EGCG-induced selective death of cancer cells through autophagy-dependent regulation of the p62-mediated antioxidant survival pathway.

The effects of EGCG on the selective death of cancer cells by modulating antioxidant pathways through autophagy were explored in various normal and cancer cells. EGCG positively regulated the p62-KEAP1-NRF2-HO-1 pathway in normal cells, while negatively regulating it in cancer cells, leading to selective apoptotic death of cancer cells. In EGCG-treated MRC5 cells (EGCG-MRC5), autophagic flux was blocked, which was accompanied by the formation of p62-positive aggregates. However, EGCG-treated HeLa cells (EGCG-HeLa) showed incomplete autophagic flux and no aggregate formation. The levels of P-ULK1 S556 and S758 increased in EGCG-MRC5 through AMPK-mTOR cooperative interaction. In contrast, EGCG treatment in HeLa cells led to AMPK-induced mTOR inactivation, resulting in abrogation of P-ULK1 S556 and S758 levels. AMPK knockout in EGCG-HeLa restored positive regulation of the p62-mediated pathway, which was accompanied by increased P-mTOR S2448 and P-ULK1 S758 levels. Knockdown of 67LR in EGCG-HeLa abolished AMPK activity but did not restore the p62-mediated pathway. Surprisingly, both AMPK knockout and 67LR knockdown in EGCG-HeLa markedly increased cell viability, despite differential regulation of the antioxidant enzyme HO-1. In conclusion, EGCG induces the selective death of cancer cells through the modulation of at least two autophagy-dependent and independent regulatory pathways: negative regulation involves the mTOR-ULK1 (S556 and S758)-p62-KEAP1-NRF2-HO-1 axis via AMPK activation, whereas positive regulation occurs through the 67LR-AMPK axis.

Transport Mechanism for Nuclear Localization of Irradiation-Activated EGFR Measured by Single-Molecule Pull-Down Assay.

Nuclear transport of epidermal growth factor receptor (EGFR) is considered to be a key cause of radiation resistance in cancer therapy. Here, we showed that irradiation-activated EGFR binds to the nuclear transport protein karyopherin alpha (KPNA) rather than karyopherin subunit beta 1 (KPNB1), through a single-molecule pull-down assay, which allows measurement of the binding affinity by single proteins in cell lysate without an additional purification step. We also obtained kinetic parameters for the binding between the phosphorylated nuclear localization signal (NLS) peptide of EGFR (645RRRHIVRKRpTLRR657) and KPNA. This observation may help developing small molecules to modulate nuclear transport, which potentially reduces the radiation resistance during irradiation therapy.

Inhibition of NUPR1-Karyopherin ß1 Binding Increases Anticancer Drug Sensitivity.

BACKGROUND: Nuclear protein-1 (NUPR1, also known as p8/Com-1) is a transcription factor involved in the regulation of cellular stress responses, including serum starvation and drug stimulation. METHODS: We investigated the mechanism of NUPR1 nuclear translocation involving karyopherin ß1 (KPNB1), using a single-molecule binding assay and confocal microscopy. The cellular effects associated with NUPR1-KPNB1 inhibition were investigated by gene expression profiling and cell cycle analysis. RESULTS: The single-molecule binding assay revealed that KPNB1 bound to NUPR1 with a binding affinity of 0.75 nM and that this binding was blocked by the aminothiazole ATZ-502. Following doxorubicin-only treatment, NUPR1 was translocated to the nucleus in more than 90% and NUPR1 translocation was blocked by the ATZ-502 combination treatment in MDA-MB-231 with no change in NUPR1 expression, providing strong evidence that NUPR1 nuclear translocation was directly inhibited by the ATZ-502 treatment. Inhibition of KPNB1 and NUPR1 binding was associated with a synergistic anticancer effect (up to 19.6-fold) in various cancer cell lines. NUPR1-related genes were also downregulated following the doxorubicin-ATZ-502 combination treatment. CONCLUSION: Our current findings clearly demonstrate that NUPR1 translocation into the nucleus requires karyopherin ß1 binding. Inhibition of the KPNB1 and NUPR1 interaction may constitute a new cancer therapeutic approach that can increase the drug efficacy while reducing the side effects.

