Benzophenones and biflavonoids from Garcinia livingstonei fruits.

A series of 13 known compounds, including seven benzophenones [guttiferone A (1), guttiferone K (2), xanthochymol (3), guttiferone E (4), cycloxanthochymol (5), isoxanthochymol (6), and gambogenone (7)], five biflavonoids [amentoflavone (8), 3,8''-biapigenin (9), (+)-volkensiflavone (10), (+)-morelloflavone (11), and (+)-fukugiside (12)], and the xanthone derivative alloathyriol (13), were identified from the fruits of Garcinia livingstonei (Clusiaceae). This is the first time that compounds 2-7, 9, 12, and 13 have been reported in this species. The cytotoxicity of benzophenones 1 and 2 was assessed for their effect on HCT-116, HT-29, and SW-480 human colon cancer cell lines. Both compounds exhibited strong activity against HCT-116 and HT-29 cell lines with IC(50) values between 5 and 10 microM, and somewhat weaker activity with SW-480 cells (IC(50) values ranging from 18 to 25 microM).

(-)-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.

Histidine triad nucleotide-binding protein 1 up-regulates cellular levels of p27KIP1 by targeting ScfSKP2 ubiquitin ligase and Src.

The one or more underlying mechanisms of the tumor suppressing activity of the histidine triad nucleotide-binding protein 1 (HINT1) are not well defined. In this study we found that HINT1 regulates cellular levels of the cyclin-dependent kinase inhibitor p27(KIP1) through multiple mechanisms. Increased expression of HINT1 increases cellular levels of p27(KIP1), and HINT1 knockdown with small hairpin RNA leads to decreased cellular levels of p27(KIP1). HINT1 does not affect the transcription of p27(KIP1), but it does inhibit proteasomal degradation of the p27(KIP1) protein. HINT1 directly interacts with the SCF(SKP2) ubiquitin ligase complex and inhibits the ubiquitylation of p27(KIP1). Src has been shown to phosphorylate p27(KIP1) and thus decrease its stability. We found that HINT1 is a negative regulator of Src transcription apparently by forming a complex with the transcription factor Sp1 on the promoter of Src. Taken together, our findings indicate that HINT1 up-regulates cellular levels of p27(KIP1) by two mechanisms: 1) it inhibits its ubiquitylation by targeting the SCF(SKP2) ubiquitin ligase complex, and 2) it inhibits the phosphorylation of p27(KIP1) by Src via inhibiting Src expression.

The HINT1 tumor suppressor regulates both gamma-H2AX and ATM in response to DNA damage.

Hint1 is a haploinsufficient tumor suppressor gene and the underlying molecular mechanisms for its tumor suppressor function are unknown. In this study we demonstrate that HINT1 participates in ionizing radiation (IR)-induced DNA damage responses. In response to IR, HINT1 is recruited to IR-induced foci (IRIF) and associates with gamma-H2AX and ATM. HINT1 deficiency does not affect the formation of gamma-H2AX foci; however, it impairs the removal of gamma-H2AX foci after DNA damage and this is associated with impaired acetylation of gamma-H2AX. HINT1 deficiency also impairs acetylation of ATM and activation of ATM and its downstream effectors, and retards DNA repair, in response to IR. HINT1-deficient cells exhibit resistance to IR-induced apoptosis and several types of chromosomal abnormalities. Our findings suggest that the tumor suppressor function of HINT1 is caused by, at least in part, its normal role in enhancing cellular responses to DNA damage by regulating the functions of both gamma-H2AX and ATM.

(-)-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.

Activation of protein kinase G Increases the expression of p21CIP1, p27KIP1, and histidine triad protein 1 through Sp1.

The anticancer role of cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase G (PKG) has become of considerable interest, but the underlying mechanisms are not fully established. In this study, we examined the effects of activation of PKG on the expression of three tumor suppressor proteins in human SW480 colon cancer cells. Our results revealed that treatment with cell permeable cGMP derivatives, or the cGMP phosphodiesterase inhibitor sulindac sulfone (exisulind, aptosyn, hereafter called exisulind) led to increased expression of the tumor suppressor proteins p21(CIP1), p27(KIP1), and Histidine triad protein 1 (HINT1), and their corresponding mRNAs. Overexpression of PKG Ibeta also caused increased expression of the p21(CIP1), p27(KIP1), and HINT1 proteins. Both the p21(CIP1) and p27(KIP1) promoters contain Sp1 binding sites and they were activated by PKG in luciferase reporter assays. Specific Sp1 sites in the p21 and p27 promoters were sufficient to mediate PKG-induced luciferase reporter activity, suggesting an interaction between Sp1 and PKG. Indeed, we found that PKG can phosphorylate Sp1 on serine residue(s) and this resulted in transcriptional activation of Sp1. Knockdown of Sp1 expression with siRNA inhibited the increased expression of p21(CIP1), p27(KIP1), and HINT1 induced by the cGMP derivative 8-pCPT-cGMP in SW480 cells. These novel effects of PKG activation on the expression of three tumor suppressor genes may explain, at least in part, the anticancer effects of activation of PKG. They also provide a rationale for further developing activators of PKG for the prevention and treatment of cancer.

Oncogene addiction.

Cancer cells contain multiple genetic and epigenetic abnormalities. Despite this complexity, their growth and survival can often be impaired by the inactivation of a single oncogene. This phenomenon, called "oncogene addiction," provides a rationale for molecular targeted therapy. The efficacy of this strategy requires novel methods, including integrative genomics and systems biology, to identify the state of oncogene addiction (i.e., the "Achilles heel") in specific cancers. Combination therapy may also be required to prevent the escape of cancers from a given state of oncogene addiction.

