Suppressing Src-Mediated EGFR Signaling by Sustained Calcium Supply Targeting Triple-Negative Breast Cancer.

Src is emerging as a promising target in triple-negative breast cancer (TNBC) treatment because it activates survival signaling linked to the epidermal growth factor receptor. In this study, the effect of calcium supply on Src degradation was investigated to confirm underlying mechanisms and anticancer effects targeting TNBC. MDA-MB-231 cells, the TNBC cell line, were used. Calcium supply was feasible through lactate calcium salt (CaLac), and the applicable calcium concentration was decided by changes in the viability with different doses of CaLac. Expression of signaling molecules mediated by calcium-dependent Src degradation was observed by Western blot analysis and immunocytochemistry, and the recovery of the signaling molecules was confirmed following calpeptin treatment. The anticancer effect was investigated in the xenograft animal model. Significant suppression of Src was induced by calcium supply, followed by a successive decrease in the expression of epithelial growth factor receptor, RAS, extracellular signal-regulated kinase, and nuclear factor kappa B. Then, the suppression of cyclooxygenase-2 contributed to a significant deactivation of the prostaglandin E2 receptors. These results suggest that calcium supply has the potential to reduce the risk of TNBC. However, as this study is at an early stage to determine clinical applicability, close consideration is needed.

Combination Antitumor Effect of Sorafenib via Calcium-Dependent Deactivation of Focal Adhesion Kinase Targeting Colorectal Cancer Cells.

Sorafenib has been recently used for the treatment of patients with advanced colorectal cancer (CRC) and is recognized for its therapeutic value. However, the continuous use of sorafenib may cause resistance in the treatment of cancer patients. In this study, we investigated whether sorafenib exerts an enhanced anticancer effect on CRC cells via the calcium-mediated deactivation of the focal adhesion kinase (FAK) signaling pathways. The appropriate dose of sorafenib and lactate calcium salt (CaLa) for a combination treatment were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Then, cell cycle analysis was performed following treatment with 2.5 µM sorafenib and/or 2.5 mM CaLa. CRC cells were found to be in the G1 phase by sorafenib treatment, and they accumulated in the sub-G1 phase with CaLa treatment. Western blots and enzyme-linked immunosorbent assays were performed to analyze the elements of the recombinant activated factor (RAF) and focal adhesion kinase (FAK) signaling cascades. Sorafenib-inhibited RAF-dependent signaling in CRC cells, however, either did not affect the expression of Akt or increased it. As the upstream signaling of FAK was suppressed by CaLa, we observed that the expression of the sub-signaling phospho (p) AKT and p-mammalian target of rapamycin was also suppressed. Treatment with a combination of sorafenib and CaLa enhanced the antitumor activity of CRC cells. The % viability of CRC cells was significantly decreased compared to the single treatment with sorafenib or CaLa, and the accumulation of Sub G1 of CRC cells was clearly confirmed. The migration ability of CRC cells was significantly reduced. The findings of this study indicate that sorafenib will show further improved antitumor efficacy against CRC due to overcoming resistance through the use of CaLa.

Antitumor Effect of Calcium-Mediated Destabilization of Epithelial Growth Factor Receptor on Non-Small Cell Lung Carcinoma.

Despite the development of numerous therapeutics targeting the epithelial growth factor receptor (EGFR) for non-small cell lung carcinoma (NSCLC), the application of these drugs is limited because of drug resistance. Here, we investigated the antitumor effect of calcium-mediated degradation of EGFR pathway-associated proteins on NSCLC. First, lactate calcium salt (LCS) was utilized for calcium supplementation. Src, α-tubulin and EGFR levels were measured after LSC treatment, and the proteins were visualized by immunocytochemistry. Calpeptin was used to confirm the calcium-mediated effect of LCS on NSCLC. Nuclear expression of c-Myc and cyclin D1 was determined to understand the underlying mechanism of signal inhibition following EGFR and Src destabilization. The colony formation assay and a xenograft animal model were used to confirm the in vitro and in vivo antitumor effects, respectively. LCS supplementation reduced Src and α-tubulin expression in NSCLC cells. EGFR was destabilized because of proteolysis of Src and α-tubulin. c-Myc and cyclin D1 expression levels were also reduced following the decrease in the transcriptional co-activation of EGFR and Src. Clonogenic ability and tumor growth were significantly inhibited by LSC treatment-induced EGFR destabilization. These results suggest that other than specifically targeting EGFR, proteolysis of associated molecules such as Src or α-tubulin may effectively exert an antitumor effect on NSCLC via EGFR destabilization. Therefore, LCS is expected to be a good candidate for developing novel anti-NSCLC therapeutics overcoming chemoresistance.

