(-)-Epigallocatechin gallate downregulates EGF receptor via phosphorylation at Ser1046/1047 by p38 MAPK in colon cancer cells.

We previously reported that (-)-epigallocatechin gallate (EGCG) in green tea alters plasma membrane organization and causes internalization of epidermal growth factor receptor (EGFR), resulting in the suppression of colon cancer cell growth. In the present study, we investigated the detailed mechanism underlying EGCG-induced downregulation of EGFR in SW480 colon cancer cells. Prolonged exposure to EGCG caused EGFR degradation. However, EGCG required neither an ubiquitin ligase (c-Cbl) binding to EGFR nor a phosphorylation of EGFR at tyrosine residues, both of which are reportedly necessary for EGFR degradation induced by epidermal growth factor. In addition, EGCG induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), a stress-inducible kinase believed to negatively regulate tumorigenesis, and the inhibition of p38 MAPK by SB203580, a specific p38 MAPK inhibitor, or the gene silencing using p38 MAPK-small interfering RNA (siRNA) suppressed the internalization and subsequent degradation of EGFR induced by EGCG. EGFR underwent a gel mobility shift upon treatment with EGCG and this was canceled by SB203580, indicating that EGCG causes EGFR phosphorylation via p38 MAPK. Moreover, EGCG caused phosphorylation of EGFR at Ser1046/1047, a site that is critical for its downregulation and this was also suppressed by SB203580 or siRNA of p38 MAPK. Taken together, our results strongly suggest that phosphorylation of EGFR at serine 1046/1047 via activation of p38 MAPK plays a pivotal role in EGCG-induced downregulation of EGFR in colon cancer cells.

HINT1 inhibits beta-catenin/TCF4, USF2 and NFkappaB activity in human hepatoma cells.

In this study we explored the relevance of Hint, a novel tumor suppressor gene, to human hepatoma. The human hepatoma cell lines Hep3B and HepG2 express very low levels of the HINT1 protein but the Huh7 cells express a relatively high level. In Hep3B and HepG2 cells, but not in Huh7 cells, the promoter region of Hint1 is partially methylated and treatment with 5-azadcdeoxycytidine increased expression of the HINT1 protein and Hint1 mRNA in Hep3B and HepG2 cells. Increased expression of HINT1 in HepG2 cells markedly inhibited their growth. It also inhibited the transcriptional activities of beta-catenin/TCF4, and USF2, and inhibited the expression of endogenous cyclin D1 and TGFbeta2. Furthermore, HINT1 co-immunoprecipitated with USF2 in extracts of Hep2 cells. HINT1 also inhibited NFkappaB transcription factor reporter activity and inhibited translocation of the endogenous p65 protein to the nucleus of HepG2 cells. Therefore, decreased expression of the Hint1 gene through epigenetic silencing may play a role in enhancing the growth of a subset of human hepatoma by increasing the expression of genes controlled by the transcription factors beta-catenin, USF2, and NFkappaB.

Hint1 inhibits growth and activator protein-1 activity in human colon cancer cells.

There is accumulating evidence that histidine triad (HIT) nucleotide-binding protein 1 (HINT1), a member of the evolutionary highly conserved HIT protein super family, is a novel tumor suppressor. However, the mechanism of action of HINT1 with respect to tumor suppression is not known. In the present study, we found that a series of human colon cancer cell lines displayed various levels of expression of HINT1, with a very low level in SW480 cells. This cell line also displayed partial methylation of the promoter region of the Hint1 gene, and treatment of these cells with 5-azadeoxycitidine increased expression of Hint1 mRNA and protein. Therefore, the decreased expression of HINT1 in SW480 cells seems to be due to epigenetic silencing. Increased expression of HINT1 in these cells, using a retrovirus vector (pLNCX2) that encodes either wild-type (WT) Hint1 or a point mutant (His(112)/Asn(112)) of Hint1, inhibited the proliferation of SW480 cells. Because of the important role of the activator protein-1 (AP-1) transcription factor in cancer cells, we examined possible effects of HINT1 on AP-1 transcription factor activity in SW480 cells transfected with an AP-1-luciferase reporter. We found that cotransfection with a pHA-Hint1 plasmid DNA significantly inhibited this activity. Studies with inhibitors indicated that AP-1 activity in SW480 cells requires the activity of c-Jun NH(2)-terminal kinase (JNK) 2 and not JNK1. Cotransfection with the Hint1 plasmid DNA also inhibited AP-1-luciferase reporter activity in WT mouse embryo fibroblast (MEF) studies, and studies with JNK1 deleted or JNK2 deleted MEFs confirmed the essential role for JNK2, but not JNK1, in mediating AP-1 activity. Recent studies indicate that the protein plenty of SH3 (POSH) provides a scaffold that enhances JNK activity. We found that cotransfection of a plasmid DNA encoding POSH stimulated the phosphorylation of c-Jun and also AP-1 reporter activity, and cotransfection with Hint1 inhibited both of these activities. Furthermore, coimmunoprecipitation studies provided evidence that HINT1 forms an in vivo complex with POSH and JNK. These results suggest that HINT1 inhibits AP-1 activity by binding to a POSH-JNK2 complex, thus inhibiting the phosphorylation of c-Jun. This effect could contribute to the tumor suppressor activity of HINT1.

