Characterization of human gastric carcinoma-related methylation of 9 miR CpG islands and repression of their expressions in vitro and in vivo.

BACKGROUND: Many miR genes are located within or around CpG islands. It is unclear whether methylation of these CpG islands represses miR transcription regularly. The aims of this study are to characterize gastric carcinoma (GC)-related methylation of miR CpG islands and its relationship with miRNA expression. METHODS: Methylation status of 9 representative miR CpG islands in a panel of cell lines and human gastric samples (including 13 normal biopsies, 38 gastritis biopsies, 112 pairs of GCs and their surgical margin samples) was analyzed by bisulfite-DHPLC and sequencing. Mature miRNA levels were determined with quantitative RT-PCR. Relationships between miR methylation, transcription, GC development, and clinicopathological characteristics were statistically analyzed. RESULTS: Methylation frequency of 5 miR CpG islands (miR-9-1, miR-9-3, miR-137, miR-34b, and miR-210) gradually increased while the proportion of methylated miR-200b gradually decreased during gastric carcinogenesis (Ps < 0.01). More miR-9-1 methylation was detected in 62%-64% of the GC samples and 4% of the normal or gastritis samples (18/28 versus 2/48; Odds ratio, 41.4; P < 0.01). miR-210 methylation showed high correlation with H. pylori infection. miR-375, miR-203, and miR-193b methylation might be host adaptation to the development of GCs. Methylation of these miR CpG islands was consistently shown to significantly decrease the corresponding miRNA levels presented in human cell lines. The inverse relationship was also observed for miR-9-1, miR-9-3, miR-137, and miR-200b in gastric samples. Among 112 GC patients, miR-9-1 methylation was an independent favourable predictor of overall survival of GC patients in both univariate and multivariate analysis (P < 0.02). CONCLUSIONS: In conclusion, alteration of methylation status of 6 of 9 tested miR CpG islands was characterized in gastric carcinogenesis. miR-210 methylation correlated with H. pylori infection. miR-9-1 methylation may be a GC-specific event. Methylation of miR CpG islands may significantly down-regulate their transcription regularly.

Involvement of hepatopoietin Cn in the development of human hepatocellular carcinoma.

Hepatopoietin Cn (HPPCn) is a novel nuclear protein with the ability to promote liver regeneration. In the present study, we investigated the expression profile of HPPCn and its functional activity in human hepatocellular carcinoma (HCC) cell line and tissue samples. HPPCn expression was detected in HCC cell lines and 54 paired HCC carcinomas by immunochemical staining and Western blotting. The functional activity of HPPCn in cell lines was evaluated by MTT and colony formation assays and with nude mouse model. The correlation of HPPCn expression with clinicopathological characteristics of 54 HCC patients was also analyzed. Our results showed that HPPCn protein was prominently located within the nuclei of hepatocytes and the expression level was evidently increased in HepG2 and Bel7402 cell lines compared with L02 normal hepatocytes. HPPCn silencing by small interfering RNA greatly suppressed HepG2 cell proliferation and colony formation capacity and the inhibitory effect was also observed in a Balb/c-null mouse model. The silencing HPPCn expression effectively enhanced the apoptosis of HepG2 cells. In addition, HPPCn expression was detected in 48 of 54 (89%) human HCC tissues in sharp contrast with the corresponding non-tumor liver tissues. HPPCn protein was mainly accumulated in the tumor nucleus. The elevated expression of HPPCn protein in tumors was significantly associated with poor tumor cellular differentiation and present of vascular invasion. Patients with higher HPPCn expression in tumors had significantly shorter overall survival (OS) of both all of patients and the patients at the early stage. On multivariate Cox analysis, elevated expression of HPPCn in tumors was found to be an independent prognostic factor for OS. Therefore, these data suggest that HPPCn expression might be involved in the development of HCC and could be served as a promising biomarker.

Methylation of GATA-4 and GATA-5 and development of sporadic gastric carcinomas.