Enhancement of the Antitumor Effect of Methotrexate on Colorectal Cancer Cells via Lactate Calcium Salt Targeting Methionine Metabolism.

Methionine (Met) is involved in one-carbon de novo nucleotide synthesis and is an essential amino acid for cell survival. The impact of lactate calcium salt (CaLa) on the Met metabolism was investigated to evaluate the enhanced antitumor effect of methotrexate (MTX) on colorectal cancer (CRC) cells. Met dependency relating to homocysteine (Hcy) and betaine was investigated in human CRC cells (HCT-116 and HT-29) using a viability assay and liquid chromatography-mass spectrometry. Expression of betaine transporter-1 (BGT-1) following treatment with MTX alone or with CaLa was determined by Western blot. Enhanced antitumor effect due to malfunction of Met synthesis was confirmed. CRC cell viability decreased in Met-restricted medium, but was maintained after Hcy and betaine treatment while overcoming Met restriction. BGT-1 expression was downregulated following the treatment of dose-increased CaLa, whereas there was no effect on BGT-1 expression after MTX treatment. CaLa in combination with MTX induced reduced Met synthesis when CRC cell viability was reduced. The results indicated that CaLa-mediated BGT-1 downregulation inhibits Met synthesis by disrupting betaine homeostasis. CaLa raised the antitumor effect of MTX via secondary role in the inhibition of the de novo nucleotide synthesis. Combination therapy of MTX and CaLa could maximize the effectiveness of CRC treatment.

Synthetic strategy for increasing solubility of potential FLT3 inhibitor thieno[2,3-d]pyrimidine derivatives through structural modifications at the C2 and C6 positions.

Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic progenitor cell. In AML, a mutation in FLT3 is commonly occurs and is associated with poor prognosis. We have previously reported that thieno[2,3-d]pyrimidine derivative compound 1 exhibited better antiproliferative activity against MV4-11 cells which harbor mutant FLT3 than AC220, which is a well-known FLT3 inhibitor, and has good microsomal stability. However, compound 1 had poor solubility. We then carried out further structural modification at the C2 and the C6 positions of thieno[2,3-d]pyrimidine scaffold. Compound 13b, which possesses a thiazole moiety at the C2 position, exhibited better antiproliferative activity than compound 1 and showed increased solubility and moderate microsomal stability. These results indicate that compound 13b could be a promising potential FLT inhibitor for AML chemotherapy.

Ninjurin1 suppresses metastatic property of lung cancer cells through inhibition of interleukin 6 signaling pathway.

Nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a cell surface molecule that can mediate homophilic adhesion and promote neurite outgrowth from cultured dorsal root ganglion (DRG) neurons. Interestingly, Ninj1 overexpressed in human cancer; however, its role in metastasis is not clear. This study showed that inhibition of Ninj1 promotes lung cancer metastasis through interleukin 6 (IL-6)/STAT3 signaling. Ninj1 levels were relatively low in highly motile lung cancer cells. While inhibition of Ninj1 enhanced cell migration in lung cancer cells, overexpression of Ninj1 significantly suppressed it. We found that inhibition of Ninj1 significantly increased expression and secretion of IL-6 in A549 cells. We also found that inhibition of IL-6 decreased intercellular adhesion molecule 1 (ICAM-1) expression. In addition, inhibition of Ninj1 significantly increased cell motility and invasiveness of lung cancer cells. In an in vivo model, we found that Ninj1 suppression did not affect tumor growth but induced significant increase in incidence of lung metastasis, and sizes and number of tumor nodules. Taken together, our data clearly demonstrate that Ninj1 suppresses migration, invasion and metastasis of lung cancer via inhibition of the IL-6 signaling pathway in vitro and in vivo.