The inhibitory effect of (-)-epigallocatechin gallate on activation of the epidermal growth factor receptor is associated with altered lipid order in HT29 colon cancer cells.

(-)-Epigallocatechin gallate (EGCG), a major biologically active constituent of green tea, inhibits activation of the epidermal growth factor (EGF) receptor (EGFR) and downstream signaling pathways in several types of human cancer cells, but the precise mechanism is not known. Because several plasma membrane-associated receptor tyrosine kinases (RTK) including EGFR are localized in detergent-insoluble ordered membrane domains, so-called "lipid rafts," we examined whether the inhibitory effect of EGCG on activation of the EGFR is associated with changes in membrane lipid order in HT29 colon cancer cells. First, we did cold Triton X-100 solubility assays. Phosphorylated (activated) EGFR was found only in the Triton X-100-insoluble (lipid raft) fraction, whereas total cellular EGFR was present in the Triton X-100-soluble fraction. Pretreatment with EGCG inhibited the binding of Alexa Fluor 488-labeled EGF to the cells and also inhibited EGF-induced dimerization of the EGFR. To examine possible effects of EGCG on membrane lipid organization, we labeled the cells with the fluorescent lipid analogue 1, 1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, which preferentially incorporates into ordered membrane domains in cells and found that subsequent treatment with EGCG caused a marked reduction in the Triton X-100-resistant membrane fraction. Polyphenon E, a mixture of green tea catechins, had a similar effect but (-)-epicatechin (EC), the biologically inactive compound, did not significantly alter the Triton X-100 solubility properties of the membrane. Furthermore, we found that EGCG but not EC caused dramatic changes in the function of bilayer-incorporated gramicidin channels. Taken together, these findings suggest that EGCG inhibits the binding of EGF to the EGFR and the subsequent dimerization and activation of the EGFR by altering membrane organization. These effects may also explain the ability of EGCG to inhibit activation of other membrane-associated RTKs, and they may play a critical role in the anticancer effects of this and related compounds.

(-)-Epigallocatechin gallate causes internalization of the epidermal growth factor receptor in human colon cancer cells.

We recently found that the inhibitory effect of (-)-epigallocatechin gallate (EGCG) on epidermal growth factor (EGF) binding to the epidermal growth factor receptor (EGFR) is associated with alterations in lipid organization in the plasma membrane of colon cancer cells. Since changes in lipid organizations are thought to play a role in the trafficking of several membrane proteins, in this study we examined the effects of EGCG on cellular localization of the EGFR in SW480 cells. Treatment of the cells for 30 min with as little as 1 microg/ml of EGCG caused a decrease in cell surface-associated EGFRs and this was associated with internalization of EGFRs into endosomal vesicles. Similar effects were seen with a green fluorescent protein (GFP)-EGFR fusion protein. As expected, the EGFR protein was phosphorylated at tyrosine residues, ubiquitinated and partially degraded when the cells were treated with EGF, but treatment with EGCG caused none of these effects. The loss of EGFRs from the cell surface induced by treating the cells with EGF for 30 min persisted for at least 2 h. However, the loss of EGFRs from the cell surface induced by temporary exposure to EGCG was partially restored within 1-2 h. These studies provide the first evidence that EGCG can induce internalization of EGFRs into endosomes, which can recycle back to the cell surface. This sequestrating of inactivated EGFRs into endosomes may explain, at least in part, the ability of EGCG to inhibit activation of the EGFR and thereby exert anticancer effects.

Celecoxib-induced growth inhibition in SW480 colon cancer cells is associated with activation of protein kinase G.

