Human Stomach Cancer Cells Research Articles

Determination of binding constant of transcription factor myc-max/max-max and E-box DNA: the effect of inhibitors on the binding.

The truncated myc and max proteins, only containing basic regions and helix-loop-helix/zipper (b/HLH/Zip) regions were over-expressed in E. coli and used for the determination of the binding constant and of the inhibitory mechanism on myc-max (or max-max)-DNA complex formation. The association kinetic constants (k(1) and k(-1)) of truncated max-max or myc-max dimer and DNA were determined as k(1)=(1.7+/-0.6)x10(5) M(-1) s(-1), k(-1)=(3.4+/-1.2)x10(-2) s(-1) for max-max and DNA or k(1)=(2.1+/-0.7)x10(5) M(-1) s(-1), k(-1)=(3.2+/-1.4)x10(-2) s(-1) for myc-max and DNA. The equilibrium binding constant (K(1)) was determined using these kinetic parameters [K(XXD)=(7.8+/-2.6)x10(6) M(-1) for max-max and DNA or K(XYD)=(6.9+/-2.2)x10(6) M(-1) for myc-max and DNA]. The binding constants of myc-max or max-max dimer formation were K(XX)=(2.6+/-0.9)x10(5) M(-1) or K(XY)=(1.3+/-0.4)x10(4) M(-1), respectively. When truncated proteins were used, the max-max dimer formation was easier than the myc-max dimer formation, contrary to the physiologically determined case. This leads us to deduce that domains other than b/HLH/Zip are very important for the transcriptional regulatory activity in physiological conditions. The truncated myc and max proteins, which were expressed in E. coli and contained only b/HLH/Zip regions were also used for the screening of inhibitors of myc-max-DNA complex formation. A synthesized curcuminoid, 1,7-bis(4-methyl-3-nitrophenyl)-1,6-heptadiene-3,5-dione (curcuminoid 004), showed the most potent inhibition out of the synthesized curcuminoids, in competition with DNA. The dissociation constant of max-max dimer and the inhibitor was 9 microM, when investigated using in vitro expressed b/HLH/Zip dimer proteins. The curcuminoid 004 showed an inhibitory effect on the binding of myc-max protein to the E-box element in SNU16 cells, and suppressed the expression of myc target genes including ornithine decarboxylase (ODC), cdc25a and c-myc in myc over-expressed human stomach cancer cell line SNU16.

Anti-cancer effects of COX-2 inhibitors and their correlation with angiogenesis and invasion in gastric cancer.

To observe the anti-cancer effects of COX-2 inhibitors and investigate the relationship between COX-2 inhibitors and angiogenesis, infiltration or metastasis in SGC7901 cancer xenografts. Thirty athymic mice xenograft models with human stomach cancer cell SGC7901 were established and divided randomly into 3 groups of 10 each. Sulindac, one non-specific COX inhibitor belonging to non-steroidal anti-inflammatory drugs (a series of COX inhibitors known as NSAIDs) and celecoxib, one selective COX-2 inhibitor (known as SCIs) were orally administered to mice of treatment groups. Immunohistochemistry was used to examine the expression of PCNA, CD44v6 and microvessel density (MVD). Apoptosis was detected by using TUNEL assay. Tumors in sulindac and celecoxib groups were significantly smaller than those in control group from the second week after drug administration (P<0.01). In treatment group, the cell proliferation index was lower (P<0.05) and apoptosis index was higher (P<0.05) than those in control groups. Compared with the controls, microvessel density was reduced (P<0.01) and expression of CD44v6 on tumor cells was weakened (P<0.05) in treatment groups. COX-2 inhibitors have anticancer effects on gastric cancer. They play important roles in angiogenesis and infiltration or metastasis of stomach carcinoma. The anticancer effects of COX-2 inhibitors may include inducing apoptosis, suppressing proliferation, reducing angiogenesis and weakening invasiveness.