Identification of Targets of the HIF-1 Inhibitor IDF-11774 Using Alkyne-Conjugated Photoaffinity Probes.

We developed a hypoxia-inducible factor-1 (HIF-1) inhibitor, IDF-11774, as a clinical candidate for cancer therapy. To understand the mechanism of action of IDF-11774, we attempted to isolate target proteins of IDF-11774 using bioconjugated probes. Multifunctional chemical probes containing sites for click conjugation and photoaffinity labeling were designed and synthesized. After fluorescence and photoaffinity labeling of proteins, two-dimensional electrophoresis (2DE) was performed to isolate specific molecular targets of IDF-11774. Heat shock protein (HSP) 70 was identified as a target protein of IDF-11774. We revealed that IDF-11774 inhibited HSP70 chaperone activity by binding to its allosteric pocket, rather than the ATP-binding site in its nucleotide-binding domain (NBD). Moreover, IDF-11774 reduced the oxygen consumption rate (OCR) and ATP production, thereby increasing intracellular oxygen tension. This result suggests that the inhibition of HSP70 chaperone activity by IDF-11774 suppresses HIF-1α refolding and stimulates HIF-1α degradation. Taken together, these findings indicate that IDF-11774-derived chemical probes successfully identified IDF-11774's target molecule, HSP70, and elucidated the mode of action of IDF-11774 in inhibiting HSP70 chaperone activity and stimulating HIF-1α degradation in cancer cells.

Investigation of the functional difference between the pathological itching and neuropathic pain-induced rat brain using manganese-enhanced MRI.

BACKGROUND: There is a remarkable similarity in the central sensitization of itch and pain. However, the interactions between itch and pain are only partially understood. PURPOSE: To investigate the functional activity of cerebral regions to provide clear information on the neuronal pathways related to both pathological itching (PI) and neuropathic pain (NP). MATERIAL AND METHODS: Sprague-Dawley rats were used in this study. PI was induced via neonatal capsaicin treatment, and scratching behavior was counted. NP was induced via lumbar spinal nerve 5 (L5) ligation, and mechanical allodynia was measured. The activated cerebral regions in the control, PI, and NP rats were measured using a 4.7 T magnetic resonance imaging (MRI) system and manganese-enhanced MRI (MEMRI). Subsequently, the cerebral activation regions were identified, and the signal intensity was compared. RESULTS: Cerebral activities of the PI-induced rats were found in three regions -7.10 and -4.20 mm, and two regions -2.45 mm from the bregma. In the NP-induced rats, cerebral activities were found in two regions 7.10 and -2.45 mm, and one region -4.20 mm from the bregma. Comparing the PI and NP rats, the cerebral activities were different in one region -7.10 mm and -2.45 mm, and two regions -4.20 mm from the bregma. The different regions were the midbrain area, the geniculate complex, the hypothalamic area, and the amygdala area. CONCLUSION: Our MEMRI investigation indicates functionally different activity of cerebral regions due to the effect of PI or NP. These findings provide clear information of the signal transduction in the brain regarding PI or NP that share a similar neuronal pathway.

p53 Modulates Notch Signaling in MCF-7 Breast Cancer Cells by Associating With the Notch Transcriptional Complex Via MAML1.