Although it is often assumed that the antitumor effects of nonsteroidal anti-inflammatory drugs (NSAIDs) are due to inhibition of cyclooxgenase (COX) activity, specifically COX-2, there is accumulating evidence that COX-2 independent mechanisms can also play an important role. Studies with sulindac sulfone (Aptosyn) and related derivatives have revealed a novel pathway of tumor growth inhibition and apoptosis mediated by activation of the guanosine 3',5' monophosphate (cGMP)-dependent enzyme protein kinase G (PKG). The present study indicates that concentrations of the NSAIDs celecoxib, indomethacin, and meclofenamic acid that inhibit growth of SW480 human colon cancer cells inhibit subcellular cGMP-phosphodiesterase (PDE) enzymatic activity and in intact cells induce a two- to threefold increase in intracellular levels of cGMP. This is associated with phosphorylation of the protein VASP, a marker of PKG activation, activation of JNK1 and a decrease in cellular levels of cyclin D1; effects seen with other agents that cause activation of PKG in these cells. On the other hand even a high concentration of the COX-2 specific inhibitor rofecoxib (500 microM) did not inhibit growth of SW480 cells. Nor did rofecoxib inhibit cGMP-PDE activity or cause other changes related to PKG activation in these cells. Since activation of the PKG pathways by celecoxib, indomethacin, and meclofenamic acid in this cell culture system required high concentrations of these compounds, it remains to be determined whether activation of this pathway contributes to the in vivo antitumor effects of specific NSAIDs.

Protein kinase C beta enhances growth and expression of cyclin D1 in human breast cancer cells.

Although alterations in the expressions of protein kinase C (PKC) have been implicated in breast carcinogenesis, the roles of specific isoforms in this process remain elusive. In the present study, we examined the specific roles of PKCbeta1 and beta2 in growth control in human breast cancer cell lines. The PKCbeta-specific inhibitor LY379196 significantly inhibited growth of the breast cancer cell lines MCF-7, MDA-MB-231, and BT474, but not the normal mammary epithelial cell line MCF-10F. Treatment of MCF-7 cells with LY379196 caused an increase in the fraction of cells in the G(1) phase of the cell cycle. To explore the roles of PKCbeta1 and beta2, we used cDNA expression vectors that encode wild-type and constitutively activated or dominant negative mutants of these two proteins. When compared with vector controls, derivatives of MCF-7 cells that stably overexpress wild-type PKCbeta1 or PKCbeta2 displayed a slight increase in growth rate; derivatives that stably express the constitutively active mutants of PKCbeta1 or PKCbeta2 displayed a marked increase in growth rate; and derivatives that stably express a dominant negative mutant of PKCbeta1 or beta2 displayed inhibition of growth. The derivatives of MCF-7 cells that stably express the constitutively activated mutants of PKCbeta1 or beta2 were more resistant to growth inhibition by LY379196 than the vector control MCF-7 cells. Immunoblot analysis indicated that MCF-7 cells that stably overexpress wild-type or constitutively activated mutants of PKCbeta1 or beta2 had higher cellular levels of cyclin D1 than vector control cells, whereas cells that express a dominant negative mutant had decreased levels of cyclin D1. The derivatives that stably express the constitutively activated mutants of PKCbeta1 or beta2 also displayed increased cyclin D1 promoter activity in transient transfection luciferase reporter assays, and this induction of activity requires activator protein 1. Constitutively activated PKCbeta1 and beta2 also enhanced the transcription of c-fos in transient transfection luciferase reporter assays. Thus, PKCbeta1 and beta2 may play important positive roles in the growth of at least a subset of human breast cancers. Therefore, inhibitors of these isoforms may be useful in breast cancer chemoprevention or therapy.

Synergistic effects of RXR alpha and PPAR gamma ligands to inhibit growth in human colon cancer cells--phosphorylated RXR alpha is a critical target for colon cancer management.