Synthetic CDCA Derivatives-Induced Apoptosis of Stomach Cancer Cell Line SNU-1 Cells

This study was conducted to explore whether CDCA derivatives induce apoptosis in a stomach cancer cell line, and to dissect the detailed mechanism underlying apoptosis. The human stomach cancer cell line, SNU-1, cells were treated with the synthetic CDCA derivatives, HS-1199 and HS-1200. DNA and mitochondrial stains were used to detect apoptotic cells by fluorescence imaging or flow cytometry. The caspase-3 activity was measured by Western blotting. Both the HS-1199 and HS-1200 induced decreased viabilities of the SNU-1 cells, in time-dependent manners. The CDCA derivatives demonstrated various apoptosis hallmarks, such as mitochondrial changes (reduction of MMP, cytochrome c release, and Smac/ DIABLO translocation), activation of caspase-3 (resulting in the degradation of PARP and DFF45), DNA fragmentation and nuclear condensation. The CDCA derivatives, HS-1199 and HS-1200, both induced apoptosis of the SNU-1 gastric cancer cells in caspase- and mitochondria-dependent fashions. Many important issues relating to their therapeutic applications remain to be elucidated.

Orphan nuclear receptor Nur77 translocates to mitochondria in the early phase of apoptosis induced by synthetic chenodeoxycholic acid derivatives in human stomach cancer cell line SNU-1.

Apoptosis-inducing activity of synthetic CDCA derivatives, HS-1199 and HS-1200, on gastric cancer cell line SNU-1 cells was explored. CDCA derivatives demonstrated various apoptosis hallmarks, such as mitochondrial changes, activation of caspase, DNA fragmentation, and nuclear condensation. Importantly, the orphan receptor Nur77 (TR3) was shown to translocate from the nucleus to mitochondria at the early time points after CDCA derivatives treatment. These data support the theory that CDCA derivatives-induced apoptosis of SNU-1 gastric cancer cell lines is mediated by mitochondria and caspase, and, at least in part, by Nur77.

龍葵對人類胃癌細胞（SC-M1）胺基乙醯轉移酵素（NAT）的影響

Objectives. Gastric cancer is one of the leading causes of death in Taiwan and Solanum nigrum is one of the medicinal plants used in cancer therapy. However, there is no available information that addresses the mechanism by which Solanum nigrum affects human stomach cancer cells. Methods. We examined the dose effect of crude Solanum nigrum extract on the degree of N-acetylation of 2-aminofluorene (2-AF) in SC-M1 cell cytosol and intact cell cultures by acetyl Co-A recycling mixture assay and high pressure liquid chromatography (HPLC). Results. The crude extract of Solanum nigrum reducesed NAT activity in SC-M1 cytosol and intact cell cultures. Conclusions. Solanum nigrum affects malignant diseases by reducing NAT activity in SC-M1 cells.

Coordination modes vs. antitumor activity: synthesis and antitumor activity of novel platinum(II) complexes of N-substituted amino dicarboxylic acids

The trans-(±)-1,2-diaminocyclohexaneplatinum(II) complexes of multidentate l-glutamate (Glu) and l-aspartate (Asp) were prepared and their antitumor activity was examined in relation with their coordination modes. All these complexes were obtained as a mixture of (O,O′)- and (O,N)-chelate isomers due to rapid isomerization of the initially formed (O,O′)-isomer to the thermodynamically more stable (O,N)-isomer. The (O,O′)/(O,N)-isomeric mixture with the mole ratio of 80/20 exhibited excellent antitumor activity while the pure (O,N)-isomer was only marginally active. Therefore, in order to prevent the linkage isomerization of the active (O,O′)-isomer to the inactive (O,N)-isomer, we have designed N-substituted amino dicarboxylic acids as a leaving group and prepared a new series of complexes, [Pt(dach)(RGlu)] and [Pt(dach)(RAsp)] (dach=trans-(±)-1,2-diaminocyclohexane; R=acetyl (Ac), propionyl (Pro), pivaloyl (Piv), carbobenzyloxy (Cbz) or phthaloyl (Phth)) and characterized by means of elemental analyses, and 1H NMR, 195Pt NMR and IR spectroscopies. The N-substituted amino dicarboxylate ligands were found to coordinate to platinum(II) ion through only the (O,O′)-chelation mode, and their Pt(II) complexes were chemically stable in aqueous solution. The present Pt(II) complexes of N-substituted amino dicarboxylic acids showed excellent antitumor activity against both murine leukemia L1210 and human tumor cells. Especially, the highly hydrophobic N-phthaloylglutamate complex, [Pt(dach)(PhthGlu)], exhibited an outstanding in vitro activity (IC50=2.22 μM) on the human stomach cancer cells which are not responsive to cisplatin and carboplatin.