AIM: To understand the implication of GATA-4 and GATA-5 methylation in gastric carcinogenesis. METHODS: Methylation status of GATA-4 and GATA-5 CpG islands in human gastric mucosa samples, including normal gastric biopsies from 45 outpatients, gastric dysplasia [low-grade gastric intraepithelial neoplasia (GIN), n = 30; indefinite, n = 77], and 80 paired sporadic gastric carcinomas (SGC) as well as the adjacent non-neoplastic gastric tissues was analyzed by methylation specific polymerase chain reaction (MSP) and confirmed by denatured high performance liquid chromatography (DHPLC). Immunohistochemical staining was used to detect protein expression. The correlation between GATA-4 and GATA-5 methylation and clinicopathological characteristics of patients including Helicobacter pylori (H. pylori) infection was analyzed. RESULTS: GATA-4 and GATA-5 methylation was frequently observed in SGCs (53.8% and 61.3%, respectively) and their corresponding normal tissues (41.3% and 46.3%) by MSP. The result of MSP was consistent with that of DHPLC. Loss of both GATA-4 and GATA-5 proteins was associated with their methylation in SGCs (P = 0.01). Moreover, a high frequency of GATA-4 and GATA-5 methylation was found in both gastric low-grade GIN (57.1% and 69.0%) and indefinite for dysplasia (42.9% and 46.7%), respectively. However, GATA-4 and GATA-5 methylation was detected only in 4/32 (12.5%) and 3/39 (7.7%) of normal gastric biopsies. GATA-4 methylation in both normal gastric mucosa and low-grade GIN was also significantly associated with H. pylori infection (P = 0.023 and 0.027, two-sides). CONCLUSION: Epigenetic inactivation of GATA-4 (and GATA-5) by methylation of CpG islands is an early frequent event during gastric carcinogenesis and is significantly correlated with H. pylori infection.

Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas.

BACKGROUND: Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation. METHODS: Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0. RESULTS: From the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A approximately Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%). CONCLUSIONS: Most Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively.

[Biological properties of mesenchymal stem cells derived from bone marrow of patients with multiple myeloma].

The study was purposed to explore the role of mesenchymal stem cells (MSCs) in the pathogenesis of bone disease particularly observed in multiple myeloma (MM), the biological features of marrow derived MSCs from patients with MM have been investigated. Marrow aspirates were harvested from 11 newly diagnosed patients with MM and 5 normal adults and MSCs were isolated and culture-expanded by the cell properties of adherence to plastic flasks, The phenotype was analyzed by flow cytometric technique. The proliferation of MSCs was observed by MTT assay and their differentiation capacities into osteoblasts and adipoblasts were assessed with lineage-specific histochemical staining. The concentrations of IL-6 and SCF in the culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). MSC culture supernatants were collected and MTT assay was performed to evaluate their support on the proliferation of an MM cell line SKO007 cells. The results showed that bone marrow-derived MSCs from MM patients were homogeneously positive for CD29, CD73, CD166 and HLA-ABC and negative for hematopoietic cell marker CD45 and endothelial cell marker CD31, the phenotype of which was similar to that of marrow counterparts from normal adults. MTT assay indicated that MSCs from MM patients or normal adults proliferated at similar rates. MSCs from MM patients occupied in vitro osteogenic and adipogenic capacity as those from normal adults. The levels of IL-6 and SCF in culture supernatant were greatly up-regulated in MM patients by ELISA assay. Furthermore, MSC culture supernatants from MM bone marrow displayed enhanced activity to promote the proliferation of SKO007 cells. It is concluded that marrow-derived MSCs from bone marrow of MM patients are normal in their proliferation and differentiation capacities, and myeloma bone disease may not be ascribed to the differentiation of MSCs while the elevated secretion of IL-6 and SCF may provide necessary cues for the survival of malignant myeloma cells.

[Prognostic value of thymidylate synthase, topoisomerase-1 and Ki-67 in advanced colorectal cancer patients on irinotecan and fluorouracil treatment].

