TGF-ß Suppresses COX-2 Expression by Tristetraprolin-Mediated RNA Destabilization in A549 Human Lung Cancer Cells.

PURPOSE: Overexpression of cyclooxygenase 2 (COX-2) is thought to promote survival of transformed cells. Transforming growth factor ß (TGF-ß) exerts anti-proliferative effects on a broad range of epithelial cells. In the current study, we investigated whether TGF-ß can regulate COX-2 expression in A549 human lung adenocarcinoma cells, which are TGF-ß-responsive and overexpress COX-2. MATERIALS AND METHODS: Western blotting, Northern blotting, and mRNA stability assays were performed to demonstrate that COX-2 protein and mRNA expression were suppressed by TGF-ß. We also evaluated the effects of tristetraprolin (TTP) on COX-2 mRNA using RNA interference. RESULTS: We demonstrated that COX-2 mRNA and protein expression were both significantly suppressed by TGF-ß. An actinomycin D chase experiment demonstrated that COX-2 mRNA was more rapidly degraded in the presence of TGF-ß, suggesting that TGF-ß-induced inhibition of COX-2 expression is achieved via decreased mRNA stability. We also found that TGF-ß rapidly and transiently induced the expression of TTP, a well-known mRNA destabilizing factor, before suppression of COX-2 mRNA expression was observed. Using RNA interference, we confirmed that increased TTP levels play a pivotal role in the destabilization of COX-2 mRNA by TGF-ß. Furthermore, we showed that Smad3 is essential to TTP-dependent down-regulation of COX-2 expression in response to TGF-ß. CONCLUSION: The results of this study show that TGF-ß down-regulated COX-2 expression via mRNA destabilization mediated by Smad3/TTP in A549 cells.

A-kinase anchoring protein 12 regulates the completion of cytokinesis.

A-kinase anchoring protein 12 (AKAP12) gene is frequently inactivated in human gastric cancer and in several other cancers due to promoter hypermethylation. However, the biological function of AKAP12 in tumorigenesis remains to be identified. Aneuploidy, a hallmark of cancer cells, is often caused by abnormal cell division. In the present study, AKAP12 was found to localize to the cell periphery during interphase and to the actomyosin contractile ring during cytokinesis. Furthermore, AKAP12 depletion using small interfering RNA increased the number of multinucleated cells, and disrupted the completion of cytokinesis. Interestingly, the inhibition of myosin light chain kinase (MLCK), a key regulator of actomyosin contractility, removed AKAP12 from the cell periphery during interphase and from the contractile ring during cytokinesis, suggesting that AKAP12 might be a downstream effector of MLCK. Our findings implicate AKAP12 in the regulation of cytokinesis progression, and suggest a novel role for AKAP12 tumor suppressor.

Overexpression of A-kinase anchoring protein 12A activates sterol regulatory element binding protein-2 and enhances cholesterol efflux in hepatic cells.

A-kinase anchoring protein 12 (AKAP12) is known to function as a scaffold protein and as a putative tumor suppressor. However, little is known about the biological role of AKAP12 in hepatic cells. In this study, we performed micro-array analysis to identify the downstream pathway of AKAP12A, and found that AKAP12A overexpression up-regulates the expressions of several cholesterol-associated genes including HMG-CoA reductase and LDL receptor, which have been reported to be controlled by sterol regulatory element binding protein-2 (SREBP-2). It was found that AKAP12A activates SREBP-2 in hepatic cells, as demonstrated by the presence of its cleavage product, whereas the activation of sterol regulatory element binding protein-1 was not remarkably changed. Moreover, AKAP12A-induced SREBP-2 activation was found to depend on SREBP cleavage-activating protein (SCAP), as inhibition of SCAP by RNAi or sterols blocked SREBP-2 activation in response to AKAP12A overexpression. Interestingly, the hydrophobic amine U18666A caused dramatic movement of AKAP12A from the plasma membrane to cytosol and lysosomal membranes. Moreover, cholesterol depletion from the plasma membrane (using methyl-beta-cyclodextrin) caused a shift of AKAP12A from the plasma membrane to the cytoplasm. Cholesterol binding assay revealed that the N-terminal region of AKAP12A binds directly to cholesterol in vitro. Furthermore, AKAP12A overexpression enhanced [3H]-cholesterol efflux to extracellular acceptors, suggesting that AKAP12A may activate SREBP-2 by increasing cholesterol efflux. In conclusion, the present study suggests that AKAP12A is a novel regulator of cellular cholesterol metabolism.