HPLC and ToF‒SIMS Analyses of Toxicodendron vernicifluum Tree Sap Mixed with Other Natural Lacquers.

Lacquer sap has been used by humans from antiquitywhen it was treated as a luxury item because of its desirable physical properties. In modern times, although access barriers are lower, lacquer is still considered to be rare and valuable. Thus, low quality, inexpensive Vietnamese and Myanmarese lacquers and cashew nutshell liquid are frequently added to the costly Toxicodendron vernicifluum lacquer sap from Korea, China, and Japan. However, these blended lacquers can diminish the quality of artisan works. The Toxicodendron vernicifluum lacquer saps mixed with other natural lacquers were characterized using time-of-flight secondary-ion mass spectrometry (ToF-SIMS) and high-performance liquid chromatography (HPLC). ToF-SIMS provided the chemical structure of the lacquer monomer, copolymerized dimers, trimers, etc. HPLC provided quantitative analysis of the components of a randomly mixed lacquer. These techniques can be used to control the quality of commercial lacquer sap for the Asian lacquer industry and the traditional conservation of ancient objects.

Quantitative Analysis of Blended Asian Lacquers Using ToF-SIMS, Py-GC/MS and HPLC.

Asian lacquer is a special polymeric material tapped from lacquer trees. The tree's sap is a complex mixture of compounds, such as catechol lipids, polysaccharides, glycoproteins, enzymes, and water. Researchers have not yet quantitatively analyzed blended lacquers. We evaluated the compositions of Japanese and Vietnamese lacquers, and blends of the two, using time-of-flight secondary ion mass spectrometry (ToF-SIMS), pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), and high-performance liquid chromatography (HPLC). ToF-SIMS provided quantitative results for blended lacquers; provided structural information on polymeric lacquer films; and indicated the presence of dimers of urushiol-urushiol, urushiol-laccol, and laccol-laccol derivatives. We used Py-GC/MS and HPLC to obtain linear calibration curves. The specific peak intensity was a linear function of the ratio of Japanese to Vietnamese lacquer in the blends. For an unknown mixture, all three techniques gave essentially the same results. These quantitative methods will be useful for improving the physical properties of polymeric lacquer films, and evaluating the lacquer quality in industry and historic conservation.

A Small Organic Molecule Blocks EGFR Transport into the Nucleus by the Nonclassical Pathway Resulting in Repression of Cancer Invasion.

In addition to the traditional epidermal growth factor receptor (EGFR) signaling pathways, nuclear EGFR has been shown to control multiple cellular functions, including cell proliferation and invasion. It has been reported that EGFR is transported into the nucleus after forming a complex with KPNA/KPNB1 or KPNB1. Herein, it is shown that EGFR can interact with both KP and KPNA, but EGF-activated EGFR mostly binds with KPNB1 through the pull-down assay. Also, a small organic molecule (1), an effective binder of KPNB1, inhibits the interaction between EGFR and KPNB1 in the nonclassical transport pathway, but not KPNA. Furthermore, treatment of cancer cells with 1 noticeably blocks the nuclear entry of EGFR, which results in significant suppression of invasion by lung cancer H1299 cells. These findings show that 1 is an effective inhibitor of EGFR/KPNB1 interactions in vitro, it may be used in cellular studies as a tool to determine the role of nuclear EGFR, and it is a drug candidate.

Determination of the binding site of 2-aminothiazole derivative with importin ß1 by UV-crosslinking experiment.

Importin ß1 (KPBN1) appears to be overexpressed in several cancer cells and siRNA-induced inhibition of KPNB1 shows significant inhibition of cancer cell proliferation, but do not affect normal cells. These results indicate that KPNB1 is a potential target and inhibition of KPNB1 can be used as a novel therapeutic approach for the treatment of cancer. Recently, we identified the aminothiazole derivative 1 as a KPNB1-targeted anticancer agent. Herein, we report that compound 1 binds strongly to KPNB1, in a pocket centered around serine-476, as shown by UV-crosslinking and tandem mass spectrometry experiments, and supported using a model derived from molecular docking.