TREK-1 is a novel molecular target in prostate cancer.

TREK-1 is a two-pore domain (K(2P)) potassium channel that carries a leak current that is time- and voltage-independent. Recently, potassium channels have been related to cell proliferation and some K(2P) family channels, such as TASK-3, have been shown to be overexpressed in specific neoplasms. In this study, we addressed the expression of TREK-1 in prostatic tissues and cell lines, and we have found that this potassium channel is highly expressed in prostate cancer but is not expressed in normal prostate nor in benign prostatic hyperplasia. Furthermore, expression of TREK-1 correlates strongly with the grade and the stage of the disease, suggesting a causal link between channel expression and abnormal cell proliferation. In vitro studies showed that TREK-1 is highly expressed in PC3 and LNCaP prostate cancer cell lines but is not detectable in normal prostate epithelial cells (NPE). In this report, we show that overexpression of TREK-1 in NPE and Chinese hamster ovary (CHO) cells leads to a significant increase in proliferation. Moreover, the increased cell proliferation rate of PC3 cells and TREK-1 overexpressing CHO cells could be reduced when TREK-1 current was reduced by overexpression of a dominant-negative TREK-1 mutant or when cells were exposed to a TREK-1 inhibitor. Taken together, these data suggest that TREK-1 expression is associated with abnormal cell proliferation and may be a novel marker for and a molecular target in prostate cancer.

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.

EGCG inhibits activation of the insulin-like growth factor (IGF)/IGF-1 receptor axis in human hepatocellular carcinoma cells.

The receptor tyrosine kinase (RTK) insulin like growth factor-1 (IGF-1)/IGF-1 receptor (IGF-1R) axis plays an important role in the development of hepatocellular carcinoma (HCC). EGCG inhibits activation of the various types of RTKs and that this is associated with inhibition of multiple downstream signaling pathways. In this study we examined the effects of EGCG on activity of the IGF/IGF-1R axis in HepG2 human HCC cells which express constitutive activation of this axis. The level of phosphorylated (i.e. activated) form of the IGF-1R protein (p-IGF-1R) was increased in a series of human HCC cell lines when compared with the Hc normal human hepatocytes. EGCG preferentially inhibited growth of HepG2 cells when compared with Hc cells. Treatment of HepG2 cells with EGCG induced apoptosis and caused a decrease in the p-IGF-1R protein and its downstream signaling molecules including the p-ERK, p-Akt, p-Stat-3, and p-GSK-3ß proteins, both in the absence or presence of ligand stimulation. EGCG also decreased the levels of both IGF-1 and IGF-2 proteins and mRNAs, but increased the levels of the IGFBP-3 protein. These findings suggest that EGCG can overcome the stimulatory effects of IGFs on the IGF-1R dependent signaling pathway, thus expanding the roles of EGCG as an inhibitor of critical RTKs involved in HCC cell proliferation. These results provide further evidence that EGCG may be useful in the chemoprevention or treatment of liver cancer.

Cytotoxic chalcones and antioxidants from the fruits of a Syzygium samarangense (Wax Jambu).

Bioassay-guided fractionation of the methanolic extracts of the pulp and seeds of the fruits of Syzygium samarangense Merr. & Perry (Blume) led to the identification of four cytotoxic compounds and eight antioxidants on the basis of HPLC-PDA analysis, MS, and various NMR spectroscopic techniques. Three C-methylated chalcones, 2',4'-dihydroxy-3',5'-dimethyl-6'-methoxychalcone (1), 2',4'-dihydroxy-3'-methyl-6'-methoxychalcone (stercurensin, 2), and 2',4'-dihydroxy-6'-methoxychalcone (cardamonin, 3), were isolated and displayed cytotoxic activity (IC(50) = 10, 35, and 35 µM, respectively) against the SW-480 human colon cancer cell line. Also a number of known antioxidants were obtained including six quercetin glycosides: reynoutrin (4), hyperin (5), myricitrin (6), quercitrin (7), quercetin (9), and guaijaverin (10), one flavanone: (S)-pinocembrin (8), and two phenolic acids: gallic acid (11) and ellagic acid (12).

The growth inhibitory effect of actein on human breast cancer cells is associated with activation of stress response pathways.

Previous studies indicate that the triterpene glycoside actein from the herb black cohosh inhibits growth of human breast cancer cells. This study seeks to identify genes altered in human breast cancer cells by treatment with actein, using gene expression analysis. We treated MDA-MB-453 human breast cancer cells with actein at 2 doses, 20 or 40 microg/mL, for 6 or 24 hr. We identified 5 genes that were activated after each of the treatments that are known to play a role in cellular responses to diverse stresses, including the DNA damage and unfolded protein responses. In addition, four genes that mediate the integrated stress response (ISR), including activating transcription factor 4, were induced under at least one of the 4 treatment conditions. We used hierarchical clustering to define clusters comprising patterns of gene expression. Two ISR genes, activating transcription factor 3 (ATF3) and DNA damage- inducible transcript 3, and lipid biosynthetic genes were activated after exposure to actein at 40 microg/mL for 6 hr, whereas the cell cycle genes cyclin E2 and cell division cycle 25A were repressed. Our results suggest that actein induces 2 phases of the ISR, the survival phase and the apoptotic phase, depending on the dose and duration of treatment. We confirmed the results of gene expression analysis with real-time RT-PCR for 18 selected genes and Western blot analysis for ATF3. Since actein activated transcription factors that enhance apoptosis, and repressed cell cycle genes, it may be useful in the prevention and therapy of breast cancer.

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.

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.

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.

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.

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.