Identification of selective inhibitors for diffuse-type gastric cancer cells by screening of annotated compounds in preclinical models.

BACKGROUND: Diffuse-type gastric cancer (DGC) exhibits rapid disease progression and poor patient prognosis. We have previously established an E-cadherin/p53 double conditional knockout (DCKO) mouse line as the first genetically engineered one, which morphologically and molecularly recapitulates human DGC. In this study, we explored low-molecular-weight drugs selectively eliminating mouse and human DGC cells. METHODS: We derived mouse gastric cancer (GC) cell lines from DGC of the DCKO mice demonstrating enhanced tumourigenic activity in immunodeficient mice and acquired tolerance to cytotoxic anti-cancer agents. RESULTS: We performed a synthetic lethal screening of 1535 annotated chemical compounds, and identified 27 candidates selectively killing the GC cell lines. The most potent drug mestranol, an oestrogen derivative, and other oestrogen receptor modulators specifically attenuated cell viability of the GC cell lines by inducing apoptosis preceded by DNA damage. Moreover, mestranol could significantly suppress tumour growth of the GC cells subcutaneously transplanted into nude mice, consistent with longer survival time in the female DCKO mice than in the male. Expectedly, human E-cadherin-mutant and -low gastric cancer cells showed higher susceptibility to oestrogen drugs in contrast to E-cadherin-intact ones in vitro and in vivo. CONCLUSIONS: These findings may lead to the development of novel therapeutic strategies targeting DGC.

Plasma microRNA-103, microRNA-107, and microRNA-194 levels are not biomarkers for human diffuse gastric cancer.

PURPOSE: Diffuse-type gastric cancer (DGC) carries a poor prognosis. Effective screening is one measure that might improve the prognosis of this disease. An E-cadherin/p53 double-conditional knockout (DCKO) mouse line recapitulates human DGC morphologically and molecularly. Three circulating microRNAs (miRNA) (miR-103, miR-107, miR-194) in DCKO mice have been identified as biomarkers for DGC. We sought to evaluate whether these circulating miRNAs could be used for the detection of human DGC. METHODS: Subjects were 50 patients with DGC. Controls were first-time outpatients at Aichi Cancer Center Hospital, age- and sex-matched, without a cancer diagnosis. Total RNA containing miRNA was extracted from the plasma samples and then reverse-transcribed. The levels of miRNAs in plasma samples were quantitatively determined by real-time RT-PCR. Spiked-in cel-miR-39 was analyzed as a normalization control. RESULTS: Levels of the three plasma microRNA levels in DGC cases with or without an intestinal component were not significantly different from those in control subjects. The areas under the receiver operating characteristic curve of miR-103, miR-107, and miR-194 were 0.548, 0.563, and 0.512, respectively. CONCLUSIONS: In contrast to the DCKO mouse model, plasma miR-103, miR-107, and miR-194 levels are not altered in DGC and are not suitable for human DGC screening.

[Clinical utility of Pourmedia GBS agar on screening for vaginal colonization of Group B Streptococcus].

Group B Streptococcus (GBS) are normal flora of the vagina and intestinal, but if the pregnant woman was infected with GBS in the vagina, miscarriage or premature would occur or the newborn would be developed to severe GBS infection. It is recommended that the inspection of GBS on all pregnant women by Japan Society of Obstetrics and Gynecology (JSOG) and Center for Disease Control and Prevention (CDC). We examined the comparison of detection rate between Pourmedia GBS agar (Eiken Chemical Co., Ltd.) and Nissui Separated Plate Sheep Blood Agar/BTB Lactose Agar medium (Nissui Pharmaceutical Co., Ltd.) on 112 sample. The positive rate of Pourmedia GBS agar was 21.4% (24/112 samples), Whereas Nissui Separated Plate Sheep Blood Agar/BTB Lactose Agar medium was 17.8% (20/112 samples). It was found that the detection rate was improved by using Pourmedia GBS agar on GBS screening test of vaginal swab.

