International Journal of Molecular Medicine

Tang, Hui ; Guo, Qiang ; Zhang, Chao ; Zhu, Jun ; Yang, Hui ; Zou, Yun-Lian ; Yan, Yan ; Hong, Dong ; Sou, Tao ; Yan, Xin-Min

doi: 10.3892/ijmm_00000508pmid: 20878084

The colorectal adenoma-carcinoma sequence describes the stepwise progression from normal to dysplastic epithelium and then to carcinoma. Only a small proportion of colorectal adenomas (CRAs) progress to colorectal carcinomas (CRCs). Endoscopic intervention is currently being used on patients with high grade dysplasia CRAs, with diameters of >1 cm, or villous components of >25% who are at higher risk than other CRA sufferers. During the process, biopsy samples are taken for conventional histological diagnosis, but poor pathomorphological sensitivity and specificity greatly limit the diagnostic accuracy. Unfortunately, there are no reliable molecular criteria available that can predict the potential development of CRA to CRC. Gene expression profiles of normal colorectal mucosa (NOR), CRA and different Dukes' stages of CRC biopsy specimens, which represent the gradual progress of the CRA to CRC sequence, were determined by Affymetrix technology. Representative regulated genes were further analyzed by quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Intersectional analyses of discriminative expression signatures of CRC vs. CRA and CRA vs. NOR allowed the identification of an intermediate signature of 463 probe sets (psets) that mark the NOR↷ CRA↷CRC progression. This signature represents a reservoir of candidate markers for the early diagnosis of higher-risk CRA, thus allowing for timely therapeutic intervention and more selective treatment. A further 279 CRC-specific psets pointing to the malignant transition from CRA to CRC were identified and these could represent potential therapeutic targets for CRC. The reliability of the results was further confirmed by qRT-PCR and IHC analyses of the 4-gene sets randomly selected from the 463 psets.

Jedinak, Andrej ; Dudhgaonkar, Shailesh ; Jiang, Jiahua ; Sandusky, George ; Sliva, Daniel

doi: 10.3892/ijmm_00000509pmid: 20878085

Colorectal cancer is one of the leading causes of cancer deaths in both men and women in the world. However, colon cancer can be prevented to some extent by consumption of edible natural products with chemopreventive properties. Therefore, we investigated, whether edible mushroom Pleurotus ostreatus (PO) has chemopreventive effect on inflammation-associated colon carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) and promoted by dextran sodium sulfate (DSS). PO treatment, at both doses (100 and 500 mg/kg), significantly reduced by 50 and 78% the number of aberrant crypt foci and the multiplicity of colon neoplasms by 43 and 89%, respectively. However, incidence of colon tumors and high grade dysplasia was reduced by 50 and 63% only in the dose 500 mg/kg of PO, respectively. Colon shortening and dysplastic index was significantly reduced by PO treatment in dose-dependent manner. The immunohistochemistry of colons revealed that treatment with PO suppressed expression of cyclin D1, Ki-67, COX-2 and F4/80. In summary, our data suggest that PO may prevent inflammation-associated colon carcinogenesis with exposure to PhIP through combined modulatory mechanisms of inflammation and tumor growth via suppression of COX-2, F4/80, Ki-67 and cyclin D1 expression in mice.

Lee, Byung Cheol; Kim, Myun Soo; Cho, Daeho Soo; Choi, Sang Ho; Kim, Tae Sung

doi: 10.3892/ijmm_00000510pmid: 20878086

In a previous study, we reported that a gene mutation of repeat in toxin E (RtxE), a transporter of cytotoxic factors, resulted in a significant impairment of epithelial cell cytotoxicity in Vibrio vulnificus, and that the expression of the rtxE gene was induced by the exposure to the host cells. In this study, we evaluated and compared the effects of co-culture supernatants from V. vulnificus-infected INT-407 cells and either the V. vulnificus wild-type or rtxE mutant on the production of interleukin (IL)-8, a pro-inflammatory cytokine, as well as its underlying mechanisms in human intestinal epithelial cells. INT-407 cells were co-cultured with the wild-type V. vulnificus or the rtxE mutant strain to obtain the conditioned supernatants. IL-8 production and nuclear factor (NF)-κB activation from the INT-407 cells treated with each supernatant, were investigated. The co-culture supernatants from the rtxE mutant V. vulnificus-infected INT-407 cells significantly induced lower levels of IL-8 production and promoter activation, NF-κB DNA binding activity, and NF-κB minimal promoter activation in human intestinal epithelial cells, than those from the wild-type V. vulnificus-infected INT-407 cells. Importantly, the reduced IL-8 production and NF-κB activity of the V. vulnificus rtxE mutant, were restored by co-culture supernatants from the rtxE-complemented V. vulnificus. On the whole, these results show that the rtxE gene of V. vulnificus performs a critical role in the secretion of factors from bacteria and host cells, which are involved in IL-8 production via the NF-κB activation pathway in host cells.

