It has been discovered that proteasome inhibitors are able to induce tumor growth arrest or cell death and that tea consumption is correlated with cancer prevention. Here, we show that ester bond-containing tea polyphenols, such as (؊)؊epigallocatechin-3-gallate (EGCG), potently and specifically inhibit the chymotrypsin-like activity of the proteasome in vitro (IC 50 ‫؍‬ 86 -194 nM) and in vivo (1-10 M) at the concentrations found in the serum of green tea drinkers. Atomic orbital energy analyses and high performance liquid chromatography suggest that the carbon of the polyphenol ester bond is essential for targeting, thereby inhibiting the proteasome in cancer cells. This inhibition of the proteasome by EGCG in several tumor and transformed cell lines results in the accumulation of two natural proteasome substrates, p27Kip1 and IB-␣, an inhibitor of transcription factor NF-B, followed by growth arrest in the G 1 phase of the cell cycle. Furthermore, compared with their simian virus-transformed counterpart, the parental normal human fibroblasts were much more resistant to EGCG-induced p27Kip1 protein accumulation and G 1 arrest. Our study suggests that the proteasome is a cancer-related molecular target of tea polyphenols and that inhibition of the proteasome activity by ester bond-containing polyphenols may contribute to the cancer-preventative effect of tea.

Purpose: Signal transducer and activator of transcription 3 (Stat3) protein is persistently activated in breast cancer and promotes tumor cell survival. To gain a better understanding of the role of constitutive Stat3 signaling in breast cancer progression, we evaluated the expression profile of potential Stat3-regulated genes that may confer resistance to apoptosis. Experimental Design: Stat3 signaling was blocked with antisense oligonucleotides in human MDA-MB-435s breast cancer cells and Affymetrix GeneChip microarray analysis was done. The candidate Stat3 target gene Survivin was further evaluated in molecular assays using cultured breast cancer cells and immunohistochemistry of breast tumor specimens. Results: Survivin, a member of the inhibitor of apoptosis protein family, was identified as a potential Stat3-regulated gene by microarray analysis. This was confirmed in Survivin gene promoter studies and chromatin immunoprecipitation assays showing that Stat3 directly binds to and regulates the Survivin promoter. Furthermore, direct inhibition of Stat3 signaling blocked the expression of Survivin protein and induced apoptosis in breast cancer cells. Direct inhibition of Survivin expression also induced apoptosis. Increased Survivin protein expression correlates significantly (P = 0.001) with elevated Stat3 activity in primary breast tumor specimens from high-risk patients who were resistant to chemotherapy treatment. Conclusions: We identify Survivin as a direct downstream target gene of Stat3 in human breast cancer cells that is critical for their survival in culture. Our findings suggest that activated Stat3 signaling contributes to breast cancer progression and resistance to chemotherapy by, at least in part, inducing expression of the antiapoptotic protein, Survivin.

Src family kinases (SFK) are currently being investigated as targets for treatment strategies in various cancers. The novel SFK/Abl inhibitor, dasatinib (BMS-354825), is a promising therapeutic agent with oral bioavailability. Dasatinib has been shown to inhibit growth of Bcr-Abl-dependent chronic myeloid leukemia xenografts in nude mice. Dasatinib also has been shown to have activity against cultured human prostate and breast cancer cells. However, the molecular mechanism by which dasatinib acts on epithelial tumor cells remains unknown. In this study, we show that dasatinib blocks the kinase activities of the SFKs, Lyn, and Src, in human prostate cancer cells at low nanomolar concentrations. Moreover, focal adhesion kinase and Crk-associated substrate (p130(CAS)) signaling downstream of SFKs are also inhibited at similar concentrations of dasatinib. Consistent with inhibition of these signaling pathways, dasatinib suppresses cell adhesion, migration, and invasion of prostate cancer cells at low nanomolar concentrations. Therefore, dasatinib has potential as a therapeutic agent for metastatic prostate cancers harboring activated SFK and focal adhesion kinase signaling.

Stat3 protein has an important role in oncogenesis and is a promising anticancer target. Indirubin, the active component of a traditional Chinese herbal medicine, has been shown previously to inhibit cyclin-dependent kinases, resulting in cell cycle arrest. Here, we show that the indirubin derivatives E564, E728, and E804 potently block constitutive Stat3 signaling in human breast and prostate cancer cells. In addition, E804 directly inhibits Src kinase activity (IC50 ‫؍‬ 0.43 M) in an in vitro kinase assay. Levels of tyrosyl phosphorylation of c-Src are also reduced in cultured cells 30 min after E804 treatment. Tyrosyl phosphorylation of Stat3, which is known to be phosphorylated by c-Src, was decreased, and constitutive Stat3 DNA binding-activity was suppressed in cells 30 min after E804 treatment. The antiapoptotic proteins Mcl-1 and Survivin, which are encoded in target genes of Stat3, were downregulated by indirubin derivatives, followed by induction of apoptosis. These results demonstrate that E804 directly blocks the Src-Stat3 signaling pathway, suggesting that the antitumor activity of indirubin compounds is at least partially due to inhibition of this pathway.Src ͉ signal transducer and activator of transcription 3