Insulin-like growth factor 2 hypomethylation of blood leukocyte DNA is associated with gastric cancer risk.

To determine whether or not the methylation status of blood leukocyte DNA can be used as a surrogate marker of the risk for cancer, we quantitatively determined the methylation levels of insulin-like growth factor 2 (IGF2) and TUSC3 in 299 gastric cancer cases, and 299 age- and gender-matched controls. The IGF2 methylation levels in blood leukocyte DNA of the cases were lower than those of the healthy controls and there was a significant trend of increasing gastric cancer risk with decreasing methylation level of IGF2. Patients with hypermethylated IGF2 in blood leukocyte DNA showed a significantly better survival rate than those with hypomethylated IGF2, indicating that the IGF2 methylation level in blood leukocyte DNA can be a possible marker not only of the risk for but also of the prognosis of gastric cancer. In contrast, the TUSC3 methylation level in blood leukocyte DNA was higher in the cases than in the healthy controls, but the difference was not significant. The past lifestyle and clinicopathological characteristics of the participants were analyzed for any relationship with the methylation level. With aging and smoking, methylation of IGF2 and TUSC3 decreased and increased in blood leukocyte DNA, respectively. These results indicate that the methylation level of IGF2 in blood leukocyte DNA may be used as an important surrogate marker of the risk for gastric cancer.

DNA methylation status is inversely correlated with green tea intake and physical activity in gastric cancer patients.

Epigenetic silencing of genes by aberrant DNA methylation is recognized as a crucial component of the mechanism underlying tumorigenesis. However, the relationship between DNA methylation and the past lifestyle in cancer patients remains largely unknown. We examined the methylation statuses of 6 tumor-related genes, CDX2 (homeobox transcription factor), BMP-2 (bone morphogenetic protein 2), p16 (INK4A), CACNA2D3 (calcium channel-related), GATA-5 (transcription factor) and ER (estrogen receptor), in 106 primary gastric carcinomas by methylation-specific PCR and compared them with the past lifestyles of the patients. The methylation frequencies of the genes were 23.6, 21.7, 9.4, 32.4, 40.8 and 59.1%, respectively. Significant association was found between a decreased intake of green tea and methylation of CDX2 and BMP-2. More physical activity was correlated with a lower methylation frequency of CACNA2D3. Of these 6 genes, the methylation statuses of CDX2, BMP-2 and p16 revealed a significant interrelationship and those of CACNA2D3, GATA-5 and ER did likewise. Thus, some epidemiological factors, such as green tea intake, could be important as to determination of the methylation statuses of selected genes and may influence the development of cancer, including that of the stomach.

Methylation of the calcium channel-related gene, CACNA2D3, is frequent and a poor prognostic factor in gastric cancer.

BACKGROUND & AIMS: The calcium channel voltage-dependent alpha2delta subunit consists of 4 genes, CACNA2D1 to CACNA2D4, of which CACNA2D2 and CACNA2D3 are located on 3p21.3 and 3p21.1, respectively. Here, we examined the relation between alpha2delta subunit gene alterations and gastric carcinogenesis. METHODS: The expression and methylation status of the alpha2delta subunit genes were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and methylation-specific PCR in gastric cancers (GCs). The effects of CACNA2D3 expression were examined by cell proliferation and adhesion assays, and they predicted target gene alterations. RESULTS: Aberrant methylation of CACNA2D1 and CACNA2D3 mostly corresponded to their expression status in GC cell lines. CACNA2D1/3 methylation was detected in 10 (12.5%) and 24 (30%) of the 80 GC cases, respectively, but no CACNA2D2 methylation was seen in 32 cases. CACNA2D3 methylation was more frequently found in diffuse type than in intestinal type (16/38 [42.1%] vs 8/42 [19.0%]; P = .025) GCs. Among the 53 patients with advanced GCs, patients with cancers showing CACNA2D3 methylation had a significantly shorter survival time than patients without this methylation (P = .003). Exogenous CACNA2D3 expression strongly inhibited cell growth and adhesion and up-regulated p21 and p27 expression in HEK-293T and NUGC4 cells. Inverse effects were seen by CACNA2D3 small interfering RNA treatment in the CACNA2D3-positive cell lines, indicating that CACNA2D3 may have tumor suppressive functions. CONCLUSIONS: Loss of CACNA2D3 expression through aberrant promoter hypermethylation may contribute to gastric carcinogenesis, and CACNA2D3 methylation is a useful prognostic marker for patients with advanced GC.