Cytotoxic diterpenes from Scoparia dulcis.

Four new labdane-derived diterpenes, iso-dulcinol (1), 4-epi-scopadulcic acid B (2), dulcidiol (4), and scopanolal (5), together with two known diterpenes, dulcinol/scopadulciol (3) and scopadiol (6), were isolated from the aerial parts of Scoparia dulcis. The structures were determined by extensive NMR studies. The crude extracts as well as the pure diterpenes showed cytotoxicity against a panel of six human stomach cancer cell lines.

Inhibition of inducible NF-κB activity reduces chemoresistance to 5-fluorouracil in human stomach cancer cell line

5-fluorouracil (5-FU) is used for the treatment of stomach and colon cancer, but many tumors are resistant to this chemotherapeutic agent. 5-FU induces apoptosis of several cancer cell lines, while some chemotherapeutic agents are known to activate the transcriptional factor NF-κB, which strongly suppresses apoptosis in vitro. In the present study, we investigated the relationship between activation of NF-κB and chemoresistance to 5-FU in human stomach cancer cell lines, NUGC3 (5-FU sensitive) and NUGC3/5FU/L (5-FU resistant). Treatment with 5-FU for 9–12 h caused activation of inducible NF-κB in NUGC3/5FU/L cells but not in NUGC3 cells. 5-FU also resulted in an increase in the number of TUNEL-positive cells and enhanced caspase-3 activity 3- to 5-fold in NUGC3 cells but not NUGC3/5FU/L cells. Moreover we also demonstrated that the inhibition of inducible NF-κB activation by using a NF-κB decoy could induce apoptosis and reduce chemoresistance against 5-FU. Our results suggest that 5-FU chemoresistance can be overcome by inhibition of inducible NF-κB activation, and that the use of the NF-κB decoy combined with 5-FU treatment is a new molecular and gene therapeutic strategy aimed at treatment of human stomach cancers resistant to 5-FU.

Src/ERK but not phospholipase D is involved in keratinocyte growth factor-stimulated secretion of matrix metalloprotease-9 and urokinase-type plasminogen activator in SNU-16 human stomach cancer cell.

We investigated the signaling pathway for keratinocyte growth factor (KGF)-induced invasion using human stomach cancer cell line, SNU-16. Alterations in the activities of Src, extracellular signal-regulated kinase (ERK), and phospholipase D (PLD) were measured using [gamma-(32)P] ATP for autophosphorylation of Src, phospho-specific ERK antibody, and [9,10-(3)H] myristic acid, respectively, while herbimycin A, PD98059 and butan-1-ol were used to inhibit their activities. Matrix metalloproteases (MMPs) and urokinase-type plasminogen activator (uPA) were quantified with zymography and Matrigel-coated Transwell was employed to estimate the invasiveness of SNU-16 cells. Src, ERK, and PLD were activated in response to KGF treatment, and inhibition of these enzymes - by their specific inhibitors - decreased KGF-induced invasion in a dose-dependent manner. However, only inhibition of Src and ERK could block KGF-stimulated secretion of uPA and MMP-9. Src, ERK, and PLD are suggested as mediators of KGF-induced invasion in SNU-16. uPA and MMP-9 are considered as downstream targets of Src and ERK whereas PLD is thought to utilize different pathways.