BACKGROUND AND AIMS: The activation of the peroxisome proliferator-activated receptor gamma (PPAR gamma) that forms heterodimers with retinoid X receptors (RXRs) elicits an antineoplastic effect on colorectal cancer. It was previously reported that the accumulation of the non-functional phosphorylated form of RXR alpha (p-RXR alpha) interfered with its signalling and promoted the growth of hepatoma cells. In this study the effects of p-RXR alpha on the ability of RXR alpha and PPAR gamma ligands to inhibit growth in colon cancer cells was examined. METHODS: The effects of the combination of the PPAR gamma ligand ciglitazone and the RXR alpha lignad 9-cis-retinoic acid (RA) on inhibition of cell growth in Caco2 human colon cancer cells which express high levels of p-RXR alpha protein were examined. RESULTS: The RXR alpha protein was phospholylated and also accumulated in human colon cancer tissue samples as well as human colon cancer cell lines. When the phosphorylation of RXR alpha was inhibited by the MEK inhibitor PD98059 or by transfection with a point-mutated RXR alpha, which mimicked the unphosphorylated form, the combination of 9-cisRA and ciglitazone synergistically inhibited the cell growth and induced apoptosis. The combined treatment with these agents also caused a decrease in the expression levels of both cyclo-oxygenase-2 (COX-2) and c-Jun proteins and mRNAs. Reporter assays indicated that this combination induced the transcriptional activity of the peroxisome proliferator-responsive element promoter and also inhibited that of the AP-1 promoter. CONCLUSION: A malfunction of RXR alpha due to phosphorylation is associated with colorectal cancer. Therefore, the inhibition of phosphorylation of RX R alpha and the activation of the RXR-PPAR gamma heterodimer by their respective ligands may be useful in the chemoprevention and/or treatment of colorectal cancer.

Dip1 inhibits growth and gene transcription in MCF-7 breast cancer cells.

In previous studies we identified a novel gene Dipl, also designated CCNDBP1, which encodes a 42kDa helix-loop-helix (HLH) nuclear protein. Although this protein was originally identified by its ability to bind to cyclin D1 its precise biochemical functions are not known. In the present study we carried out mechanistic studies on Dip1 focusing on the human breast cancer cell line MCF-7. We found that overexpression of Dip1 in MCF-7 cells inhibited colony formation and cell proliferation. Reporter assays in MCF-7 cells indicated that Dip1 strongly inhibited the transcriptional activities of the cyclin D1, c-fos, NF-kappaB, SRE and p21cP1 promoters. Furthermore studies with truncated and mutant forms of the cyclin D1 promoter suggest that Dip1 does not act on specific transcriptional elements. Assays with mutant and truncated forms of Dip1 indicated that both the LXXLL motif and the HLH domain play important, but not exclusive, roles in these inhibitory effects. Dip1 co-immunoprecipitated with the histone deacetylase (HDAC) proteins HDAC1 and HDAC3. Nevertheless Dip1 markedly inhibited the stimulation of cyclin D1 promoter activity obtained with trichostatin A [1], an inhibitor of HDAC. Taken together these findings suggest that Dip1 functions as a general repressor of transcription. Although the precise mechanism by which Dip1 inhibits gene transcription and the growth of MCF-7 cells remain to be determined, the present results suggest that Dip1 is a candidate tumor suppressor gene.

Signal transducers and activators of transcription 3 up-regulates vascular endothelial growth factor production and tumor angiogenesis in head and neck squamous cell carcinoma.

Overexpression of vascular endothelial growth factor (VEGF) is associated with angiogenic phenotypes and poor prognosis of numerous tumors, including head and neck squamous cell carcinoma (HNSCC). However, the precise mechanism that causes VEGF overexpression in HNSCC remains unknown. Since there is evidence that a transcriptional factor, signal transducers and activators of transcription 3 (Stat3), is constitutively activated in HNSCC and this activation is significantly associated with aggressive phenotypes of this disease, we investigated the roles of Stat3 activation on VEGF production and tumor angiogenesis in HNSCC both in vitro and in clinical samples. VEGF promoter assays with YCU-H891 cells demonstrated that dominant negative Stat3 significantly inhibited VEGF promoter activity in the full length (-2279 to +54) and two truncated forms of VEGF promoter luciferase-reporter construct (-1179 to 54) or (-1014 to +54), which retain the putative Stat3 responsive elements (-849 to -842). However, this was not seen in the shorter construct (-794 to +54), which lacks the putative Stat3 responsive elements. In the derivative of YCU-891 cells that stably express dominant negative Stat3 protein, cellular levels of VEGF mRNA and VEGF protein were significantly inhibited. In the 51 clinical samples obtained from the patients with tongue carcinoma, the expression levels of phosphorylated (activated) form of Stat3 protein were significantly correlated with VEGF (P<0.05) production and intratumoral microvessel density IMVD (P<0.01). These results strongly indicate that Stat3 directly up-regulates VEGF transcription and thereby promotes angiogenesis in HNSCC. Inhibition of Stat3 activity may provide a new anti-angiogenic therapy in HNSCC.

Gene expression analysis of the mechanisms whereby black cohosh inhibits human breast cancer cell growth.