Relationship between CDX2 gene methylation and dietary factors in gastric cancer patients.

Epigenetic gene silencing through DNA methylation is one of the important steps in the mechanism underlying tumorigenesis, including in the stomach. Past lifestyle factors of cancer patients, such as intake of vegetables, are very important in affecting gastric carcinogenesis. However, the relationship between DNA methylation and past dietary habits in cancer patients remains largely unknown. The CDX2 homeobox transcription factor plays a key role in intestinal development, but CDX2 is also expressed in most of the intestinal metaplasia and part of the carcinomas of the stomach. We analyzed the methylation status of the CDX2 5' CpG island in gastric cancer cell lines by methylation-specific PCR (MSP), and then CDX2 mRNA was found to be activated after 5-aza-2'-deoxycytidine treatment of the methylation-positive cells. We further examined the methylation status of CDX2 in primary gastric carcinomas by MSP and compared it with the past lifestyle of the patients, including dietary habits. Methylation of CDX2 was found in 20 (34.5%) of the 58 male patients and one (6.7%) of the 15 female patients. Since the methylation frequency was low in the female patients, the analysis was performed only on the male cases. CDX2 methylation was correlated with the decreased intake of green tea and cruciferous vegetables, and also with full or overeating habits. These findings are consistent with epidemiological observations on gastric cancer. We also analyzed the methylation status of p16/INK4a and hMLH1, but their frequencies were not associated with dietary factors or other lifestyle factors. Thus, diet could be an important factor determining the methylation status of genes such as CDX2 and the resultant aberrant expression of genes involved in carcinogenesis.

Absorption, tissue distribution, excretion, and metabolism of clothianidin in rats.

Absorption, distribution, excretion, and metabolism of clothianidin [(E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine] were investigated after a single oral administration of [nitroimino-(14)C]- or [thiazolyl-2-(14)C]clothianidin to male and female rats at a dose of 5 mg/kg of body weight (bw) (low dose) or 250 mg/kg of bw (high dose). The maximum concentration of carbon-14 in blood occurred 2 h after administration of the low oral dose for both labeled clothianidins, and then the concentration of carbon-14 in blood decreased with a half-life of 2.9-4.0 h. The orally administered carbon-14 was rapidly and extensively distributed to all tissues and organs within 2 h after administration, especially to the kidney and liver, but was rapidly and almost completely eliminated from all tissues and organs with no evidence of accumulation. The orally administered carbon-14 was almost completely excreted into urine and feces within 2 days after administration, and approximately 90% of the administered dose was excreted via urine. The major compound in excreta was clothianidin, accounting for >60% of the administered dose. The major metabolic reactions of clothianidin in rats were oxidative demethylation to form N-(2-chlorothiazol-5-ylmethyl)-N'-nitroguanidine and the cleavage of the carbon-nitrogen bond between the thiazolylmethyl moiety and the nitroguanidine moiety. The part of the molecule containing the nitroguanidine moiety was transformed mainly to N-methyl-N'-nitroguanidine, whereas the thiazol moiety was further metabolized to 2-(methylthio)thiazole-5-carboxylic acid. With the exception of the transiently delayed excretion of carbon-14 at the high-dose level, the rates of biokinetics, excretion, distribution, and metabolism of clothianidin were not markedly influenced by dose level and sex.