SMAD4 Controls Cancer Cell Metabolism by Regulating Methylmalonic Aciduria Cobalamin Deficiency (cbl) B Type.

Suppressor of mothers against decapentaplegic homolog (SMAD) 4 is a pluripotent signaling mediator that regulates myriad cellular functions, including cell growth, cell division, angiogenesis, apoptosis, cell invasion, and metastasis, through transforming growth factor ß (TGF-ß)-dependent and -independent pathways. SMAD4 is a critical modulator in signal transduction and functions primarily as a transcription factor or cofactor. Apart from being a DNA-binding factor, the additional SMAD4 mechanisms in tumor suppression remain elusive. We previously identified methyl malonyl aciduria cobalamin deficiency B type (MMAB) as a critical SMAD4 binding protein using a proto array analysis. This study confirmed the interaction between SMAD4 and MMAB using bimolecular fluorescence complementation (BiFC) assay, proximity ligation assay (PLA), and conventional immunoprecipitation. We found that transient SMAD4 overexpression down-regulates MMAB expression via a proteasome-dependent pathway. SMAD4-MMAB interaction was independent of TGF-ß signaling. Finally, we determined the effect of MMAB downregulation on cancer cells. siRNA-mediated knockdown of MMAB affected cancer cell metabolism in HeLa cells by decreasing ATP production and glucose consumption as well as inducing apoptosis. These findings suggest that SMAD4 controls cancer cell metabolism by regulating MMAB.

Macrosphelide A Exhibits a Specific Anti-Cancer Effect by Simultaneously Inactivating ENO1, ALDOA, and FH.

Aerobic glycolysis in cancer cells, also known as the Warburg effect, is an indispensable hallmark of cancer. This metabolic adaptation of cancer cells makes them remarkably different from normal cells; thus, inhibiting aerobic glycolysis is an attractive strategy to specifically target tumor cells while sparing normal cells. Macrosphelide A (MSPA), an organic small molecule, is a potential lead compound for the design of anti-cancer drugs. However, its role in modulating cancer metabolism remains poorly understood. MSPA target proteins were screened using mass spectrometry proteomics combined with affinity chromatography. Direct and specific interactions of MSPA with its candidate target proteins were confirmed by in vitro binding assays, competition assays, and simulation modeling. The siRNA-based knockdown of MSPA target proteins indirectly confirmed the cytotoxic effect of MSPA in HepG2 and MCF-7 cancer cells. In addition, we showed that MSPA treatment in the HEPG2 cell line significantly reduced glucose consumption and lactate release. MSPA also inhibited cancer cell proliferation and induced apoptosis by inhibiting critical enzymes involved in the Warburg effect: aldolase A (ALDOA), enolase 1 (ENO1), and fumarate hydratase (FH). Among these enzymes, the purified ENO1 inhibitory potency of MSPA was further confirmed to demonstrate the direct inhibition of enzyme activity to exclude indirect/secondary factors. In summary, MSPA exhibits anti-cancer effects by simultaneously targeting ENO1, ALDOA, and FH.

An Immune-Magnetophoretic Device for the Selective and Precise Enrichment of Circulating Tumor Cells from Whole Blood.

Here, we validated the clinical utility of our previously developed microfluidic device, GenoCTC, which is based on bottom magnetophoresis, for the isolation of circulating tumor cells (CTCs) from patient whole blood. GenoCTC allowed 90% purity, 77% separation rate, and 80% recovery of circulating tumor cells at a 90 µL/min flow rate when tested on blood spiked with epithelial cell adhesion molecule (EpCAM)-positive Michigan Cancer Foundation-7 (MCF7) cells. Clinical studies were performed using blood samples from non-small cell lung cancer (NSCLC) patients. Varying numbers (2 to 114) of CTCs were found in each NSCLC patient, and serial assessment of CTCs showed that the CTC count correlated with the clinical progression of the disease. The applicability of GenoCTC to different cell surface biomarkers was also validated in a cholangiocarcinoma patient using anti-EPCAM, anti-vimentin, or anti-tyrosine protein kinase MET (c-MET) antibodies. After EPCAM-, vimentin-, or c-MET-positive cells were isolated, CTCs were identified and enumerated by immunocytochemistry using anti-cytokeratin 18 (CK18) and anti-CD45 antibodies. Furthermore, we checked the protein expression of PDL1 and c-MET in CTCs. A study in a cholangiocarcinoma patient showed that the number of CTCs varied depending on the biomarker used, indicating the importance of using multiple biomarkers for CTC isolation and enumeration.