BACKGROUND: Previous studies indicate that specific extracts and the pure triterpene glycoside actein obtained from black cohosh inhibit growth of human breast cancer cells. Our aim is to identify alterations in gene expression induced by treatment with a methanolic extract (MeOH) of black cohosh. MATERIALS AND METHODS: We treated MDA-MB-453 human breast cancer cells with the MeOH extract at 40 microg/ml and collected RNA at 6 and 24 h; we confirmed the microarray results with real-time RT-PCR for 18 genes. RESULTS: At 6 h after treatment there was significant increase in expression of ER stress (GRP78), apoptotic (GDF15), lipid biosynthetic (INSIG1 and HSD17B7) and Phase 1 (CYP1A1) genes and, at 24 h, decrease in expression of cell cycle (HELLS and PLK4) genes. CONCLUSION: Since the MeOH extract activated genes that enhance apoptosis and repressed cell cycle genes, it may be useful in the prevention and therapy of breast cancer.

Sulindac sulfide and exisulind inhibit expression of the estrogen and progesterone receptors in human breast cancer cells.

In previous studies, we found that sulindac sulfide and exisulind (sulindac sulfone, Aptosyn) cause growth inhibition, arrest cells in the G1 phase of the cell cycle, and induce apoptosis in human breast cancer cell lines. These effects were associated with decreased expression of cyclin D1. The present study focuses on the effects of sulindac sulfide and exisulind on hormone signaling components in breast cancer cells. We found that estrogen receptor (ER)-positive and progesterone receptor (PR)-positive T47D breast cancer cells were somewhat more sensitive to growth inhibition by sulindac sulfide or exisulind than ER-negative PR-negative MB-MDA-468 breast cancer cells. Further studies indicated that sulindac sulfide and exisulind caused marked down-regulation of expression of the ER and PR-A and PR-B in T47D cells. However, neither compound caused a major change in expression of the retinoic acid receptor alpha (RARalpha), RARbeta, or RARalpha in T47D cells. Sulindac sulfide and exisulind also caused a decrease in expression of the ER in estrogen-responsive MCF-7 breast cancer cells. Both compounds also markedly inhibited estrogen-stimulated activation of an estrogen-responsive promoter in transient transfection reporter assays. Treatment of T47D cells with specific protein kinase G (PKG) activators did not cause a decrease in ER or PR expression. Therefore, although sulindac sulfide and exisulind can cause activation of PKG, the inhibitory effects of these two compounds on ER and PR expression does not seem to be mediated by PKG. Our findings suggest that the growth inhibition by sulindac sulfide and exisulind in ER-positive and PR-positive human breast cancer cells may be mediated, in part, by inhibition of ER and PR signaling. Thus, these and related compounds may provide a novel approach to the prevention and treatment of human breast cancers, especially those that are ER positive.

Antisense to cyclin D1 inhibits vascular endothelial growth factor-stimulated growth of vascular endothelial cells: implication of tumor vascularization.

PURPOSE: Our aim was to determine the effects of cyclin D1 inhibition on tumor-associated neovascularization and endothelial cell growth. EXPERIMENTAL DESIGN: We have generated adenovirus system for antisense to cyclin D1 (AS CyD1) and evaluated in vitro and in vivo effects. Small interfering RNA against cyclin D1 was also used to analyze cyclin D1 inhibition-associated vascular endothelial growth factor (VEGF) regulation. RESULTS: The xenografts treated with adenoviral AS CyD1 showed less vessel density and displayed smaller tumor size in colon cancer cell lines HCT116 and DLD1. In vitro studies indicated that AS CyD1 decreased VEGF protein expression in DLD1 but not in HCT116. Cyclin D1 small interfering RNA caused a decrease in VEGF expression at protein and RNA levels in DLD1. A modest decrease was noted in the VEGF promoter activity, with inactivation of the STAT3 transcription factor through dephosphorylation. On the hand, the cyclin D1 inhibition plus STAT3 inhibitor markedly decreased VEGF expression in HCT116, although VEGF did not change by the STAT3 inhibitor alone. In cultures of human umbilical vein endothelial cells (HUVEC), VEGF augmented cyclin D1 expression and cell growth. AS CyD1 significantly inhibited HUVEC growth even in the presence of VEGF. AS CyD1 also significantly suppressed in vitro tube formation in VEGF-treated HUVEC and in vivo macroaneurysm formation in VEGF-treated Matrigel plug. CONCLUSIONS: Our results suggest that cyclin D1 may play a role in the maintenance of VEGF expression and that AS CyD1 could be potentially useful for targeting both cancer cells and their microenvironment of tumor vessels.