Trastuzumab inhibits the growth of human gastric cancer cell lines with HER2 amplification synergistically with cisplatin.

HER2 has been found to be amplified in 10-20% of gastric cancers, and is correlated with poor outcome. The aims of this study were to recognize HER2 amplification in gastric cancer cell lines via fluorescence in situ hybridization and to evaluate the growth inhibitory effect of trastuzumab in HER2-amplified cell lines. To elucidate the mechanism of the growth inhibition, we performed cell cycle analysis and immunoblotting of downstream molecules. We also conducted drug interaction studies of trastuzumab with other chemotherapeutic agents. HER2 amplification was newly identified only in SNU-216 cells, and trastuzumab moderately inhibited the growth of SNU-216 cells and positive controls. Trastuzumab-mediated G1 arrest occurred with increased expression of p27(KIP1) and decreased cyclins. Phosphorylation of HER2 and downstream molecules, STAT3, AKT, and ERK, was also inhibited by trastuzumab. Treatment of SNU-216 cells with trastuzumab plus cisplatin resulted in a synergistic inhibitory effect, whereas treatment of SNU-216 cells with trastuzumab plus 5-FU, or trastuzumab plus oxaliplatin produced an additive effect. These results suggest that trastuzumab combined with chemotherapeutic agents can be active against gastric cancer with HER2 amplification.

Transcriptional induction of DLC-1 gene through Sp1 sites by histone deacetylase inhibitors in gastric cancer cells.

We previously reported that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, induced DLC-1 mRNA expression and accumulated acetylated histones H3 and H4 associated with the DLC-1 promoter in DLC-1 non-expressing gastric cancer cells. In this study, we demonstrated the molecular mechanisms by which TSA induced the DLC-1 gene expression. Treatment of the gastric cancer cells with TSA activates the DLC-1 promoter activity through Sp1 sites located at -219 and -174 relative to the transcription start site. Electrophoretic mobility-shift assay (EMSA) revealed that Sp1 and Sp3 specifically interact with these Sp1 sites and showed that TSA did not change their binding activities. The ectopic expression of Sp1, but not Sp3, enhances the DLC-1 promoter responsiveness by TSA. Furthermore, the TSA-induced DLC-1 promoter activity was increased by p300 expression and reduced by knockdown of p300. These results demonstrated the requirement of specific Sp1 sites and dependence of Sp1 and p300 for TSA-mediated activation of DLC-1 promoter.

Inhibitors of histone deacetylases induce tumor-selective cytotoxicity through modulating Aurora-A kinase.

The molecular basis of the antitumor selectivity of histone deacetylase inhibitors (HDIs) remains unclear. Centrosomal Aurora-A kinase regulates chromosomal segregation during mitosis. The overexpression or amplification of Aurora-A leads to genetic instability, and its inhibition has shown significant antitumor effects. In this paper, we report that structurally related hydroxamate LAQ824 and SK-7068 induce tumor-selective mitotic defects by depleting Aurora-A. We found that HDI-treated cancer cells, unlike nontransformed cells, exhibit defective mitotic spindles. After HDI, Aurora-A was selectively downregulated in cancer cells, whereas Aurora-B remained unchanged in both cancer and nontransformed cells. LAQ824 or SK-7068 treatment inhibited histone deacetylase (HDAC) 6 present in Aurora-A/heat shock protein (Hsp) 90 complex. Inhibition of HDAC6 acetylated Hsp90 and resulted in dissociation of acetylated Hsp90 from Aurora-A. As a result, Hsp70 binding to Aurora-A was enhanced in cancer cells, leading to proteasomal degradation of Aurora-A. Overall, these provide a novel molecular basis of tumor selectivity of HDI. LAQ824 and SK-7068 might be more effective HDIs in cancer cells with Aurora-A overexpression.

Potential advantages of DNA methyltransferase 1 (DNMT1)-targeted inhibition for cancer therapy.