Inhibition of Importin ß1 With a 2-Aminothiazole Derivative Resulted in G2/M Cell-cycle Arrest and Apoptosis.

BACKGROUND: The design and synthesis of novel chemotherapeutic agents that can induce apoptosis and cell-cycle arrest has emerged as an attractive approach for the treatment of cancer, because they can limit possible nonspecific effects of compound treatment. Previous studies established that the expression of KPNB1 was increased in several cancer cells and transformed cell lines and inhibition of KPNB1 using siRNA significantly inhibited cervical tumour proliferation, but did not affect normal cervical epithelium. Recently, we reported that a KPNB1 inhibitor, the 2-aminothiazole derivative 1, possesses strong anti-proliferative effects against several cancer cells in the nanomolar concentration range. RESULTS: Treatment with compound 1 interferes with cell-cycle progression in the G2/M phase, as detected by flow cytometry analysis and results in apoptosis by the intrinsic pathway. Fluorescence microscopic analysis of mitotic cells predominantly mitotic abnormal cells with monopolar spindles and treatment with compound 1 did not affect polymerization of microtubules. CONCLUSION: Compound 1, as a KPNB1 inhibitor, might be a good target for future development of anticancer agents showing the activities of apoptosis and cell cycle arrest.

Identification of KPNB1 as a Cellular Target of Aminothiazole Derivatives with Anticancer Activity.

We found that aminothiazole derivative (E)-N-(5-benzylthiazol-2-yl)-3-(furan-2-yl)acrylamide (1) has strong anticancer activity, and undertook proteomics approaches to identify the target protein of compound 1, importin ß1 (KPNB1). A competitive binding assay using fluorescein-labeled 1 showed that 1 has strong binding affinity for KPNB1 (Kd : ∼20 nm). Furthermore, through western blotting assays for KPNB1, KPNA2, EGFR, ErbB2, and STAT3, we confirmed that 1 has inhibitory effects on the importin pathway. KPBN1 appears to be overexpressed in several cancer cells, and siRNA-induced inhibition of KPNB1 shows significant inhibition of cancer cell proliferation, while leaving non-cancerous cells unaffected. Therefore, compound 1 is a promising new lead for the development of KPNB1-targeted anticancer agents. Fluorescein-labeled 1 could be a useful quantitative probe for the development of novel KPNB1 inhibitors.

Selective death of cancer cells by preferential induction of reactive oxygen species in response to (-)-epigallocatechin-3-gallate.

(-)-Epigallocatechin-3-gallate (EGCG) induces apoptosis in cancer cells without adversely affecting normal cells. Understanding the cancer-specific cytotoxic activity of EGCG is very important in defining the mechanism of tumorigenesis and identifying superb chemotherapeutic agents against cancer. We comparatively assayed human telomerase reverse transcriptase (hTERT)-mediated apoptosis by EGCG-induced reactive oxygen species (ROS) in normal cells and cancer cells. EGCG showed differential levels of ROS induction between the cell types; ROS, especially hydrogen peroxide, was highly induced in cancer cells, while it was not in normal cells. In addition, the higher level of ROS down-regulated hTERT via binding of CCCTC binding factor (CTCF) to the core promoter region of hTERT, which repressed hTERT expression. CTCF binding was epigenetically controlled by the demethylation of the previously hypermethylated site for CTCF, which was induced by down-regulation of DNA methyltransferase 1 (DNMT1). In contrast, hTERT down-regulation was not observed in normal cells. These results suggest that preferential death of cancer cells by EGCG could be caused by the cancer-specific induction of ROS and epigenetic modulation of expression of apoptosis-related genes, such as hTERT.

Novel thiazolidinedione derivatives with anti-obesity effects: dual action as PTP1B inhibitors and PPAR-γ activators.