Activation of protein kinase G up-regulates expression of 15-lipoxygenase-1 in human colon cancer cells.

Recent studies indicate that the induction of apoptosis in human colon cancer cells by certain nonsteroidal antiinflammatory drugs involves increased expression of 15-LOX-1 and synthesis of its major product 13-S-hydroxyoctadecadienoic acid (13-S-HODE). Evidence was obtained that this occurs via a cyclooxygenase-2 (COX-2)-independent mechanism, but the actual mechanism of induction of 15-LOX-1 by these compounds is not known. There is extensive evidence that treatment of SW480 human colon cancer cells with sulindac sulfone (Exisulind, Aptosyn) or the related derivative OSI-461, both of which inhibit cyclic GMP (cGMP)-phosphodiesterases but lack COX-2 inhibitory activity, causes an increase in intracellular levels of cGMP, thus activating protein kinase G (PKG), which then activates pathways that lead to apoptosis. Therefore, in the present study, we examined the effects of various agents that cause increased cellular levels of cGMP on the expression of 15-LOX-1 in SW480 human colon cancer cells. Treatment of the cells with Exisulind, sulindac sulfide, OSI-461, the guanylyl cyclase activator YC-1, or the cell-permeable cGMP compound 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP) caused an increase in cellular levels of 15-LOX-1. Exisulind, OSI-461, and 8-pCPT-cGMP also increased mRNA levels of 15-LOX-1, suggesting that the effects were at the level of transcription. The cGMP-phosphodiesterase inhibitors and YC-1 increased the production of 13-S-HODE, which is the linoleic acid metabolite of 15-LOX-1. Treatment of SW480 cells with the PKG inhibitor Rp-8-pCPT-cGMP blocked Exisulind-induced 15-LOX-1 expression. Furthermore, derivatives of SW480 cells that were engineered to stably overexpress wild-type PKG Ibeta displayed increased cellular levels of 15-LOX-1 when compared with vector control cells. Taken together, these results provide evidence that the cGMP/PKG pathway can play an important role in the induction of 15-LOX-1 expression by nonsteroidal antiinflammatory drugs and related agents.

The sulindac derivatives OSI-461, OSIP486823, and OSIP487703 arrest colon cancer cells in mitosis by causing microtubule depolymerization.

Exisulind (sulindac sulfone) and three highly potent derivatives, OSI-461 (CP461), OSIP486823 (CP248), and OSIP487703, inhibit growth and induce apoptosis in SW480 human colon cancer cells, with IC(50)s of 200, 2, 0.1, and 0.003 micromol/L, respectively. The latter three compounds, but not exisulind, induce marked M-phase cell cycle arrest in these cells. This effect seems to be independent of the known ability of these compounds to cause activation of protein kinase G. When tested at twice their IC(50) concentration for growth inhibition, OSI-461, OSIP486823, and OSIP487703 cause depolymerization of microtubules in interphase cells, inhibit spindle formation in mitotic cells, and induce multinucleated cells. In vitro tubulin polymerization assays indicate that all three compounds interact with tubulin directly to cause microtubule depolymerization and/or inhibit de novo tubulin polymerization. These results suggest that the dual effects of OSI-461, OSIP486823, and OSIP487703 on impairment of microtubule functions and protein kinase G activation may explain the potent antiproliferative and apoptotic effects of these compounds in cancer cells.

(-)-Epigallocatechin gallate and polyphenon E inhibit growth and activation of the epidermal growth factor receptor and human epidermal growth factor receptor-2 signaling pathways in human colon cancer cells.