Development of an Equine Antitoxin by Immunizing the Halla Horse with the Receptor-Binding Domain of Botulinum Neurotoxin Type A1.

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are the most toxic substances known. However, the number of currently approved medical countermeasures for these toxins is very limited. Therefore, studies on therapeutic antitoxins are essential to prepare for toxin-related emergencies. Currently, more than 10,000 Halla horses, a crossbreed between the native Jeju and Thoroughbred horses, are being raised in Jeju Island of Korea. They can be used for equine antitoxin experiments and production of hyperimmune serum against BoNT/A1. Instead of the inactivated BoNT/A1 toxoid, Halla horse was immunized with the receptor-binding domain present in the C-terminus of heavy chain of BoNT/A1 (BoNT/A1-HCR) expressed in Escherichia coli. The anti-BoNT/A1-HCR antibody titer increased rapidly by week 4, and this level was maintained for several weeks after boosting immunization. Notably, 20 µL of the week 24 BoNT/A1-HCR(-immunized) equine serum showed an in vitro neutralizing activity of over 8 international unit (IU) of a reference equine antitoxin. Furthermore, 20 µL of equine serum and 100 µg of purified equine F(ab')2 showed 100% neutralization of 10,000 LD50 in vivo. The results of this study shall contribute towards optimizing antitoxin production for BoNT/A1, which is essential for emergency preparedness and response.

Salivary glutamate is elevated in individuals with chronic migraine.

Background Glutamate has been implicated in migraine pathogenesis, and is elevated in the plasma, cerebrospinal fluid, and saliva in migraineurs. However, no comparison of glutamate levels among chronic migraine, episodic migraine and controls has been reported. The aim is to compare salivary glutamate levels of individuals with chronic migraine with those of individuals with episodic migraine and healthy controls. Methods We investigated salivary glutamate level of 46 women with chronic migraine, 50 women with episodic migraine, and 19 healthy controls via enzyme linked immunosorbent assay. Results The salivary glutamate level of the chronic migraine group (median and interquartile range, 20.47 [15.27-30.15] pmol/mg total protein) was significantly higher than those of the episodic migraine (16.17 [12.81-20.15] pmol/mg total protein, p = 0.008) and control (12.18 [9.40-16.24] pmol/mg total protein, p = 0.001) groups. The salivary glutamate level of the episodic migraine group was marginally elevated from that of the control group (post hoc p = 0.016). Thresholds of 16.58 and 17.94 pmol/mg total protein optimize the sensitivity and specificity to differentiate chronic migraine participants from healthy controls and episodic migraine participants, respectively. Conclusions Salivary glutamate level was elevated in chronic migraine participants. These data suggest that salivary glutamate level could be an indicator of CM.

Upregulation of SMAD4 by MZF1 inhibits migration of human gastric cancer cells.

SMAD4 is a tumor suppressor that is frequently inactivated in many types of cancer. The role of abnormal expression of SMAD4 has been reported in developmental processes and the progression of various human cancers. The expression level of SMAD4 has been related to the survival rate in gastric cancer patients. However, the molecular mechanism underlying transcriptional regulation of SMAD4 remains largely unknown. In the present study, we characterized the promoter region of SMAD4 and identified myeloid zinc finger 1 (MZF1), as a putative transcription factor. MZF1 directly bound to a core region of the SMAD4 promoter and stimulated transcriptional activity. We also found that the expression of MZF1 influences the migration ability of gastric adenocarcinoma cells. Collectively, our results showed that MZF1 has a role in cellular migration of gastric cancer cells via promoting an increase in intracellular SMAD4 levels. This study might provide new evidence for the molecular basis of the tumor suppressive effect of the MZF1-SMAD4 axis, a new therapeutic target in advanced human gastric cancer.

Development and validation of a highly sensitive LC-MS/MS method for the determination of acacetin in human plasma and its application to a protein binding study.

A highly sensitive bioanalytical method for the quantification of acacetin in human plasma was developed and comprehensively validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A minimal volume of human plasma sample (20 µL) was prepared by simple deproteinization with 80 µL of acetonitrile. Chromatographic separation was performed using Kinetex C18 column with an isocratic mobile phase consisting of water and acetonitrile (20:80, v/v) containing 0.1 % formic acid at a flow rate of 0.3 mL/min over a total run time of 2.0 min. Mass spectrometric detection was performed using multiple reaction-monitoring modes at the mass/charge transitions m/z 285.22 â†’ 242.17 for acacetin and m/z 277.59 â†’ 175.04 for chlorpropamide (internal standard). The calibration curve was linear over the range of 0.1-500 ng/mL with a lower limit of quantitation of 0.1 ng/mL. The coefficients of variation for both intra- and inter-day validation were less than 11.9 %, and the intra- and inter-day accuracy ranged from 96.8 to 108 %. Mean recovery of acacetin in human plasma was within the range of 91.5-95.6 %. This validated LC-MS/MS method was successfully applied to a human plasma protein binding study that indicated extensive and concentration-independent protein binding of acacetin in human plasma.