Unsaturated fatty acids bind Myc–Max transcription factor and inhibit Myc–Max–DNA complex formation

Oncoprotein Myc, hetero-dimerized with Max through a b/HLH/Zip region, is a transcription factor that governs important cellular processes such as cell cycle entry, proliferation and differentiation. We found that linoleic acid, isolated from Pollen Typhae, and other unsaturated fatty acids have strong inhibitory effects on the binding of Myc–Max heterodimer to an E-box DNA site (CA(C/T)GTG). The interaction of a fatty acid with a protein dimer, not with DNA, is assumed to block the entire Myc–Max–DNA complex formation. Unsaturated fatty acids also showed cytotoxicity against a SNU16 human stomach cancer cell line and conjugated linoleic acid suppressed mRNA expression of several myc-target genes; ornithine decarboxylase, p53, cdc25a in the SNU16 cells.

Specific induction of apoptosis by 1,8-cineole in two human leukemia cell lines, but not a in human stomach cancer cell line

We have investigated the effects of 1,8-cineole [the main component of essential oil prepared from bay-leaves Laurus nobilis L.)] on DNA of human leukemia cell lines, Molt 4B, HL-60 and stomach cancer KATO III cells. Specific induction of apoptosis by 1,8-cineole was observed in human leukemia Molt 4B and HL-60 cells, but not in human stomach cancer KATO III cells. Morphological changes showing apoptotic bodies were observed in the human leukemia HL-60 cells treated with 1,8-cineole. The fragmentations of DNA by cineole to oligonucleosomal-sized fragments that is a characteristic of apoptosis were concentration- and time-dependent in Molt 4B and HL-60 cells, but not in KATO III cells. The present study shows that the suppression growth by 1,8-cineole in the leukemia cell lines results from the induction of apoptosis by this compound.

Selective induction of apoptosis by ar-turmerone isolated from turmeric (Curcuma longa L) in two human leukemia cell lines, but not in human stomach cancer cell line

We have investigated the effects of ar-turmerone isolated from turmeric (Curcuma longa L) on DNA of human leukemia cell lines, Molt 4B, HL-60 and stomach cancer KATO III cells. It was found that selective induction of apoptosis by ar-turmerone was observed in human leukemia Molt 4B and HL-60 cells, but not in human stomach cancer KATO III cells. Morphological changes showing apoptotic bodies were observed in the human HL-60 and Molt 4B cells treated with ar-turmerone. The fragmentation of DNA by ar-turmerone to oligonucleosomal-sized fragments that is a characteristic of apoptosis was observed to be concentration- and time-dependent in Molt 4B and HL-60 cells, but not in KATO III cells. The data of the present study show that the suppression by ar-turmerone of growth of these leukemia cell lines results from the induction of apoptosis by this compound.

Activity-guided fractionation of green tea extract with antiproliferative activity against human stomach cancer cells.

Epidemiological studies have suggested that the consumption of green tea provides protection against stomach cancer. Fractionation of green tea extract, guided by antiproliferative activity against human stomach cancer (MK-1) cells, has resulted in the isolation of six active flavan-3-ols, epicatechin (EC), epigallocatechin (EGC), epigallocatechin gallate (EGCg), gallocatechin (GC), epicatechin gallate (ECg), gallocatechin gallate (GCg), together with inactive glycosides of kaempferol and quercetin. Among the six active flavan-3-ols, EGCg and GCg showed the highest activity, EGC, GC, ECg followed next, and the activity of EC was lowest. These data suggest that the presence of the three adjacent hydroxyl groups (pyrogallol or galloyl group) in the molecule would be a key factor for enhancing the activity. Since reactive oxygen species play an important role in cell death induction, radical scavenging activity was evaluated using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical. The order of scavenging activity was ECg > or = EGCg > or = EGC > or = GC > or = EC. The compounds having a galloyl moiety showed more potent activity. The contribution of the pyrogallol moiety in the B-ring to the scavenging activity seemed to be less than that of the galloyl moiety.

A potential use of a synthetic retinoid TAC-101 as an orally active agent that blocks angiogenesis in liver metastases of human stomach cancer cells.