PURPOSE: (-)-Epigallocatechin gallate (EGCG) inhibits activation of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor-2 (HER2) and multiple downstream signaling pathways in cancer cell lines. In this study we compared the cellular and molecular effects of EGCG with a well-standardized decaffeinated green tea catechin mixture Polyphenon E (Poly E) on human colon cancer cell lines. EXPERIMENTAL DESIGN AND RESULTS: Both EGCG and Poly E preferentially inhibited growth of the Caco2, HCT116, HT29, SW480, and SW837 colon cancer cells when compared with the FHC normal human fetal colon cell line. The EGFR and HER2 proteins were overexpressed and constitutively activated in all of the colon cancer cell lines when compared with the FHC cell line. Treatment of HT29 cells with EGCG or Poly E caused an increase of cells in G1 and induced apoptosis. Both EGCG and Poly E caused a decrease in the phosphorylated forms of EGFR and HER2 proteins, and subsequently caused a decrease in the phosphorylated forms of the extracellular signal-regulated kinase and Akt proteins. Similar effects of these compounds were seen when the cells were stimulated with transforming growth factor alpha. Reporter assays indicated that both EGCG and Poly E inhibited the transcriptional activity of the activator protein 1 (AP-1), c-fos, nuclear factor kappaB, and cyclin D1 promoters. The combination of only 1 microg/mL of epicatechin plus 10 microg/mL of EGCG displayed synergistic effects on growth inhibition and induction of apoptosis. Furthermore, when treatment was prolonged for 96 hours, 1 microg/mL of EGCG or Poly E was sufficient to inhibit growth, reduce activation of EGFR and HER2, and induce apoptosis. CONCLUSION: Our findings suggest that EGCG or Poly E may be useful in the chemoprevention and/or treatment of colon cancer. Poly E contains about 60% EGCG, yet pure EGCG and Poly E had similar potencies (expressed as microg/ml). Poly E may be preferable because it is easier to prepare and this mixture of catechins may exert synergistic effects.

Activation of protein kinase G is sufficient to induce apoptosis and inhibit cell migration in colon cancer cells.

The activation of protein kinase G (PKG) by cGMP has become of considerable interest as a novel molecular mechanism for the induction of apoptosis in cancer cells, because sulindac sulfone (exisulind, Aptosyn) and certain derivatives that inhibit cGMP-phosphodiesterases and thereby increase cellular levels of cGMP appear to induce apoptosis via this mechanism. However, other effects of these compounds have not been excluded, and the precise mechanism by which PKG activation induces apoptosis has not been elucidated in detail. To directly examine the effects of PKG on cell growth and apoptosis, we generated a series of mutants of PKG Ialpha: PKG IalphaS65D, a constitutively activated point mutant; PKG IalphaDelta, a constitutively activated N-terminal truncated mutant; and PKG IalphaK390R, a dominant-negative point mutant. A similar series of mutants of PKG Ibeta were also constructed (Deguchi et al., Mol. Cancer Ther., 1: 803-809, 2002). The present study demonstrates that when transiently expressed in SW480 colon cancer, the constitutively activated mutants of PKG Ibeta, and to a lesser extent PKG Ialpha, inhibit colony formation and induce apoptosis. We were not able to obtain derivatives of SW480 cells that stably expressed these constitutively activated mutants, presumably because of toxicity. However, derivatives that stably overexpressed wild-type PKG Ibeta displayed growth inhibition, whereas derivatives that stably expressed the dominant-negative mutant (KR) of PKG Ibeta grew more rapidly and were more resistant to Aptosyn-induced growth inhibition than vector control cells. Stable overexpression of PKG Ibeta was associated with decreased cellular levels of beta-catenin and cyclin D1 and increased levels of p21(CIP1). Reporter assays indicated that activation of PKG Ibeta inhibits the transcriptional activity of the cyclin D1 promoter. We also found that transient expression of the constitutively activated mutants of PKG Ibeta inhibited cell migration. Taken together, these results indicate that activation of PKG Ibeta is sufficient to inhibit growth and cell migration and induce apoptosis in human colon cancer cells and that these effects are associated with inhibition of the transcription of cyclin D1 and an increase in the expression of p21(CIP1).

Overexpression of cyclin D1 contributes to malignancy by up-regulation of fibroblast growth factor receptor 1 via the pRB/E2F pathway.

Overexpression of cyclin D1 due to gene rearrangement, gene amplification, or simply increased transcription occurs frequently in several types of human cancers. However, overexpression of cyclin D1 in cell culture system is insufficient, by itself, to cause malignant transformation. In the present study, we found that when rodent fibroblasts that overexpress cyclin D1, but not normal fibroblasts, were treated with basic fibroblast growth factor (bFGF), there was enhanced cell cycle progression, extracellular signal-regulated kinase 2 activation, induction of anchorage-independent growth, and enhanced invasion of a Matrigel barrier. These enhanced responses to bFGF appear to be due to increased expression of fibroblast growth factor receptor 1, at both the mRNA and protein levels, in the cyclin D1-overexpressing cells. We obtained evidence that this increase in fibroblast growth factor receptor 1 expression is mediated through cyclin D1 activation of the pRB/E2F pathway. Taken together, these results suggest that in vivo cyclin D1 overexpression can enhance tumor progression, at least in part, by potentiating the stimulatory efforts of bFGF, which is often produced by stromal cells, and the growth of adjacent tumor cells.