The deoxyribonucleic acid (DNA) methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) has been used as a drug in a part of cancer therapy. However, because of its incorporation into DNA during DNA synthesis, 5-aza-dC can cause DNA damage, mutagenesis, and cytotoxicity. In view of the adverse effects of 5-aza-dC, DNMT-targeted inhibition may be a more effective approach than treatment with 5-aza-dC. To address the possibility of DNMT-targeted cancer therapy, we compared the effects of treatment with small interfering ribonucleic acids (siRNAs) specific for DNMT1 or DNMT3b and treatment with 5-aza-dC on transcription, cell growth, and DNA damage in gastric cancer cells. We found that DNMT1-targeted inhibition induced the re-expression and reversed DNA methylation of five (CDKN2A, RASSF1A, HTLF, RUNX3, and AKAP12B) out of seven genes examined, and 5-aza-dC reactivated and demethylated all seven genes. In contrast, DNMT3b siRNAs did not show any effect. Furthermore, the double knockdown of DNMT1 and DNMT3b did not show a synergistic effect on gene re-expression and demethylation. In addition, DNMT1 siRNAs showed an inhibitory effect of cell proliferation in the cancer cells and the induction of cell death without evidence of DNA damage, whereas treatment with 5-aza-dC caused DNA damage as demonstrated by the comet assay. These results provide a rationale for the development of a DNMT1-targeted strategy as an effective epigenetic cancer therapy.

DLC-1, a GTPase-activating protein for Rho, is associated with cell proliferation, morphology, and migration in human hepatocellular carcinoma.

DLC-1 (deleted in liver cancer-1) is a tumor suppressor gene for hepatocellular carcinoma and other cancers. To characterize its functions, we constructed recombinant adenovirus encoding the wild-type DLC-1 and examined its effects on behaviors of a hepatocellular carcinoma cell line (SNU-368), which does not express DLC-1. Here, we found that restoration of DLC-1 expression in the SNU-368 cells caused an inhibition of cell proliferation with an increase of a subG1 population. Furthermore, DLC-1 overexpression induced disassembly of stress fibers and extensive membrane protrusions around cells on laminin-1. DLC-1 overexpression also inhibited cell migration and dephosphorylated focal adhesion proteins such as focal adhesion kinase (FAK), Cas (p130Cas; Crk-associated substrate), and paxillin. These observations suggest that DLC-1 plays important roles in signal transduction pathway regulating cell proliferation, cell morphology, and cell migration by affecting Rho family GTPases and focal adhesion proteins.

Histone deacetylase inhibitor enhances 5-fluorouracil cytotoxicity by down-regulating thymidylate synthase in human cancer cells.

Thymidylate synthase (TS) overexpression is a key determinant of 5-fluorouracil (5-FU) resistance in human cancer cells. TS is also acutely up-regulated with 5-FU treatment, and, thus, novel strategies targeting TS down-regulation seem to be promising in terms of modulating 5-FU resistance. Here, we report that histone deacetylase inhibitors can reverse 5-FU resistance by down-regulating TS. By using cDNA microarrays and validation experiments, we found that trichostatin A reduced the expression of both TS mRNA and TS protein. Cotreatment with trichostatin A and cycloheximide restored TS mRNA expression, suggesting that TS mRNA is repressed through new protein synthesis. On the other hand, TS protein expression was significantly reduced by lower doses of trichostatin A (50 nmol/L). Mechanistically, TS protein was found to interact with heat shock protein (Hsp) complex, and trichostatin A treatment induced chaperonic Hsp90 acetylation and subsequently enhanced Hsp70 binding to TS, which led to the proteasomal degradation of TS protein. Of note, combined treatment with low-dose trichostatin A and 5-FU enhanced 5-FU-mediated cytotoxicity in 5-FU-resistant cancer cells in accordance with TS protein down-regulation. We conclude that a combinatorial approach using histone deacetylase inhibitors may be useful at overcoming 5-FU resistance.

Antitumor activity of SK-7041, a novel histone deacetylase inhibitor, in human lung and breast cancer cells.