Benzylidene-2,4-thiazolidinedione derivatives with substitutions at both the ortho and para-positions of the phenyl group were synthesized as PTP1B inhibitors with IC(50) values in a low micromolar range. Compound 18l, the lowest, bore an IC(50) of 1.3 µM. In a peroxisome proliferator-activated receptor-γ (PPAR-γ) promoter reporter gene assay, 18l was found to activate the transcription of the reporter gene with potencies comparable to those of troglitazone, rosiglitazone, and pioglitazone. In vivo efficacy of 18l as an anti-obesity and hypoglycemic agent was evaluated in a mouse model system. Compound 18l significantly suppressed weight gain and significantly improved blood parameters such as TG, total cholesterol and NEFA without overt toxic effects.

A novel organogermanium protected atopic dermatitis induced by oxazolone.

Atopic dermatitis (AD) is a chronic inflammatory disease of the skin that is often associated with other atopic diseases, such as asthma and allergic rhinitis. Although topical steroids have widely been prescribed for patients with AD, skin abnormalities are frequently observed after prolonged steroid treatment. In this study, a novel water-soluble organogermanium compound (Ge-Vit) was prepared because organogermanium is a known INF-gamma inducer. The Ge-Vit treatment decreased the basal TEWL and IgE production and attenuated the disruption of the skin barrier function in a murine model of chronic contact dermatitis. The histological examination further supported the anti-AD activities. These results suggested that Ge-Vit can be a useful drug candidate for treating atopic dermatitis.

Synthesis and biological evaluation of water-soluble organogermanium.

Interferon (INF) is an effective drug in treating several human diseases. Ge-132, which is the most common and well-studied organic germanium, has been reported to induce INF-gamma and has undergone clinical trials with promising preclinical results. However, it has been reported that long-term ingestion or high doses of organic Ge-132 causes similar toxic effects as GeO(2) because Ge-132 can be easily contaminated with significant amounts of inorganic germanium during the preparation. In this study, we synthesized the water-soluble organogermanium compound (Ge-OH) without possible contamination with toxic inorganic germanium and showed that Ge-OH is a better INF-gamma inducer than Ge-132 by an animal study.

TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116.

Trichostatin A (TSA), an inhibitor of histone deacetylase, is a well-known antitumor agent that effectively and selectively induces tumor growth arrest and apoptosis. Recently, it was reported that hTERT is one of the primary targets for TSA-induced apoptosis in cancer cells but the mechanism of which has not yet been elucidated. In the present study, to better understand the epigenetic regulation mechanism responsible for the repression of hTERT by TSA, we examined expression of hTERT in the HCT116 colon cancer cell line after treatment with TSA and performed site-specific CpG methylation analysis of the hTERT promoter. We found that TSA-induced the demethylation of site-specific CpGs on the promoter of hTERT, which was caused by down-regulation of DNA methyltransferase 1 (DNMT1). Among the demethylated region, the 31st-33rd CpGs contained a binding site for CTCF, an inhibitor of hTERT transcription. ChIP analysis revealed that TSA-induced demethylation of the 31st-33rd CpGs promoted CTCF binding on hTERT promoter, leading to repression of hTERT. Taken together, down-regulation of DNMT1 by TSA caused demethylation of a CTCF binding site on the hTERT promoter, the result of which was repression of hTERT via recruitment of CTCF to the promoter.

Thiazolidinedione derivatives as PTP1B inhibitors with antihyperglycemic and antiobesity effects.

Benzylidene-2,4-thiazolidinedione derivatives with substitutions on the phenyl ring at the ortho or para positions of the thiazolidinedione (TZD) group were synthesized as PTP1B inhibitors with IC50 values in a low micromolar range. Compound 3e, the lowest, bore an IC50 of 5.0 microM. In vivo efficacy of 3e as an antiobesity and hypoglycemic agent was evaluated in a mouse model system. Significant improvement of glucose tolerance was observed. This compound also significantly suppressed weight gain and significantly improved blood parameters such as TG, total cholesterol and NEFA. Compound 3e was also found to activate peroxisome proliferator-activated receptors (PPARs) indicating multiple mechanisms of action.

