Dual modulation of JNK and Akt signaling pathways by chaetoglobosin K in human lung carcinoma and ras-transformed epithelial cells.

Chaetoglobosin K (ChK) is a natural product that inhibits anchorage-dependent and anchorage-independent growth of ras-transformed cells, prevents tumor-promoter disruption of cell-cell communication, and reduces Akt activation in tumorigenic cells. This study demonstrates how ChK modulates the JNK pathway in ras-transformed and human lung carcinoma cells and investigates regulatory mechanisms controlling ChK's effect on the Akt and JNK signaling pathways. Human lung carcinoma and ras-transformed epithelial cell lines treated with ChK or vehicle for varying times were assayed for cell growth or extracted for total proteins for western blot analysis using phosphorylation site-specific antibodies to monitor changes in activation of JNK, Akt, and other signaling enzymes. Results show that ChK inhibited both Akt and JNK phosphorylation at key activation sites in ras-transformed cells as well as human lung carcinoma cells. Downstream effectors of both kinases were accordingly affected. Direct upstream kinases of JNK were not affected by ChK. Wortmannin and LY294002, two PI3 kinase inhibitors, inhibited Akt but not JNK phosphorylation in ras-transformed cells. This report establishes the dual inhibitory effect of ChK on both the Akt and JNK signaling pathways in ras-transformed epithelial and human carcinoma cells. The unique effect of ChK on these two key pathways involved in carcinogenesis earmarks ChK for further studies to determine its molecular target(s) and in vivo anti-tumor potential.

A Cell-Cell Communication Marker for Identifying Targeted Tumor Therapies.

Cell-cell communication through gap junctions is aberrant or absent in a majority of human cancer cells, compared to cells in corresponding normal tissues. This and other evidence has led to the hypothesis that gap junction channels, comprised of connexin proteins, are important in growth control and cancer progression. The major goal of this ongoing study was to identify bioactive compounds that specifically upregulate gap junction channel-mediated cell-cell communication as potential anti-tumor therapies. Control of cell-cell communication is linked to growth regulatory intracellular signaling pathways; we therefore further aimed to identify signaling pathways modulated by these compounds in order to assess their potential as targeted anti-tumor therapies. Compounds were screened for their ability to upregulate gap junction-mediated cell-cell communication by using a fluorescent dye transfer assay to measure cell-cell communication between tumor promoter-treated astroglial cells or ras-transformed epithelial cells. Western blotting using connexin-specific and phosphorylation site-specific antibodies was used to monitor phosphorylation changes in signaling pathway proteins. Our results identified three compounds that upregulate gap junction-mediated cell-cell communication in our screening assays, chaetoglobosin K(ChK), 4-phenyl-3-butenoic acid (PBA) and the methyl ester of PBA (PBA-Me). Further analyses demonstrated that in tumorigenic cells, ChK downregulates phosphorylation of Akt kinase, an enzyme in the PI3-kinase signaling pathway that is found to be upregulated in a number of human cancers, on a key activation site. However, ChK did not inhibit PI-3 kinase in vitro as did the classic PI-3 kinase inhibitor, Wortmannin. PBA and PBA-Me were found to upregulate phosphorylation of p38 MAPK on a key activation site in tumorigenic cells, which is downregulated in several human cancer cell types. ChK and PBA also decreased activation of SAPK/JNK, another kinase found to be upregulated in a number of human cancers. These studies highlight the potential of monitoring gap junction intercellular communication for identifying experimental anti-tumor compounds.

p38 MAPK activation, JNK inhibition, neoplastic growth inhibition, and increased gap junction communication in human lung carcinoma and Ras-transformed cells by 4-phenyl-3-butenoic acid.

Human lung neoplasms frequently express mutations that down-regulate expression of various tumor suppressor molecules, including mitogen-activated protein kinases such as p38 MAPK. Conversely, activation of p38 MAPK in tumor cells results in cancer cell cycle inhibition or apoptosis initiated by chemotherapeutic agents such as retinoids or cisplatin, and is therefore an attractive approach for experimental anti-tumor therapies. We now report that 4-phenyl-3-butenoic acid (PBA), an experimental compound that reverses the transformed phenotype at non-cytotoxic concentrations, activates p38 MAPK in tumorigenic cells at concentrations and treatment times that correlate with decreased cell growth and increased cell-cell communication. H2009 human lung carcinoma cells and ras-transformed rat liver epithelial cells treated with PBA showed increased activation of p38 MAPK and its downstream effectors which occurred after 4 h and lasted beyond 48 h. Untransformed plasmid control cells showed low activation of p38 MAPK compared to ras-transformed and H2009 carcinoma cells, which correlates with the reduced effect of PBA on untransformed cell growth. The p38 MAPK inhibitor, SB203580, negated PBA's activation of p38 MAPK downstream effectors. PBA also increased cell-cell communication and connexin 43 phosphorylation in ras-transformed cells, which were prevented by SB203580. In addition, PBA decreased activation of JNK, which is upregulated in many cancers. Taken together, these results suggest that PBA exerts its growth regulatory effect in tumorigenic cells by concomitant up-regulation of p38 MAPK activity, altered connexin 43 expression, and down-regulation of JNK activity. PBA may therefore be an effective therapeutic agent in human cancers that exhibit down-regulated p38 MAPK activity and/or activated JNK and altered cell-cell communication.

Protective effect of the natural product, chaetoglobosin K, on lindane- and dieldrin-induced changes in astroglia: identification of activated signaling pathways.

PURPOSE: The purpose of the present study was to identify the biochemical mechanism(s) of the preventative and reversal effects of Chaetoglobosin K (ChK), a bioactive natural product, on inhibition of gap junction-mediated communication and connexin phosphorylation by the tumor promoting organochlorine compounds, lindane, and dieldrin. MATERIALS AND METHODS: A fluorescent dye transfer assay was used to quantify gap junction-mediated communication and sensitivity to lindane and dieldrin. Analyses of connexin 43, PKC, ERK, GSK-3beta, Raf, and Akt kinase phosphorylation were performed by Western blotting. RESULTS: Pre-incubation of astroglial cells with 10 microM ChK prevented inhibition of dye transfer by lindane and dieldrin, which correlates with stabilization of the connexin 43 P2 isoform, and addition of ChK after lindane or dieldrin reversed the inhibitory effect, which correlated with re-appearance of the P2 isoform. Using phosphorylation site-specific antibodies, we demonstrated that lindane, dieldrin, and ChK all activated p44/42 ERK, but only ChK activated Akt kinase. ChK also activated a downstream effector of Akt, GSK-3beta, and activation of both kinases was inhibited by Wortmannin. Wortmannin also blocked ChK's ability to prevent dieldrin-induced inhibition of gap junction-mediated communication between RG-2 cells. CONCLUSION: ChK's protective effects, both preventative and reversal, on lindane and dieldrin inhibition of gap junction-mediated communication are associated with stabilization and reappearance of the connexin 43 P2 phosphoform and may be mediated by the Akt pathway.