OBJECTIVE: To investigate the prognostic value of thymidylate synthase (TS), topoisomerase-1 (Topo-1), and proliferating index Ki-67 in advanced colorectal cancer patients on irinotecan (CPT-11) in combination with fluorouracil treatment (5-Fu). METHODS: The biomarker expression of TS, Topo-1 and Ki-67 in 78 patients detected immunohistochemically were correlated with the clinical outcome. RESULTS: The expressions of those biomarkers were not correlated with clinical therapeutic response, but with time to progression (TTP) and/or overall survival (OS). Patients with low expression of TS had significantly longer TTP (P < 0.05) and in OS (P < 0.05). The low expression of Ki-67 was also significantly predictive of longer survival (P < 0.05). As compared with any biomarker, the combination of any two biomarkers still possessed no predictive value to therapeutic response, but an enhanced predictive value to prognosis. The median time to progression in patients with low expression of TS, or Ki-67, or both were 9, 8 and 17 months, respectively; in patients with low expression of TS, or Topo-1, or both were 9, 9 and 13 months; in patients with low expression of Topo-1, or Ki-67, or both were 8, 9 and 11 months. TTP was significantly longer in patients with low expression of two biomarkers as compared with those with high expression (P = 0.031). CONCLUSION: TS, Topo-1, and Ki-67 are not predictive for chemotherapy response to CPT-11 combined with 5-Fu, but valuable in predicting prognosis. The combination of any two biomarkers can provide more powerful prognostic information for advanced colorectal cancer patients.

Antitumor effect of Gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor, combined with cytotoxic agent on murine hepatocellular carcinoma.

AIM: To investigate the inhibitory effect of gefitinib combined with cytotoxic agent cisplatin (CDDP) on hepatocellular carcinoma (HCC). METHODS: Female Kunming mice and H22 hepatocarcinoma cells were used. Gefitinib at daily dose of 100 mg/kg body weight (BW) or lecithin liquid was given by gastrogavage once a day for 5 or 10 successive days. CDDP or normal saline (NS) was administered intraperitoneally (i.p.) once a day for 5 successive days. Mice were randomly divided into control group (lecithin, or NS, i.p.), CDDP group (daily dose, 1.2 mg/kg BW; d1-5, or d6-10), Gefitinib (d1-5, or d6-10, or d1-10), and Gefitinib combined with CDDP groups. The inhibitory rate (IR) of tumor, net BW, spleen index (SI), thymus index (TI) and the amount of peripheral blood cells of mice were detected on the 12th experiment day. RESULTS: The growth of HCC in mice was inhibited by Gefitinib alone (IR: 41% in d1-10 group and 30% in d1-5 group, respectively) or CDDP alone (IR: 32-54% in d1-5 group or d6-10 group). The highest inhibitory effect (IR: 56%) on HCC growth was observed in Gefitinib (d1-10) combined with CDDP (d1-5) group. Higher inhibition was also observed in CDDP (d1-5) followed by Gefitinib (d6-10) group than that in Gefitinib (d1-5) followed by CDDP (d6-10) group (IR: 61% vs 36%, P < 0.01) in the independent study. Net BW, SI, TI and the amount of blood cells of mice in Gefitinib alone group were not significantly different from those in control groups. CONCLUSION: Gefitinib can significantly inhibit the growth of murine H22 hepatocellular carcinoma. If Gefitinib is used after CDDP treatment in animal experiments, the inhibitory effect could be enhanced.

[The effect of IRESSA on H22 mouse hepatocellular carcinoma].

OBJECTIVE: To investigate the effect of IRESSA (gefitinib, ZD1839) on H22 murine hepatocellular carcinoma. METHODS: Mice bearing H22 hepatocellular carcinoma were randomly divided into oral control group, Normal saline (NS) control group, cisplatin (CDDP) d1-5 group, CDDP d6-10 group, IRESSA group, IRESSA combined with CDDP early (IRESSA + CDDP d1-5) group, and IRESSA combined with CDDP lately (IRESSA + CDDP d6-10) group. IRESSA was given by daily gastrogavage for 10 days (day 1-day 10) at 100 mg/kg in body weight (BW). CDDP was given by daily intraperitoneal injection for 5 days (day 1-day 5, or day 6-day 10) at 1.2 mg/kg in BW. The growth inhibiting rate (IR) of tumor, change of BW, spleen index (SI), and amounts of blood leucocyte or hemoglobin were detected. RESULTS: IR of tumor in IRESSA group was not significantly difference with that in CDDP d1-5 group, CDDP d6-10 group, IRESSA + CDDP d1-5 group (P > 0.05). IR of tumor in IRESSA group, CDDP d1-5 group, CDDP d6-10 group, IRESSA and IRESSA + CDDP d1-5 group were 41%, 54%, 46%, and 56%, respectively. IR of tumor in IRESSA + CDDP d6-10 group (26%) was significantly lower than that in CDDP d6-10 group (P < 0.05) or in IRESSA + CDDP d1-5 group (P < 0.01). Compared with oral or NS control groups, SI and net BW in IRESSA group was not significantly difference (P > 0.05). SI and net BW in both IRESSA + CDDP d1-5 group and IRESSA + CDDP d6-10 group were lower markedly than those in IRESSA group (P < 0.01). CONCLUSION: Tumor growth of H22 bearing mice was markedly inhibited by IRESSA.

