early stage of adipogenesis, which includes mitotic clonal expansion (MCE), as evidenced by delayed cell cycle entry of preadipocytes from G1 to S phase. Furthermore, the regulation of MCE was accompanied by suppression of phosphatidylinositol 3‐kinase (PI3K) and extracellular‐regulated kinase (ERK) activity. HST was also shown to bind directly to PI3K and ERK1 in a non‐ATP competitive manner. Our results suggest that HST attenuates adipogenesis by directly targeting PI3K and ERK during MCE in 3T3‐L1 preadipocytes, underscoring the potential therapeutic application of HST in preventing obesity. J. Cell. Biochem. 116: 1361–1370, 2015.

Identification of a Dual Inhibitor of Janus Kinase 2 (JAK2) and p70 Ribosomal S6 Kinase1 (S6K1) Pathways

Background: An underlying cause of cancer therapeutic resistance is the hyperactivation of endogenous overlapping or redundant signaling pathways in cancer cells. Results: Gingerenone A selectively kills cancer cells through dual inhibition of JAK2 and S6K1. Conclusion: Co-targeting JAK2 and S6K1 induces synergistic anti-cancer effects. Significance: Investigating the targets of bioactive compounds can lead to suggestions for novel therapeutic strategies. Bioactive phytochemicals can suppress the growth of malignant cells, and investigation of the mechanisms responsible can assist in the identification of novel therapeutic strategies for cancer therapy. Ginger has been reported to exhibit potent anti-cancer effects, although previous reports have often focused on a narrow range of specific compounds. Through a direct comparison of various ginger compounds, we determined that gingerenone A selectively kills cancer cells while exhibiting minimal toxicity toward normal cells. Kinase array screening revealed JAK2 and S6K1 as the molecular targets primarily responsible for gingerenone A-induced cancer cell death. The effect of gingerenone A was strongly associated with relative phosphorylation levels of JAK2 and S6K1, and administration of gingerenone A significantly suppressed tumor growth in vivo. More importantly, the combined inhibition of JAK2 and S6K1 by commercial inhibitors selectively induced apoptosis in cancer cells, whereas treatment with either agent alone did not. These findings provide rationale for dual targeting of JAK2 and S6K1 in cancer for a combinatorial therapeutic approach.