Transducer of ERBB2.1 (TOB1) as a Tumor Suppressor: A Mechanistic Perspective.

Transducer of ERBB2.1 (TOB1) is a tumor-suppressor protein, which functions as a negative regulator of the receptor tyrosine-kinase ERBB2. As most of the other tumor suppressor proteins, TOB1 is inactivated in many human cancers. Homozygous deletion of TOB1 in mice is reported to be responsible for cancer development in the lung, liver, and lymph node, whereas the ectopic overexpression of TOB1 shows anti-proliferation, and a decrease in the migration and invasion abilities on cancer cells. Biochemical studies revealed that the anti-proliferative activity of TOB1 involves mRNA deadenylation and is associated with the reduction of both cyclin D1 and cyclin-dependent kinase (CDK) expressions and the induction of CDK inhibitors. Moreover, TOB1 interacts with an oncogenic signaling mediator, ß-catenin, and inhibits ß-catenin-regulated gene transcription. TOB1 antagonizes the v-akt murine thymoma viral oncogene (AKT) signaling and induces cancer cell apoptosis by activating BCL2-associated X (BAX) protein and inhibiting the BCL-2 and BCL-XL expressions. The tumor-specific overexpression of TOB1 results in the activation of other tumor suppressor proteins, such as mothers against decapentaplegic homolog 4 (SMAD4) and phosphatase and tensin homolog-10 (PTEN), and blocks tumor progression. TOB1-overexpressing cancer cells have limited potential of growing as xenograft tumors in nude mice upon subcutaneous implantation. This review addresses the molecular basis of TOB1 tumor suppressor function with special emphasis on its regulation of intracellular signaling pathways.

Design and synthesis of a macrosphelide A-biotin chimera.

The rational design and synthesis of a biochemical probe of natural (+)-macrosphelide A, a potent cell-cell adhesion inhibitor, was completed to aid in the identification of its biological target. The key features of the synthesis include: (1) an efficient synthesis of the macrosphelide core structure using Yamaguchi-Hirao alkynylation, (2) a cross metathesis to connect a linker unit to the allyl-macrosphelide and (3) coupling of the linker-bound macrosphelide A with a chemical biotin tag.

SMAD4 suppresses AURKA-induced metastatic phenotypes via degradation of AURKA in a TGFß-independent manner.

UNLABELLED: SMAD4 has been suggested to inhibit the activity of the WNT/ß-catenin signaling pathway in cancer. However, the mechanism by which SMAD4 antagonizes WNT/ß-catenin signaling in cancer remains largely unknown. Aurora A kinase (AURKA), which is frequently overexpressed in cancer, increases the transcriptional activity of ß-catenin/T-cell factor (TCF) complex by stabilizing ß-catenin through the inhibition of GSK-3ß. Here, SMAD4 modulated AURKA in a TGFß-independent manner. Overexpression of SMAD4 significantly suppressed AURKA function, including colony formation, migration, and invasion of cell lines. In addition, SMAD4 bound to AURKA induced degradation of AURKA by the proteasome. A luciferase activity assay revealed that the transcriptional activity of the ß-catenin/TCF complex was elevated by AURKA, but decreased by SMAD4 overexpression. Moreover, target gene analysis showed that SMAD4 abrogated the AURKA-mediated increase of ß-catenin target genes. However, this inhibitory effect of SMAD4 was abolished by overexpression of AURKA or silencing of AURKA in SMAD4-overexpressed cells. Meanwhile, the SMAD4-mediated repression of AURKA and ß-catenin was independent of TGFß signaling because blockage of TGFßR1 or restoration of TGFß signaling did not prevent suppression of AURKA and ß-catenin signaling by SMAD4. These results indicate that the tumor-suppressive function of SMAD4 is mediated by downregulation of ß-catenin transcriptional activity via AURKA degradation in a TGFß-independent manner. IMPLICATIONS: SMAD4 interacts with AURKA and antagonizes its tumor-promoting potential, thus demonstrating a novel mechanism of tumor suppression.

Synthesis and biological evaluation of a polyyne-containing sphingoid base probe as a chemical tool.