p53 and Notch-1 play important roles in breast cancer biology. Notch-1 inhibits p53 activity in cervical and breast cancer cells. Conversely, p53 inhibits Notch activity in T-cells but stimulates it in human keratinocytes. Notch co-activator MAML1 binds p53 and functions as a p53 co-activator. We studied the regulation of Notch signaling by p53 in MCF-7 cells and normal human mammary epithelial cells (HMEC). Results show that overexpression of p53 or activation of endogenous p53 with Nutlin-3 inhibits Notch-dependent transcriptional activity and Notch target expression in a dose-dependent manner. This effect could be partially rescued by transfection of MAML1 but not p300. Standard and quantitative co-immunoprecipitation experiments readily detected a complex containing p53 and Notch-1 in MCF-7 cells. Formation of this complex was inhibited by dominant negative MAML1 (DN-MAML1) and stimulated by wild-type MAML1. Standard and quantitative far-Western experiments showed a complex including p53, Notch-1, and MAML1. Chromatin immunoprecipitation (ChIP) experiments showed that p53 can associate with Notch-dependent HEY1 promoter and this association is inhibited by DN-MAML1 and stimulated by wild-type MAML1. Our data support a model in which p53 associates with the Notch transcriptional complex (NTC) in a MAML1-dependent fashion, most likely through a p53-MAML1 interaction. In our cellular models, the effect of this association is to inhibit Notch-dependent transcription. Our data suggest that p53-null breast cancers may lack this Notch-modulatory mechanism, and that therapeutic strategies that activate wild-type p53 can indirectly cause inhibition of Notch transcriptional activity.

NSC126188 induces apoptosis of prostate cancer PC-3 cells through inhibition of Akt membrane translocation, FoxO3a activation, and RhoB transcription.

We previously reported that NSC126188 caused apoptosis of cancer cells by inducing expression of RhoB. We here present that NSC126188 induces apoptosis of prostate cancer PC-3 cells by inhibiting Akt/FoxO3 signaling, which mediates RhoB upregulation. The apoptosis and Akt dephosphorylation caused by NSC126188 was not substantially relieved by overexpressing wild-type Akt but was relieved by overexpressing constitutively active Akt (CA-Akt) or myristoylated Akt (myr-Akt). Furthermore, overexpression of CA-Akt or myr-Akt downregulated RhoB expression, indicating that RhoB expression is regulated by Akt signaling. Interestingly, membrane translocation of GFP-Akt by insulin exposure was abolished in the cells pretreated with NSC126188 suggesting that NSC126188 directly interfered with translocation of Akt to the plasma membrane. In addition, NSC126188 activated FoxO3a by dephosphorylating S253 via Akt inhibition. Activated FoxO3a translocated to the nucleus and increased transcription of RhoB and other target genes. PC-3 cells transiently overexpressing FoxO3a exhibited increased RhoB expression and apoptosis in response to NSC126188. Conversely, FoxO3a knockdown reduced NSC126188-induced RhoB expression and cell death. These results suggest that RhoB may be a target gene of FoxO3a and is regulated by Akt signaling. Taken together, NSC126188 induces apoptosis of PC-3 cells by interfering with membrane recruitment of Akt, resulting in Akt dephosphorylation and FoxO3a activation, which leads to transcription of RhoB.

Pharmacokinetics and metabolism of 4-O-methylhonokiol in rats.

The purpose of this study was to characterize the pharmacokinetics and metabolism of 4-O-methylhonokiol in rats. The absorption and disposition of 4-O-methylhonokiol were investigated in male Sprague-Dawley rats following a single intravenous (2 mg/kg) or oral (10 mg/kg) dose. Its metabolism was studied in vitro using rat liver microsomes and cytosol. 4-O-Methylhonokiol exhibited a high systemic plasma clearance and a large volume of distribution. The oral dose gave a peak plasma concentration of 24.1±3.3 ng/mL at 2.9±1.9 h and a low estimated bioavailability. 4-O-Methylhonokiol was rapidly metabolized and converted at least in part to honokiol in a concentration-dependent manner by cytochrome P450 in rat liver microsomes, predicting a high systemic clearance consistent with the pharmacokinetic results. It was also shown to be metabolized by glucuronidation and sulfation in rat liver microsomes and cytosol, respectively. 4-O-Methylhonokiol showed a moderate permeability with no apparent vectorial transport across Caco-2 cells, suggesting that intestinal permeation process is not likely to limit its oral absorption. Taken together, these results suggest that the rapid hepatic metabolism of 4-O-methylhonokiol could be the major reason for its high systemic clearance and low oral bioavailability.

Human microRNA-27a* targets Prf1 and GzmB expression to regulate NK-cell cytotoxicity.