Growth inhibition of human hepatoma cells by acyclic retinoid is associated with induction of p21(CIP1) and inhibition of expression of cyclin D1.

Acyclic retinoid (ACR), a novel synthetic retinoid, can prevent the recurrence of human hepatoma after surgical resection of primary tumors, but the molecular mechanisms by which ACR exerts antitumor effects are not known. In this study, we found that ACR inhibited the growth of three human hepatoma cell lines. In HepG2 cells, this inhibition was associated with an arrest of the cell cycle in G(0)-G(1), increased cellular levels of p21(CIP1), decreased levels of the hyperphosphorylated form of the retinoblastoma protein, and decreased levels of cyclin D1, but no significant changes were seen in the levels of the p16(INK4a), p27(KIP1), cyclin-dependent kinase 4, cyclin-dependent kinase 6, glycogen synthase kinase 3beta, or beta-catenin proteins. ACR also caused a decrease in the level of cyclin D1 mRNA. Cotreatment of HepG2 human hepatoma cells with the proteasome inhibitor N-acetyl-Leu-Leu-norleu-al did not prevent the ACR-induced decrease in cyclin D1 protein, in contrast to the protective effect of N-acetyl-Leu-Leu-norleu-al on the cyclin D1 protein in cells treated with all-trans-retinoic acid. In transient transfection reporter assays, ACR, but not all-trans-retinoic acid, inhibited transcription from the cyclin D1 promoter. As reported previously in colon carcinoma cells, we found that in hepatoma cells, cyclin D1 promoter activity is markedly stimulated by the beta-catenin/T-cell factor pathway. Nevertheless, even in the presence of excess beta-catenin, ACR markedly inhibited the transcriptional activity of the cyclin D1 promoter. This is the first systematic study of the inhibitory effects of ACR, or any other retinoid compound, on beta-catenin/T-cell factor-stimulated cyclin D1 promoter activity in human tumor cells. These novel effects of ACR provide further evidence that ACR may be a valuable agent in the chemoprevention and therapy of hepatoma and possibly other human malignancies.

Induction of apoptosis by the garlic-derived compound S-allylmercaptocysteine (SAMC) is associated with microtubule depolymerization and c-Jun NH(2)-terminal kinase 1 activation.

Epidemiological and experimental carcinogenesis studies provide evidence that components of garlic (Allium sativum) have anticancer activity. We recently reported that the garlic derivative S-allylmercaptocysteine (SAMC) inhibits growth, arrests cells in G(2)-M, and induces apoptosis in human colon cancer cells (Shirin et al., Cancer Res., 61: 725-731, 2001). Because a fraction of the SAMC-treated cells are specifically arrested in mitosis, we examined the mechanism of this effect in the present study. Immunofluorescent microscopy revealed that the treatment of SW480 cells or NIH3T3 fibroblasts with 150 micro M SAMC (the IC(50) concentration) caused rapid microtubule (MT) depolymerization, MT cytoskeleton disruption, centrosome fragmentation and Golgi dispersion in interphase cells. It also induced the formation of monopolar and multipolar spindles in mitotic cells. In vitro turbidity assays indicated that SAMC acted directly on tubulin to cause MT depolymerization, apparently because it interacts with -SH groups on tubulin. To investigate the signaling pathways involved in SAMC-induced apoptosis, we assayed c-Jun NH(2)-terminal kinase (JNK) activity and found that treatment with SAMC caused a rapid and sustained induction of JNK activity. The selective JNK inhibitor SP600125 inhibited the early phase (24 h) but not the late phase (48 h and later) of apoptosis induced by SAMC. Expression of a dominant-negative mutant of JNK1 in SW480 cells inhibited apoptosis induced by SAMC at 24 h but had no protective effect at 48 h. JNK1(-/-) mouse embryonic fibroblasts were resistant to SAMC-induced apoptosis at 24 h but not at 48 h. On the other hand, the inhibition or abrogation of JNK1 activity did not inhibit the G(2)-M arrest induced by SAMC. SAMC also activated caspase-3. The general caspase inhibitor z-VAD-fmk inhibited both early and late phases of apoptosis induced by SAMC. We conclude that the garlic-derived compound SAMC exerts antiproliferative effects by binding directly to tubulin and disrupting the MT assembly, thus arresting cells in mitosis and triggering JNK1 and caspase-3 signaling pathways that lead to apoptosis.