BACKGROUND: A class of synthetic histone deacetylase (HDAC) inhibitors, which are hybrids of trichostatin A and MS-275 were previously developed. In this study, the antitumor effects of SK-7041, one of those novel HDAC inhibitors, was evaluated on lung and breast cancer cell lines. MATERIALS AND METHODS: Human lung and breast cancer cells, as well as normal human bronchial epithelial (NHBE) cells were treated with SK-7041, and results were compared with those of cells treated with suberoylanilide hydroxamic acid (SAHA). RESULTS: SK-7041 induced time-dependent histone hyperacetylation and showed more potent cytotoxicity than SAHA in cancer cells. These antiproliferative effects of SK-7041 were due to apoptotic cell death caused by G2/M-phase arrest and to a lesser extent to G1 arrest. Moreover, SK-7041 inhibited cancer cell proliferation more selectively than NHBE cell proliferation. CONCLUSIONS: These results suggest that SK-7041 may have potential anticancer activity.

The synergism between Belotecan and cisplatin in gastric cancer.

PURPOSE: We wanted to demonstrate the anti-cancer effect and interaction between belotecan and cisplatin on gastric cancer cell line and we evaluated the mechanisms of this synergistic effect in vitro. MATERIALS AND METHODS: The growth inhibitory effect of belotocan and cisplatin against several gastric cancer cell lines (SNU-5, SNU-16 and SNU-601) was estimated by tetrazolium dye assay. The effect of a combination treatment was evaluated by the isobologram method. The biochemical mechanisms for the interaction between the drugs were analyzed by measuring the formation of DNA interstrand cross-links (ICLs) and DNA topo-I activity. RESULTS: Belotecan showed synergism with cisplatin for growth inhibitory effect on the gastric cancer cell lines SNU-5, and SNU-16, but this was subadditive on the SNU-601 cell line. The formation of DNA ICLs in SNU-16 cells by cisplatin was increased by combination with belotecan, but this was not affected in SNU-601 cells. The topo-I inhibition by belotecan was enhanced at high concentrations of cisplatin in SNU-16, but not in SNU-601 cells. CONCLUSION: Belotecan and cisplatin show various combination effect against gastric cancer cells. The synergism between cisplatin and belotecan could be the result of one of the following mechanisms: the modulating effect of belotecan on the repair of cisplatin-induced DNA adducts and the enhancing effect of cisplatin on the belotecan-induced topo-I inhibitory effect.

The signaling network of transforming growth factor beta1, protein kinase Cdelta, and integrin underlies the spreading and invasiveness of gastric carcinoma cells.

Integrin-mediated cell adhesion and spreading enables cells to respond to extracellular stimuli for cellular functions. Using a gastric carcinoma cell line that is usually round in adhesion, we explored the mechanisms underlying the cell spreading process, separate from adhesion, and the biological consequences of the process. The cells exhibited spreading behavior through the collaboration of integrin-extracellular matrix interaction with a Smad-mediated transforming growth factor beta1 (TGFbeta1) pathway that is mediated by protein kinase Cdelta (PKCdelta). TGFbeta1 treatment of the cells replated on extracellular matrix caused the expression and phosphorylation of PKCdelta, which is required for expression and activation of integrins. Increased expression of integrins alpha2 and alpha3 correlated with the spreading, functioning in activation of focal adhesion molecules. Smad3, but not Smad2, overexpression enhanced the TGFbeta1 effects. Furthermore, TGFbeta1 treatment and PKCdelta activity were required for increased motility on fibronectin and invasion through matrigel, indicating their correlation with the spreading behavior. Altogether, this study clearly evidenced that the signaling network, involving the Smad-dependent TGFbeta pathway, PKCdelta expression and phosphorylation, and integrin expression and activation, regulates cell spreading, motility, and invasion of the SNU16mAd gastric carcinoma cell variant.

The endogenous ratio of Smad2 and Smad3 influences the cytostatic function of Smad3.