Perforin (Prf1) and granzyme B (GzmB) are essential effector molecules for natural killer (NK)-cell cytotoxicity, but how Prf1 and GzmB expression is regulated during arming of NK cells is poorly defined. We show that human microRNA (miR)-27a* is a negative regulator of NK-cell cytotoxicity by silencing Prf1 and GzmB expression. Human miR-27a* specifically bound to the 3' untranslated regions of Prf1 and GzmB, down-regulating expression in both resting and activated NK cells, and it functioned as a fine-tuner for homeostasis of the net amount of the effector proteins. Consistent with miR-27a* having an inhibitory role, knockdown of miR-27a* in NK cells dramatically increased cytotoxicity in vitro and decreased tumor growth in a human tumor xenograft model. Thus, NK-cell cytotoxicity is regulated, in part, by microRNA, and modulating endogenous miR-27a* levels in NK cells represents a potential immunotherapeutic strategy.

Design, synthesis and biological evaluation of novel aliphatic amido/sulfonamido-quaternary ammonium salts as antitumor agents.

RhoB, one of the upstream signaling proteins of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, is frequently mutated in human cancer. Based on a piperazine alkyl derivative that induced apoptosis via up-regulation of RhoB, we synthesized novel aliphatic amido/sulfonamido-quaternary ammonium salts and evaluated their biological activities using an in vitro growth inhibition assay and RhoB promoter assay in human cancer cells. Compound 3a was the most promising anticancer agent in the series, based upon its potent growth inhibition via RhoB-mediated signaling. These novel aliphatic amido/sulfonamido-quaternary ammonium salts may be useful as a platform for development of anticancer chemotherapeutic agents.

Neutral sphingomyelinase 2 modulates cytotoxic effects of protopanaxadiol on different human cancer cells.

BACKGROUND: Some of ginsenosides, root extracts from Panax ginseng, exert cytotoxicity against cancer cells through disruption of membrane subdomains called lipid rafts. Protopanaxadiol (PPD) exhibits the highest cytotoxic effect among 8 ginsenosides which we evaluated for anti-cancer activity. We investigated if PPD disrupts lipid rafts in its cytotoxic effects and also the possible mechanisms. METHODS: Eight ginsenosides were evaluated using different cancer cells and cell viability assays. The potent ginsenoside, PPD was investigated for its roles in lipid raft disruption and downstream pathways to apoptosis of cancer cells. Anti-cancer effects of PPD was also investigated in vivo using mouse xenograft model. RESULTS: PPD consistently exerts its potent cytotoxicity in 2 cell survival assays using 5 different cancer cell lines. PPD disrupts lipid rafts in different ways from methyl-ß-cyclodextrin (MßCD) depleting cholesterol out of the subdomains, since lipid raft proteins were differentially modulated by the saponin. During disruption of lipid rafts, PPD activated neutral sphingomyelinase 2 (nSMase 2) hydrolyzing membrane sphingomyelins into pro-apoptotic intracellular ceramides. Furthermore, PPD demonstrated its anti-cancer activities against K562 tumor cells in mouse xenograft model, confirming its potential as an adjunct or chemotherapeutic agent by itself in vivo. CONCLUSIONS: This study demonstrates that neutral sphingomyelinase 2 is responsible for the cytotoxicity of PPD through production of apoptotic ceramides from membrane sphingomyelins. Thus neutral sphingomyelinase 2 and its relevant mechanisms may potentially be employed in cancer chemotherapies.

Ethanol extract of Magnolia officinalis prevents lipopolysaccharide-induced memory deficiency via its antineuroinflammatory and antiamyloidogenic effects.