Tsukamoto, Yasuhiro ; Kataoka, Bin ; Adachi, Kazuhide ; Kato, Hidenori ; Inai, Marie ; Tsukamoto, Masaya ; Handharyani, Ekowati ; Taira, Eiichi

doi: 10.3892/ijmm_00000511pmid: 20878087

Gicerin is a cell adhesion molecule in the immunoglobulin superfamily. This molecule has homophilic and heterophilic adhesive activities, binding to the neurite out-growth factor (NOF). We have previously reported that gicerin plays an important role in the development and regeneration as well as in the metastasis of tumors through its adhesive activities, mediating cell-cell and/or cell-extracellular matrix interactions. In this study, we investigated the involvement of gicerin in a dermal autograft chicken model. Gicerin and NOF were transiently present in the regenerating epithelia after the dermal graft transplantation. The treatment with an anti-gicerin polyclonal antibody, by placing drops onto the wounds, inhibited the adhesiveness of the grafts to the marginal skin. The chimeric protein of gicerin-IgG, gicerin-Fc, and NOF proteins promoted the regeneration of the grafts. These findings suggest the potential function of gicerin in dermal autografts, and gicerin and NOF proteins could help clinical improvement after transplantations.

Aoki, Yusuke ; Fukao, Toshiyuki ; Zhang, Gaixiu ; Ohnishi, Hidenori ; Kondo, Naomi

doi: 10.3892/ijmm_00000512pmid: 20878088

The survival of motor neuron (SMN) protein forms a multiprotein complex (SMN complex) with Gemin proteins. The complex is known to play a crucial role in RNA metabolism. Several lines of evidence show that SMN is phosphorylated at serine and/or threonine residues. In this study, we hypothesized that SMN is phosphorylated at two kinds of serine residues, the Q28SDD31SD site and two SQ sites (80SQ and 163SQ). A FLAG-tagged wild-type construct (SMNfull) and three FLAG-tagged mutant constructs were made: an SMNAQ mutant with two AQ sites instead of two SQ sites at residues 80 and 163, an SMNQADDAD mutant with QADDAD instead of Q28SDD31SD, and an SMNAQ/QADDAD mutant with the two AQ sites and QADDAD. We expressed these mutants in HeLa cells and analyzed their phosphorylated bands by immunoblotting, the protein stability using cycloheximide, binding to Gemin 2 and foci formation. Mutations in Q28SDD31SD, but not in two SQ sites reduced the intensity of phosphorylation bands, indicating that Q28SDD31SD is the major phosphorylation site in SMN. Mutations in the two SQ sites and Q28SDD31SD did not affect protein stability and binding to Gemin 2. Whereas mutations in the two SQ sites did not cause apparent changes in foci formation, mutations in Q28SDD31SD resulted in a reduced number of large foci in the cytosol. We demonstrated that phosphorylation in Q28SDD31SD may be important in cytosolic foci formation.

Cheng, Chun-Yuan ; Lin, Yi-Hsiang ; Su, Chin-Cheng

doi: 10.3892/ijmm_00000513pmid: 20878089

Curcumin (diferuloylmethane), which is obtained from turmeric, the rhizome of Curcuma longa (L.), inhibits many human cancer cells. However, the molecular mechanisms responsible for curcumin-induced endoplasmic reticulum stress in human hepatic cellular carcinoma J5 cells, are not yet clearly understood. J5 cells were treated with various concentrations of curcumin for different durations. The cell viability was detected by MTT assay. The protein expressions of caspase-12, ATF6, GADD153, Calnexin, Calreticulin, PDI and Ero1-Lα, which are associated with endoplasmic reticulum stress and the unfolding protein response pathway, were examined by Western blot analysis. The cell cycle was analyzed by flow cytometry. The protein expressions of TCTP, Mcl-1, Bcl-2 and Bax, which are related to mitochondrial dysfunction, were detected by Western blot analysis. We also detected the ATF6 protein location by immunocytochemistry. The results showed that curcumin inhibits the proliferation of J5 cells in a time- and dose-dependent manner. Curcumin induced the unfolding protein response by down-regulating the protein expressions of Calnexin, PDI and Ero1-Lα and up-regulating the Calreticulin expression. Curcumin induces the GADD153 expression by cleaving caspase-12 and ATF6, and then by translocating ATF6 to the nucleus. Curcumin also down-regulates the protein expressions of TCTP, Mcl-1 and Bcl-2, in order to induce mitochondrial dysfunction. Curcumin induced cell cycle arrest at the G2/M phase by decreasing the Cdc2 expression. In conclusion, the present study showed that curcumin inhibits the proliferation of J5 cells by inducing endoplasmic reticulum stress and mito-chondrial dysfunction.