The sphingolipid metabolites have emerged as a starting point for the development of novel therapeutics for many diseases. However, details of the functions and mechanisms of sphingolipids remain unknown. To better understand the roles of sphingolipids, chemical tools with unique biological and physicochemical properties are needed. In this regard, we previously reported the synthesis of sphingoid base analogues in which the carbon chains are restricted by triple bonds. Here, we have conjugated a fluorescent dye to the polyyne analogues of the sphingoid bases to generate optical probes. Like the parent polyyne-containing sphingoid base, the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled triyne-sphingosine inhibited cancer cell growth far more effectively than did the corresponding sphingosine. NBD-triyne-sphingosine was rapidly incorporated into the cells and displayed broad cytoplasmic distribution. According to the results of a flow cytometric analysis, cancer cells fed with NBD-triyne-sphingosine showed significantly increased fluorescence intensity compared with the NBD-sphingosine treated cells. The metabolism of NBD-triyne-sphingosine was somewhat different from that of NBD-sphingosine. These results indicated that the incorporated rigid polyyne moiety in the sphingoid base altered the physicochemical properties of the sphingolipid, thereby affecting its biological behavior. The higher antiproliferative activity in the SRB assay and the significantly higher fluorescence intensity observed in the flow cytometric analysis are some of the interesting and distinct aspects of NBD-triyne-sphingosine compared to standard NBD-sphingosine probes. Thus, it is believed that the fluorescently labeled polyyne-containing sphingoid base developed in this study will be a useful chemical tool in sphingolipid research.

Low SP1 expression differentially affects intestinal-type compared with diffuse-type gastric adenocarcinoma.

Specificity protein 1 (SP1) is an essential transcription factor that regulates multiple cancer-related genes. Because aberrant expression of SP1 is related to cancer development and progression, we focused on SP1 expression in gastric carcinoma and its correlation with disease outcomes. Although patient survival decreased as SP1 expression increased (P<0.05) in diffuse-type gastric cancer, the lack of SP1 expression in intestinal-type gastric cancer was significantly correlated with poor survival (P<0.05). The knockdown of SP1 in a high SP1-expressing intestinal-type gastric cell line, MKN28, increased migration and invasion but decreased proliferation. Microarray data in SP1 siRNA-transfected MKN28 revealed that the genes inhibiting migration were downregulated, whereas the genes negatively facilitating proliferation were increased. However, both migration and invasion were decreased by forced SP1 expression in a low SP1-expressing intestinal-type gastric cell line, AGS. Unlike the intestinal-type, in a high SP1-expressing diffuse-type gastric cell line, SNU484, migration and invasion were decreased by SP1 siRNA. In contrast to previous studies that did not identify differences between the 2 histological types, our results reveal that low expression of SP1 is involved in cancer progression and metastasis and differentially affects intestinal-type compared with diffuse-type gastric adenocarcinoma.

Tob1 induces apoptosis and inhibits proliferation, migration and invasion of gastric cancer cells by activating Smad4 and inhibiting ß­catenin signaling.

Transducer of ErbB-2.1 (Tob1), a tumor suppressor protein, is inactivated in a variety of cancers including stomach cancer. However, the role of Tob1 in gastric carcinogenesis remains elusive. The present study aimed to investigate whether Tob1 could inhibit gastric cancer progression in vitro, and to elucidate its underlying molecular mechanisms. We found differential expression of Tob1 in human gastric cancer (MKN28, AGS and MKN1) cells. The overexpression of Tob1 induced apoptosis in MKN28 and AGS cells, which was associated with sub-G1 arrest, activation of caspase-3, induction of Bax, inhibition of Bcl-2 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, Tob1 inhibited proliferation, migration and invasion, which were reversed in MKN1 and AGS cells transfected with Tob1 siRNA. Overexpression of Tob1 in MKN28 and AGS cells induced the expression of Smad4, leading to the increased expression and the promoter activity of p15, which was diminished by silencing of Tob1 using specific siRNA. Tob1 decreased the phosphorylation of Akt and glycogen synthase kinase-3ß (GSK3ß) in MKN28 and AGS cells, resulting in the reduced protein expression and the transcriptional activity of ß­catenin, which in turn decreased the expression of cyclin D1, cyclin-dependent kinase-4 (CDK4), urokinase plasminogen activator receptor (uPAR) and peroxisome proliferator and activator receptor-δ (PPARδ). Conversely, silencing of Tob1 induced the phosphorylation of Akt and GSK-3ß, and increased the expression of ß­catenin and its target genes. Collectively, our study demonstrates that the overexpression of Tob1 inhibits gastric cancer progression by activating Smad4- and inhibiting ß­catenin-mediated signaling pathways.