Magnolia bark contains several compounds such as magnolol, honokiol, 4-O-methylhonokiol, obovatol, and other neolignan compounds. These compounds have been reported to have various beneficial effects in various diseases. There is sufficient possibility that ethanol extract of Magnolia officinalis is more effective in amyloidogenesis via synergism of these ingredients. Neuroinflammation has been known to play a critical role in the pathogenesis of Alzheimer's disease (AD). We investigated whether the ethanol extract of M. officinalis (10 mg/ kg in 0.05% ethanol) prevents memory dysfunction and amyloidogenesis in AD mouse model by intraperitoneal lipopolysaccharide (LPS, 250 µg/ kg/day for seven times) injection. We found that ethanol extract of M. officinalis prevented LPS-induced memory deficiency as well as inhibited the LPS-induced elevation of inflammatory proteins, such as inducible nitric oxide synthase and cyclooxygenase 2, and activation of astrocytes and microglia. In particular, administration of M. officinalis ethanol extract inhibited LPS-induced amyloidogenesis, which resulted in the inhibition of amyloid precursor protein, beta-site amyloid-precursor-protein-cleaving enzyme 1 and C99. Thus, this study shows that ethanol extract of M. officinalis prevents LPS-induced memory impairment as well as amyloidogenesis via inhibition of neuroinflammation and suggests that ethanol extract of M. officinalis might be a useful intervention for neuroinflammation-associated diseases such as AD.

Inhibitory effect of 4-O-methylhonokiol on lipopolysaccharide-induced neuroinflammation, amyloidogenesis and memory impairment via inhibition of nuclear factor-kappaB in vitro and in vivo models.

BACKGROUND: Neuroinflammation is important in the pathogenesis and progression of Alzheimer disease (AD). Previously, we demonstrated that lipopolysaccharide (LPS)-induced neuroinflammation caused memory impairments. In the present study, we investigated the possible preventive effects of 4-O-methylhonokiol, a constituent of Magnolia officinalis, on memory deficiency caused by LPS, along with the underlying mechanisms. METHODS: We investigated whether 4-O-methylhonokiol (0.5 and 1 mg/kg in 0.05% ethanol) prevents memory dysfunction and amyloidogenesis on AD model mice by intraperitoneal LPS (250 µg/kg daily 7 times) injection. In addition, LPS-treated cultured astrocytes and microglial BV-2 cells were investigated for anti-neuroinflammatory and anti-amyloidogenic effect of 4-O-methylhonkiol (0.5, 1 and 2 µM). RESULTS: Oral administration of 4-O-methylhonokiol ameliorated LPS-induced memory impairment in a dose-dependent manner. In addition, 4-O-methylhonokiol prevented the LPS-induced expression of inflammatory proteins; inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as well as activation of astrocytes (expression of glial fibrillary acidic protein; GFAP) in the brain. In in vitro study, we also found that 4-O-methylhonokiol suppressed the expression of iNOS and COX-2 as well as the production of reactive oxygen species, nitric oxide, prostaglandin E2, tumor necrosis factor-α, and interleukin-1ß in the LPS-stimulated cultured astrocytes. 4-O-methylhonokiol also inhibited transcriptional and DNA binding activity of NF-κB via inhibition of IκB degradation as well as p50 and p65 translocation into nucleus of the brain and cultured astrocytes. Consistent with the inhibitory effect on neuroinflammation, 4-O-methylhonokiol inhibited LPS-induced Aß1-42 generation, ß- and γ-secretase activities, and expression of amyloid precursor protein (APP), BACE1 and C99 as well as activation of astrocytes and neuronal cell death in the brain, in cultured astrocytes and in microglial BV-2 cells. CONCLUSION: These results suggest that 4-O-methylhonokiol inhibits LPS-induced amyloidogenesis via anti-inflammatory mechanisms. Thus, 4-O-methylhonokiol can be a useful agent against neuroinflammation-associated development or the progression of AD.

Induction of autophagy promotes preattachment development of bovine embryos by reducing endoplasmic reticulum stress.

The coupling of autophagy and endoplasmic reticulum (ER) stress has been implicated in a variety of biological processes; however, little is known regarding the involvement of the autophagy/ER stress pathway in early embryogenesis or the underlying mechanism(s). Here, we showed that the developmental competence of in vitro-produced (IVP) bovine embryos was highly dependent on the autophagy/ER stress balance. Although relative abundances of autophagy-associated gene transcripts, including LC3, Atg5, and Atg7 transcripts, were high in oocytes and throughout the early stages of preattachment development, extensive autophagosome formation was only detected in fertilized embryos. Using an inducer and inhibitor of autophagy, we showed that transient elevation of autophagic activity during early preattachment development greatly increased the blastocyst development rate, trophectoderm cell numbers, and blastomere survival; these same parameters were reduced by both inhibition and prolonged induction of autophagy. Interestingly, the induction of autophagy reduced ER stress and associated damage, while the developmental defects in autophagy-inhibited embryos were significantly alleviated by ER stress inhibitor treatment, indicating that autophagy is a negative regulator of ER stress in early embryos. Collectively, these results suggest that early embryogenesis of IVP bovine embryos depends on an appropriate balance between autophagy and ER stress. These findings may increase our understanding of important early developmental events by providing compelling evidence concerning the tight association between autophagy and ER stress, and may contribute to the development of strategies for the production of IVP bovine blastocysts with high developmental competence.