Although Smad2 and Smad3, critical transcriptional mediators of transforming growth factor-beta (TGF-beta) signaling, are supposed to play a role in the TGF-beta cytostatic program, it remains unclear whether TGF-beta delivers cytostatic signals through both Smads equally or through either differentially. Here, we report that TGF-beta cytostatic signals rely on a Smad3-, but not a Smad2-, dependent pathway and that the intensity of TGF-beta cytostatic signals can be modulated by changing the endogenous ratio of Smad3 to Smad2. Depleting endogenous Smad3 by RNA interference sufficiently interfered with TGF-beta cytostatic actions in various TGF-beta-sensitive cell lines, whereas raising the relative endogenous ratio of Smad3 to Smad2, by depleting Smad2, markedly enhanced TGF-beta cytostatic response. Consistently, Smad3 activation and its transcriptional activity upon TGF-beta stimulation were facilitated in Smad2-depleted cells relative to controls. Most significantly, a single event of increasing this ratio by Smad2 depletion was sufficient to restore TGF-beta cytostatic action in cells resistant to TGF-beta. These findings suggest a new important determinant of sensitivity to TGF-beta cytostatic signaling.

Cytotoxic effects of pemetrexed in gastric cancer cells.

Pemetrexed is a newly developed multitargeted antifolate with promising clinical activity in many solid tumors including gastric cancer. The aim of the present study was to evaluate the cytotoxicity of pemetrexed and its mode of interaction with cisplatin in gastric cancer cell lines, and to identify genes associated with sensitivity to pemetrexed. The cytotoxic activity of pemetrexed was assessed by tetrazolium-based colorimetric assay (MTT assay) and the interaction between pemetrexed and cisplatin was evaluated by the isobologram method. Western immunoblotting and real time RT-PCR analysis of thymidylate synthase (TS), folylpoly-gamma-glutamate synthetase (FPGS) and reduced folate carrier (RFC1) were performed in order to determine whether sensitivity to pemetrexed would be predictable by protein or mRNA expression levels. Pemetrexed was more cytotoxic than 5-fluorouracil, with IC50 between 17 and 310 nM in most of the gastric cancer cell lines examined and the pemetrexed/cisplatin combination resulted in additive or synergistic interaction. The protein expressions of TS, FPGS, and RFC1 were significantly associated with IC50 for 5-fluorouracil, but no such association was found for pemetrexed chemosensitivity. The mRNA expressions of RFC1, FPGS and other target and resistance related genes revealed no significant association with pemetrexed sensitivity. In conclusion, pemetrexed is active against gastric cancer cell lines and the pemetrexed/cisplatin combination showed a synergistic or additive interaction, supporting its clinical use in gastric cancer. Drug sensitivity toward pemetrexed could not be predicted by the expressions of TS, RFC1, or FPGS and we suggest that it is determined by interactions between multiple genes.

Cell adhesion status-dependent histone acetylation is regulated through intracellular contractility-related signaling activities.

Although histone acetylation is important for epigenetic gene transcription, histone acetylation regulation by extracellular cues has rarely been evidenced. Here, we examined whether and how histone acetylation is regulated by cell adhesion-mediated signaling. Gastric carcinoma cells in suspension showed a higher histone acetylation, compared with fibronectin-adherent cells. This difference was supported by a decreased histone deacetylases activity. Furthermore, trichostatin A (TSA)-mediated histone acetylation was significantly increased only in suspended, but not in fibronectin-adherent, cells. Pharmacological inhibition of intracellular contractility-related myosin light chain kinase or RhoA-kinase (ROCK) or expression of ROCK1 small interfering RNA, dominant negative RhoA, or active Rac1 decreased basal and TSA-mediated histone H3 acetylations in suspended cells,whereas inhibition of calmodulin-dependent protein kinase II or transient overexpression of wild type myosin light chain kinase enhanced the acetylations. Meanwhile, chromatin immunoprecipitation showed higher basal and TSA-enhanced associations of ROCK1 promoter regions with Lys(9)-acetylated histone 3 in suspended cells than in fibronectin-adherent cells and expression of ROCK1 was higher and further increased by TSA treatment in suspension. In addition, phosphorylation of myosin light chain was further increased by TSA in suspension and higher in anchorage-independent cells over adherently growing cells, indicating an inverse relationship between ROCK1 expression-mediated contractility and cell adhesion abilities. Cell adhesion analysis showed that pharmacological activation of intracellular contractility-related signaling activities decreased cell adhesion abilities, whereas inhibition of them increased the adhesion. Taken together, these observations suggest that cell adhesion-related signal transduction regulates histone acetylation, presumably through a close functional linkage between intracellular contractility and histone deacetylases activity/histone acetylation.