[Enhancement of antioxidant capability of cancer patients during chemotherapy by reduced glutathione].

BACKGROUND & OBJECTIVE: It is unknown how administration of reduced glutathione (GSH) affects chemotherapy of cancer patients. This study was designed to investigate the effect of GSH on lipid peroxidation, and activities of antioxidant enzyme among cancer patients with chemotherapy. METHODS: Sixty-two cancer patients with chemotherapy were enrolled randomly into AB or BA group in cross-over pattern. In AB group, combination of chemotherapy and GSH was administrated first, then following chemotherapy alone was given 21 or 28 days later. In group BA, chemotherapy alone was administrated first, then the combination therapy was given. Duration of chemotherapy was 2-5 days, 21-28 days for a cycle, depended on chemotherapy strategies. GSH was given as a 15 minute intravenous infusion at the dose of 1 500 mgx(m(2)xd)(-1) for 7 days from day 1. Serum samples were collected from the patients on the day just before the chemotherapy, the 7(th) day, and the 21(st) (if 21 days per cycle of the chemotherapy) or 28(th) day of treatment. Concentration of malondialdehyde (MDA), activity of glutathione peroxidase (GSH-Px), and total superoxide dismutase (T-SOD) of serum samples were analyzed biochemically. RESULTS: (1)Administration of chemotherapy significantly increased serum MDA level on the 7(th) day compared with that before chemotherapy (mean+/-SD,6.12+/-1.94 micromol/L versus 4.63+/-1.87 micromol/L,P< 0.01). The increased serum MDA level was restored partially (5.05+/-2.07)micromol/L on the 21(th) or 28(th) day, but still higher than that before chemotherapy (P< 0.05). (2)Serum activity of T-SOD and GSH-Px decreased on the 7(th) day (P< 0.01) and restored partially on the 21(th) or 28th day, but still lower than that before chemotherapy (T-SOD, P< 0.05;GSH-Px,P< 0.01).(3)Co-treatment of GSH prevents lipid peroxidation and depletion of antioxidant enzymes by chemotherapy partially but significantly (P< 0.01). (4)Similar results were obtained in both AB group and BA group. CONCLUSION: Chemotherapy depletes antioxidant capability of cancer patients and co- treatment of GSH might prevent such depletion.

Silencing-specific methylation and single nucleotide polymorphism of hMLH1 promoter in gastric carcinomas.

AIM: To investigate CpG methylation and single nucleotide polymorphism (SNP) of a specific promoter region of hMLH1 in primary gastric carcinoma. METHODS: Primary gastric carcinomas (n=80), their corresponding normal mucosal samples, and gastric mucosal biopsies from normal/gastritis control patients (n=54) were used. Hypermethylation at -253 nt and -251 nt in relation with the translational start site and SNP of a silencing specific region (-339 nt-46 nt) in the hMLH1 promoter were analyzed by Bst UI-combined bisulfite assay (COBRA), denaturing high performance liquid chromatogram (DHPLC), and sequencing. RESULTS: (A) The specific methylation at -253 nt and -251 nt was observed in 2 of 60 primary gastric carcinomas, but neither in all of the corresponding mucosa nor in normal/gastritis samples, by Bst UI-COBRA and DHPLC. (B) The hMLH1 promoter was methylated homogeneously in the xenograft of the primary gastric carcinoma with the methylated and unmethylated hMLH1. (C) The pattern of SNP at -93 nt of the hMLH1 promoter in 54 Chinese patients with gastric carcinoma was the same as that in the control patients: 51 % was A/G heteroalleles, 34 % and 15 % were A/A and G/G homoalleles, respectively. CONCLUSION: Biallelic inactivation of hMLH1 by epigenetic silencing existed in human primary gastric carcinoma homogeneously. Hypermethylation of hMLH1 may play a role in the early stage of development of a few gastric carcinomas. The SNP at -93 nt is not related to the susceptibility of gastric carcinomas.