No abstract

Methods for the practical, intermolecular functionalization of aliphatic C–H bonds remain a paramount goal of organic synthesis. Free radical alkane chlorination is an important industrial process for the production of small molecule chloroalkanes from simple hydrocarbons, yet applications to fine chemical synthesis are rare. Herein, we report a site-selective chlorination of aliphatic C–H bonds using readily available N-chloroamides, and apply this transformation to a synthesis of chlorolissoclimide, a potently cytotoxic labdane diterpenoid. These reactions deliver alkyl chlorides in useful chemical yields with substrate as the limiting reagent. Notably, this approach tolerates substrate unsaturation that poses major challenges in chemoselective, aliphatic C–H functionalization. The sterically- and electronically-dictated site selectivities of the C–H chlorination are among the most selective alkane functionalizations known, providing a unique tool for chemical synthesis. The short synthesis of chlorolissoclimide features a high yielding, gram-scale radical C–H chlorination of sclareolide and a three-step/two-pot process for the introduction of the β-hydroxysuccinimide that is salient to all the lissoclimides and haterumaimides. Preliminary assays indicate that chlorolissoclimide and analogues are moderately active against aggressive melanoma and prostate cancer cell lines.

As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for anti-angiogenesis treatment. However, the side effects associated with most anti-VEGF agents limit their chronic use. Identification of naturally occurring VEGF inhibitors derived from diet is a potential alternative approach, with the advantage of known safety. To isolate natural inhibitors of VEGF, we established an in vitro tyrosine kinase assay to screen for diet-based agents that suppress VEGFR2 kinase activity. We found that a water-based extract from cinnamon (cinnamon extract, CE), one of the oldest and most popular spices, was a potent inhibitor of VEGFR2 kinase activity, directly inhibiting kinase activity of purified VEGFR2 as well as mitogen-activated protein kinase- and Stat3-mediated signaling pathway in endothelial cells. As a result, CE inhibited VEGF-induced endothelial cell proliferation, migration and tube formation in vitro, sprout formation from aortic ring ex vivo and tumor-induced blood vessel formation in vivo. Depletion of polyphenol from CE with polyvinylpyrrolidone abolished its anti-angiogenesis activity. While cinnamaldehyde, a component responsible for CE aroma, had little effect on VEGFR2 kinase activity, high-performance liquid chromatography-purified components of CE, procyanidin type A trimer (molecular weight, 864) and a tetramer (molecular weight, 1152) were found to inhibit kinase activity of purified VEGFR2 and VEGFR2 signaling, implicating procyanidin oligomers as active components in CE that inhibit angiogenesis. Our data revealed a novel activity in cinnamon and identified a natural VEGF inhibitor that could potentially be useful in cancer prevention and/or treatment.

Signal Transducer and Activator of Transcription 3 (STAT3) is persistently activated and contributes to malignant progression in various cancers. Janus kinases (JAKs) phosphorylate STAT3 in response to stimulation with cytokines or growth factors. The STAT3 signaling pathway has been validated as a promising target for development of anti-cancer therapeutics. Small-molecule inhibitors of JAK/STAT3 signaling represent potential molecular-targeted cancer therapeutic agents. In this study, we investigated the role of JAK/STAT3 signaling in 6-bromoindirubin-3'-oxime (6BIO) mediated growth inhibition of human melanoma cells and assessed 6BIO as an anticancer drug candidate. We found that 6BIO is a pan-JAK inhibitor that induced apoptosis of human melanoma cells. 6BIO directly inhibited JAK family kinase activity both in vitro and in cancer cells. Apoptosis of human melanoma cells induced by 6BIO was associated with reduced phosphorylation of JAKs and STAT3 in both a dose- and time-dependent manners. Consistent with inhibition of STAT3 signaling, the anti-apoptotic protein Mcl-1 was down-regulated. In contrast to the decreased levels of phosphorylation of JAKs and STAT3, phosphorylation levels of the AKT and MAPK signaling proteins were not inhibited in cells treated with 6BIO. Importantly, 6BIO suppressed tumor growth in vivo with low toxicity in a mouse xenograft model of melanoma. Taken together, these results demonstrate that 6BIO is a novel pan-JAK inhibitor that can selectively inhibit STAT3 signaling and induced tumor cell apoptosis. Our findings support further development of 6BIO as a potential anti-cancer therapeutic agent that targets JAK/STAT3 signaling in tumor cells.