TGF-beta1-mediated activations of c-Src and Rac1 modulate levels of cyclins and p27(Kip1) CDK inhibitor in hepatoma cells replated on fibronectin.

Integrin-mediated cell adhesion transduces signals to regulate actin cytoskeleton and cell proliferation. While understanding how integrin signals cross-talk with the TGF-beta1 pathways, we observed lamellipodia formation and cyclin regulation in Hep3B cells, following TGF-beta1 treatment. To answer if integrin signaling via actin organization might regulate cell cycle progression after TGF-beta1 treatment, we analyzed cross-talk between the two receptor-mediated pathways in hepatoma cells on specific ECMs. We found that basal and TGF-beta1-mediated activation of c-Src and Rac1, expression of cyclins E and A, and suppression of p27Kip1 were significant in cells replated on fibronectin, but not in cells on collagen I, indicating a different integrin-mediated cellular response to TGF-beta1 treatment. Levels of tyrosine phosphorylation and actin-enriched lamellipodia on fibronectin were also more prominent than in cells on collagen I. Studies using pharmacological inhibitors or transient transfections revealed that the preferential TGF-beta1 effects in cells on fibronectin required c-Src family kinase activity. These observations suggest that a specific cross-talk between TGF-beta1 and fibronectin-binding integrin signal pathways leads to the activation of c-Src/Rac1/actin-organization, leading to changes in cell cycle regulator levels in hepatoma cells. Therefore, this study represents another mechanism to regulate cell cycle regulators when integrin signaling is collaborative with TGF-beta1 pathways.

Cyclooxygenase-2 inhibits novel ginseng metabolite-mediated apoptosis.

Recently, a novel intestinal bacterial metabolite of ginseng protopanaxadiol saponins, i.e., 20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (IH-901), has been reported to induce apoptosis in a variety of cancer cells. Here we show a differential effect of IH-901 on several cell types. Exposure to IH-901 for 48 hours at a supposedly subapoptotic concentration of 40 mumol/L led to both apoptotic cell death and G1 arrest in Hep3B cells, but only resulted in G1 arrest in MDA-MB-231, Hs578T, and MKN28 cells. Additionally, the treatment of MDA-MB-231, but not of Hep3B, with IH-901 up-regulated cyclooxygenase-2 (COX-2) mRNA (2 hours) and protein (6 hours), and enhanced the production of prostaglandin E2. In MDA-MB-231 cells, IH-901 induced the sustained activation of extracellular signal-regulated kinase (ERK), whereas inhibition of mitogen-activated protein/ERK kinase blocked IH-901-mediated COX-2 induction and resulted in apoptosis, suggesting the involvement of an ERK-COX-2 pathway. Combined treatment with IH-901 and nonsteroidal anti-inflammatory drugs inhibited COX-2 enzyme and induced apoptosis in MDA-MB-231 and Hs578T cells. Adenovirus-mediated COX-2 small interfering RNAs also effectively inhibited COX-2 protein expression and enhanced IH-901-mediated apoptosis without inhibiting ERK 1/2 phosphorylation, thus providing direct evidence that COX-2 is an antiapoptotic molecule. Moreover, IH-901-mediated G1 arrest resulted from an increase in p27Kip1 mRNA and protein expression followed by a decrease in CDK2 kinase activity that was concurrent with the hypophosphorylation of Rb and p130. In conclusion, IH-901 induced both G1 arrest and apoptosis, and this apoptosis could be inhibited by COX-2 induction.

Class I histone deacetylase-selective novel synthetic inhibitors potently inhibit human tumor proliferation.