Structure-based virtual screening approach to identify novel classes of Cdc25B phosphatase inhibitors.

Discovery of Cdc25B phosphatase inhibitors has been actively pursued with the aim to develop anticancer agents. We have been able to identify eight novel Cdc25B inhibitors by means of a computer-aided drug design protocol involving the virtual screening with docking simulations under consideration of the effects of ligand solvation in the binding free energy function. Structural features relevant to the interactions of the newly identified inhibitors with the active-site residues of Cdc25B are also discussed in detail.

Structure-based virtual screening approach to identify novel classes of PTP1B inhibitors.

Discovery of protein tyrosine phosphatase 1B (PTP1B) inhibitors has been actively pursued with the aim to develop therapeutics for the treatment of type 2 diabetes and obesity. We have been able to identify 9 novel PTP1B inhibitors by means of a computer-aided drug design protocol involving virtual screening with docking simulations under consideration of the effects of ligand solvation in the binding free energy function. Because the newly discovered inhibitors are structurally diverse and reveal a significant potency with IC(50) values lower than 50 microM, all of them can be considered for further development by structure-activity relationship studies. Structural features relevant to the interactions of the newly identified inhibitors with the active-site residues of PTP1B are discussed in detail.

2-O-carboxymethylpyrogallol derivatives as PTP1B inhibitors with antihyperglycemic activity.

2-O-carboxymethylpyrogallol derivatives (4-17) were synthesized, with their in vitro inhibitory activities against PTP1B and in vivo antihyperglycemic effects examined. Compound 14, the most potent among the series, showed a K(i) value of 1.1 microM against PTP1B, 7-fold lower than that against TC-PTP. When compound 14 was fed to a high-fat diet-induced diabetic mouse model, significant improvements were observed in both the fasting glucose level and glucose tolerance.

Fluorinated NSC as a Cdc25 inhibitor.

We report on the fluorinated form of NSC 95397 as a Cdc25B inhibitor, which is predicted to be only an arylator of cysteine-containing proteins, without generating reactive oxygen species.

H32, a non-quinone sulfone analog of vitamin K3, inhibits human hepatoma cell growth by inhibiting Cdc25 and activating ERK.

We previously synthesized a K-vitamin derivative, Cpd 5, which was a potent growth inhibitor of human tumor cells, including Hep3B hepatoma cells. However, being a quinone compound, Cpd 5 has the potential for generating toxic reactive oxygen species (ROS). We therefore synthesized a nonquinone sulfone derivative, H32, which has a sufone group substituting the quinone. The IC50 of H32 for Hep3B cells was found to be 2.5 microM, which was 2.5 and 3.2 times more potent than Cpd 5 and vitamin K3 respectively. It induced apoptosis in Hep3B cells but did not generate ROS when compared to Cpd 5. Interestingly, under similar culture conditions, normal rat hepatocytes were 14-fold more and 7-fold more resistant to the growth inhibitory effects of H32 than Hep3B and PLC/PRF5 cells respectively. H32 preferentially inhibited the activities of the cell cycle controlling Cdc25A phosphatase likely by binding to its catalytic cysteine. As a consequence, it induced inhibitory tyrosine phosphorylation of the Cdc25 substrate kinases Cdk2 and Cdk4 in Hep3B cells and the cells undergo an arrest in the G1 phase of the cell cycle. H32 also induced persistent phosphorylation of the MAPK protein ERK1/2, but marginal JNK1/2 and p38 phosphorylation. The ERK inhibitor U0126, added at least 30 min prior to H32, antagonized the growth inhibition induced by H32. However, the JNK and p38 inhibitors, JNKI-II and SB203580, were not able to antagonize H32 induced growth inhibition. Thus, H32 differentially inhibited growth of normal and liver tumor cells by preferentially inhibiting the actions of Cdc25 phosphatases and inducing persistent ERK phosphorylation.