DNA methylation biomarker candidates for early detection of colon cancer.

Promoter CpG island hypermethylation of tumor suppressor genes is a common hallmark of all human cancers. Many researchers have been looking for potential epigenetic therapeutic targets in cancer using gene expression profiling with DNA microarray approaches. Our recent genome-wide platform of CpG island hypermethylation and gene expression in colorectal cancer (CRC) cell lines revealed that FBN2 and TCERG1L gene silencing is associated with DNA hypermethylation of a CpG island in the promoter region. In this study, promoter DNA hypermethylation of FBN2 and TCERG1L in CRC occurs as an early and cancer-specific event in colorectal cancer. Both genes showed high frequency of methylation in colon cancer cell lines (>80% for both of genes), adenomas (77% for FBN2, 90% for TCERG1L, n = 39), and carcinomas (86% for FBN2, 99% for TCERG1L, n = 124). Bisulfite sequencing confirmed cancer-specific methylation of FBN2 and TCERG1L of promoters in colon cancer cell line and cancers but not in normal colon. Methylation of FBN2 and TCERG1L is accompanied by downregulation in cell lines and in primary tumors as described in the Oncomine™ website. Together, our results suggest that gene silencing of FBN2 and TCERG1L is associated with promoter DNA hypermethylation in CRC tumors and may be excellent biomarkers for the early detection of CRC.

Structure and property based design, synthesis and biological evaluation of γ-lactam based HDAC inhibitors: part II.

Histone deacetylases (HDACs) are involved in post-translational modification and epi-genetic expression, and have been the intriguing targets for treatment of cancer. In previous study, we reported synthesis and the biological preliminary results of γ-lactam based HDAC inhibitors. Based on the previous results, smaller γ-lactam core HDAC inhibitors are more active than the corresponding series of larger δ-lactam based analogues and the hydrophobic and bulky cap groups are required for better potency which decreased microsomal stability. Thus, γ-lactam analogues with methoxy, trifluoromethyl groups of ortho-, meta-, para-positions of cap group were prepared and evaluated their biological potency. Among them, trifluoromethyl analogues, which have larger lipophilicity, showed better HDAC inhibitory activity than other analogues. In overall, lipophilicity leads to increase hydrophobic interaction between surface of HDAC active site and HDAC inhibitor, improves HDAC inhibitory activity.

Vitamin D3 upregulated protein 1 suppresses TNF-α-induced NF-κB activation in hepatocarcinogenesis.

Vitamin D(3) upregulated protein 1 (VDUP1) is a candidate tumor suppressor, the expression of which is dramatically reduced in various tumor tissues. In this study, we found that VDUP1 expression is suppressed during human hepatic carcinogenesis, and mice lacking VDUP1 are much more susceptible to diethylnitrosamine-induced hepatocarcinogenesis compared with wild type mice. VDUP1-deficient tumors proliferated significantly more than wild type tumors and had corresponding changes in the expression of key cell cycle regulatory proteins. In addition, the hepatomitogen-induced response was associated with a considerable increase in the release of TNF-α and subsequent enhancement of NF-κB activation in VDUP1-deficient mice. When cells were treated with TNF-α, the VDUP1 level was markedly reduced, concomitant with elevated NF-κB activation. Furthermore, the overexpression of VDUP1 resulted in the robust suppression of TNF-α-activated NF-κB activity via association with HDAC1 and HDAC3. These results indicate that VDUP1 negatively regulates hepatocarcinogenesis by suppressing TNF-α-induced NF-κB activation.