We have developed previously a class of synthetic hybrid histone deacetylase (HDAC) inhibitors, which were built from hydroxamic acid of trichostatin A and pyridyl ring of MS-275. In this study we evaluated the antitumor effects of these novel hybrid synthetic HDAC inhibitors, SK-7041 and SK-7068, on human cancer cells. Both SK-7041 and SK-7068 effectively inhibited cellular HDAC activity at nanomolar concentrations and induced the time-dependent hyperacetylation of histones H3 and H4. These HDAC inhibitors preferentially inhibited the enzymatic activities of HDAC1 and HDAC2, as compared with the other HDAC isotypes, indicating that class I HDAC is the major target of SK-7041 and SK-7068. We found that these compounds exhibited potent antiproliferative activity against various human cancer cells in vitro. Growth inhibition effect of SK-7041 and SK-7068 was related with the induction of aberrant mitosis and apoptosis in human gastric cancer cells. Both compounds induced the accumulation of cells at mitosis after 6 h of treatment, which was demonstrated by accumulation of tetraploid cells, lack of G(2) cyclin/cyclin-dependent kinase inactivation, and higher mitotic index. After 12 h of treatment, apoptotic cells were increased through mitochondrial and caspase-mediated pathway. Finally, in vivo experiment showed that SK-7041 or SK-7068 was found to reduce the growth of implanted human tumors in nude mice. Therefore, based on isotype specificity and antitumor activity, SK-7041 and SK-7068 HDAC inhibitors are expected to be promising anticancer therapeutic agents and need additional clinical development.

AKAP12/Gravin is inactivated by epigenetic mechanism in human gastric carcinoma and shows growth suppressor activity.

AKAP12/Gravin, one of the A-kinase anchoring proteins (AKAPs), functions as a kinase scaffold protein and as a dynamic regulator of the beta2-adrenergic receptor complex. However, the biological role of AKAP12 in cancer development is not well understood. The AKAP12 gene encodes two major isoforms of 305 and 287 kDa (designated AKAP12A and AKAP12B, respectively, in this report). We found that these two isoforms are independently expressed and that they are probably under the control of two different promoters. Moreover, both isoforms were absent from the majority of human gastric cancer cells. The results from methylation-specific PCR (MSP) and bisulfite sequencing revealed that the 5' CpG islands of both AKAP12A and AKAP12B are frequently hypermethylated in gastric cancer cells. Treatment with DNA methyltransferase inhibitor and/or histone deacetylase inhibitor efficiently restored the expression of AKAP12 isoforms, confirming that DNA methylation is directly involved in the transcriptional silencing of AKAP12 in gastric cancer cells. Hypermethylation of AKAP12A CpG island was also detected in 56% (10 of 18) of primary gastric tumors. The restoration of AKAP12A in AKAP12-nonexpressing cells reduced colony formation and induced apoptotic cell death. In conclusion, our results suggest that AKAP12A may function as an important negative regulator of the survival pathway in human gastric cancer.

Smad2 mediates Erk1/2 activation by TGF-beta1 in suspended, but not in adherent, gastric carcinoma cells.

Integrin-mediated cell adhesion enables cells to respond to extracellular stimuli for diverse cellular functions including proliferation, leading to differential biological activities from cells in suspension. Integrins can transduce signals (directly) to intracellular molecules and also collaborate with other membrane receptor-mediated signal pathways, including TGF-beta1 pathway. TGF-beta1 induces growth inhibition in epithelial cells and is known to transduce intracellular signaling in Smad-dependent or -independent manner. Currently effects of cell adhesion status on the TGF-beta1-mediated Erk1/2 regulation and on its Smad-(in)dependency are not known. In this study, we examined effects of cell adhesion status on the TGF-beta1-mediated Erk1/2 regulation, and roles of Smad proteins on the cell adhesion-mediated effects, using a gastric carcinoma cell variant. First, we found that cell adhesion-dependent Erk1/2 activation responded differentially to TGF-beta1, depending on cell adhesion status; TGF-beta1 treatment resulted in activation of Erk1/2 in suspended cells, whereas a decrease was noted in adherent cells. This activation of Erk1/2 by TGF-beta1 in suspension was more enhanced by an overexpression of Smad2, but not of other Smads 2, 4, and 7, but abolished by a Smad2 reduction via an introduction of its siRNA. In contrast, PKB/Akt regulation by TGF-beta1 was not different in suspension or in adhesion, and Smad7, but not the other Smads, activated PKB/Akt phosphorylation on TGF-beta1 treatment, indicating a specificity of Smad2-mediated and cell adhesion status-dependent activation of Erk1/2 activity.