TAC‐101 (4‐[3,5‐bis(trimethylsilyl)benzamido]benzoic acid) is a novel, synthetic retinoid that is effective against liver metastases of human gastrointestinal cancer cells such as the human stomach carcinoma line AZ‐521 in animal models, and is currently in use in phase I cancer trials. However, the mechanism of its antimetastatic action is still poorly understood. Tumor metastasis depends on angiogenesis, and various retinoids have been found to exhibit antiangiogenic activity. Based on these findings we here examined the antiangiogenic effects of TAC‐101. Oral administration of TAC‐101 (2‐8 mg/kg/day) resulted in a drastic suppression of the AZ‐521 cell‐induced angiogenesis in a mouse dorsal air sac assay system, compared to the vehicle alone. Immunohistochemical analysis with antibody against the endothelial marker CD31 revealed a significant reduction in microvessel density in liver metastases from animals treated with TAC‐101 (8 mg/kg p.o.), compared to liver metastases from the untreated control animals. The ability of TAC‐101 (8 mg/kg p.o.) to prevent experimental liver metastasis of AZ‐521 cells in athymic nude mice was comparable with that of the known angiogenesis inhibitor TNP‐470 (30 mg/kg s.c.). TAC‐101 also affected angiogenesis in chorioallantoic membranes and some functions of endothelial cells associated with angiogenesis, whereas the retinoid failed to suppress AZ‐521 cell proliferation directly. These data suggest that the TAC‐101 is an orally active antiangiogenic agent and that this antiangiogenic property may contribute to its efficacy against liver metastasis of human stomach cancer cells.

Cytotoxicity of the hinokitiol-related compounds, gamma-thujaplicin and beta-dolabrin.

Gamma-thujaplicin and beta-dolabrin, the constituents of the wood of Thujopsis dolabrata Sieb. et Zucc. var. hondai showed strong in vitro cytotoxic effects against the human stomach cancer cell lines KATO-III and Ehrlich's ascites carcinoma. The cytotoxic effects of the two compounds against both tumor cell lines were clear when cell growth was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Gamma-thujaplicin and beta-dolabrin at 0.32 microg/ml inhibited cell growth of human stomach cancer KATO-III by 85 and 67%, and Ehrlich's ascites carcinoma by 91 and 75%, respectively. There is no large difference in cytotoxicity between these compounds, but the activity of gamma-thujaplicin was slightly more potent than that of beta-dolabrin. On the other hand, hinokitiol acetate did not show a cytotoxic effect, suggesting that at least a part of the mechanism of the cytotoxic effect of hinokitiol-related compounds is due to metal chelation between the carbonyl group at C-1 and the hydroxyl group at C-2 in the tropolone skeleton of these molecules. The acute toxicities [50% lethal dose (LD50) value: intraperitoneal injection, Van der Waedem] of gamma-thujaplicin and beta-dolabrin in mice were 277 mg/kg and 232 mg/kg, respectively.

Genomic structure and chromosomal localization of the human hepatocyte growth factor activator inhibitor type 1 and 2 genes.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are recently discovered Kunitz-type serine protease inhibitors which can be purified and cloned from human stomach cancer cell line MKN45 as specific inhibitors against hepatocyte growth factor activator (HGFA). HAI-2 was identical with the protein originally reported as placental bikunin. Both proteins contain two Kunitz inhibitor domains (KDs), of which the first domain (KD1) is mainly responsible for the inhibitory activity against HGFA, and are expressed ubiquitously in various tissues. In this study, we cloned the genes coding for these two structurally similar proteins by screening of human genomic bacterial artificial chromosome (BAC) library and their genomic structures were compared. HAI-1 and -2 genes consist of 11 and 8 exons spanning 12 kbp and 12.5 kbp, respectively. Three exons were inserted between KD1 and KD2 of each gene, of which the middle one was the low-density lipoprotein (LDL) receptor-like domain (HAI-1) and the testis specific exon (HAI-2). Apparently homologous regions between HAI-1 and -2 were not found in 5'-flanking region and neither TATA nor CAAT box was present. The genes were mapped to chromosome 15q15 (HAI-1) and 19q13.11 (HAI-2). These results suggested that although HAI-1 and -2 genes might be derived from same ancestor gene, they acquired distinctive in vivo roles during their evolution.