Xu, Yanfang ; Ruan, Shiwei ; Xie, Hong ; Lin, Jiumao

doi: 10.3892/ijmm_00000514pmid: 20878090

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), plays an important role in angiotension II (Ang II)-induced hypertensive renal injury associated with pro-inflammatory responses, tubular functional damage and cellular apotosis. In this study, we report on the role of LOX-1 in Ang II-induced oxidative functional damage and underlying signaling in human renal proximal tubular epithelial cells (HRPTEpiCs). The exposure to Ang II enhanced the expression of the NADPH oxidases (the p22phox, p47phox and Nox4 subunits), LOX-1 and the adhesion molecule, ICAM-1. It also promoted monocytic U937 cell adherences to HRPTEpiCs, increased reactive oxygen species formation and stimulated apotosis, which was concomitant with an increase in the activation of p38 and p44/42 mitogen-activated protein kinases (MAPK). Furthermore, the Ang II treatment disturbed the balance of the Bcl-2 family proteins, destabilized mitochondrial membrane potential, and subsequently triggered the release of cytochrome c into the cytosol, causing the activation of caspase-3. The NADPH oxidase inhibitors and LOX-1 small interfering RNA markedly ameliorated these detrimental effects by reducing LOX-1 expression and MAPK activation. The p38 and p44/42MAPK inhibitors also inhibited the Ang II-induced functional damage without affecting LOX-1 expression in the HRPTEpiCs. These observations suggest that LOX-1 mediates Ang II-induced renal tubular epithelial dysfunction. In addition, MAPK pathway activation occurs downstream of the Ang II/reactive oxygen species/LOX-1 cascade.

Castillo-Díaz, Silvia A.; Garay-Sevilla, María E.; Hernández-González, Martha A.; Solís-Martínez, Martha O.; Zaina, Silvio O.

doi: 10.3892/ijmm_00000515pmid: 20878091

Global DNA hypomethylation potentially leading to pro-atherogenic gene expression occurs in atherosclerotic lesions. However, limited information is available on the genomic location of hypomethylated sequences. We present a microarray-based survey of the methylation status of CpG islands (CGIs) in 45 human atherosclerotic arteries and 16 controls. Data from 10,367 CGIs revealed that a subset (151 or 1.4%) of these was hypermethylated in control arteries. The vast majority (142 or 94%) of this CGI subset was found to be unmethylated or partially methylated in atherosclerotic tissue, while only 17 of the normally unmethylated CGIs were hypermethylated in the diseased tissue. The most common functional classes among annotated genes adjacent to or containing differentially methylated CGIs, were transcription (23%) and signalling factors (16%). The former included HOX members, PROX1, NOTCH1 and FOXP1, which are known to regulate key steps of atherogenesis. Expression analysis revealed differential expression of all CGI-associated genes analysed. Sequence analysis identified novel DNA motifs with regulatory potential, associated with differentially methylated CGIs. This study is the first large-scale analysis of DNA methylation in atherosclerosis. Our data suggest that aberrant DNA methylation in atherosclerosis affects the transcription of critical regulatory genes for the induction of a pro-atherogenic cellular phenotype.

Hasegawa, Tomokazu ; Chosa, Naoyuki ; Asakawa, Takeyoshi ; Yoshimura, Yoshitaka ; Ishisaki, Akira ; Tanaka, Mitsuro

doi: 10.3892/ijmm_00000516pmid: 20878092

Although periodontal ligament (PDL) cells have previously been isolated from permanent teeth, they have not been isolated from deciduous teeth. Here, we used human telomerase reverse transcriptase (hTERT) induction to establish the first immortalized PDL cell lines derived from deciduous teeth. Cells were transfected with plasmids containing hTERT. Single-cell cloning was then performed using the limited dilution method. Reverse transcriptase polymerase chain reaction and stretch PCR were used to detect hTERT expression in the clones. In order to determine whether the clones could differentiate into osteoblasts, we stimulated the cells with ascorbic acid and β-glycerophosphate. We success-fully obtained 3 single-cell clones, and named them single cell derived from human deciduous PDL (SH) 9, 10 and 11. All the SH cells showed hTERT expression and stable proliferation after >80 population doublings and expressed the marker genes of PDL cells, including scleraxis, periostin, cementum-derived protein 23, and tenomodulin. Although all the clones expressed osteoblastic markers, only the clones from the SH 9 cell line differentiated into osteoblastic cells. This is the first report of the immortalization of PDL cells derived from deciduous teeth. These cells could be useful in studies investigating the cellular mechanisms and regenerative processes of human PDL cells.

Shiroeda, Hisakazu ; Tahara, Tomomitsu ; Shibata, Tomoyuki ; Nakamura, Masakatsu ; Yamada, Hideto ; Nomura, Tomoe ; Hayashi, Ranji ; Saito, Takashi ; Fukuyama, Tomoki ; Otsuka, Toshimi ; Yano, Hirokazu ; Ozaki, Kazuaki ; Tsuchishima, Mutsumi ; Tsutsumi, Mikihiro ; Arisawa, Tomiyasu