Identification and characterization of a deletion mutant of DNA topoisomerase I mRNA in a camptothecin-resistant subline of human colon carcinoma.

In previous studies, we established two camptothecin (CPT)‐resistant sublines, HT‐29/CPT and St‐4/CPT, from the human colon cancer cell line HT‐29 and the human stomach cancer cell line St‐4, respectively. Cellular contents of DNA topoisomerase I (topo I) in the resistant cells were eight‐fold less than those in the corresponding parental lines. In this study, we have shown expression of two species of the TOP1 mRNA in HT‐29/CPT. The longer mRNA (4.0 kb) is the wild‐type TOP1 mRNA, and the shorter mRNA (3.3 kb) proved to have a deletion of 672 bp (nucleotides 58–729 or 59–730) that caused the in‐frame deletion of amino acids 20–243 of human topo I. The deleted region is identical to exons 3–9 of the TOP1 gene. The expression level of the 3.3‐kb mRNA was similar to that of the wild‐type mRNA in HT‐29/CPT. St‐4/CPT expressed only the wild‐type TOP1 mRNA in lesser amounts than did St‐4. Mouse NIH3T3 cells transfected with the wild‐type TOP1 cDNA showed higher sensitivity to CPT than the parental cells, whereas those transfected with the deleted TOP1 cDNA showed levels similar to those of the parental cells. Expression of the exogenous TOP1 mRNA was confirmed; however, expression of the truncated topo I was not detected in cells transfected with the deleted TOP1 cDNA. These results suggest that the expression of the deleted TOP1 mRNA led to the low expression of CPT‐sensitive topo I in the resistant cells.

Upregulation of HGF activator inhibitor type 1 but not type 2 along with regeneration of intestinal mucosa.

Hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are new Kunitz-type serine protease inhibitors that were recently purified and cloned from the human stomach cancer cell line MKN45 as specific inhibitors against HGF activator. Both proteins contain two Kunitz inhibitor domains and are expressed abundantly throughout the gastrointestinal tract, in addition to the placenta, pancreas, and kidney. In this study, to assess the possible roles of HAI-1 and HAI-2 in the intestinal mucosa, we examined the expression of HAI-1 and HAI-2 during regeneration of the intestinal mucosa. Immunohistochemical studies revealed that HAI-1 but not HAI-2 was detected more strongly in regenerative epithelium than in normal epithelium, although both proteins were detected throughout the human gastrointestinal tract. During the course of acetic acid-induced experimental colitis in an in vivo mouse model, HAI-1 but not HAI-2 was upregulated in the recovery phase, suggesting that HAI-1 but not HAI-2 is associated with the regeneration of damaged colonic mucosa. Upregulation of HAI-1 may serve to downregulate the proliferative response after initial activation of MET receptor by HGF/scatter factor after an injury.

The inhibitory effect of amiloride on the growth of human stomach cancer cells in vitro

The inhibitory effect of amiloride on the growth of human stomach cancer cells in vitro

Apoptosis-inducing activity of galloyl monosaccharides in human histiocytic lymphoma U937 cells.

Three galloyl monosaccharides contained in medicinal plants were examined for apoptosis-inducing activity in human histiocytic lymphoma U937 cells. Tetragalloyl glucose (TgG) induced apoptosis as found by chromatin condensation, DNA ladder formation, and inhibition by a caspase inhibitor. Digalloyl hamamelose had moderate activity, while monogalloyl glucose was only marginally active. These findings suggest that the number and disposition of their phenolic groups are important for apoptosis induction. TgG induced apoptosis in human colon and stomach cancer cell lines as well, indicating it is potentially useful as an anti-cancer agent.