Regulatory effect of anti-gp130 functional mAb on IL-6 mediated RANKL and Wnt5a expression through JAK-STAT3 signaling pathway in FLS.

We investigated the effect on rheumatoid arthritis (RA) of an anti-gp130 monoclonal antibody (mAb) and its mechanism using RA fibroblast-like synoviocytes (FLS) and a collagen antibody-induced arthritis (CAIA) mouse model. We determined the interleukin 6 (IL-6), IL-6 receptor α (IL-6Rα), gp130, receptor activator of nuclear factor κB ligand (RANKL), matrix metalloproteinase 3 (MMP3), TIMP metallopeptidase inhibitor 1 (TIMP1), and Bcl-2 levels in RA and osteoarthritis (OA) serum and synovial fluid. RA FLS were cultured with or without IL-6/IL-6Rα; WNT5A and RANKL levels were detected. We generated an anti-gp130 mAb (M10) with higher affinity and specificity, blocked IL-6 signaling with it, and assessed its effects on the CAIA model, WNT5A and RANKL expression, and signal transducer and activator of transcription 3 (STAT3) phosphorylation. The IL-6 signaling system in patients with RA was increased; RANKL, MMP3, TIMP1, and Bcl-2 in RA bone were elevated. IL-6/IL-6Rα increased RA FLS WNT5A and RANKL expression. M10 ameliorated arthritis in the CAIA model, and inhibited RANKL, WNT5A, and Bcl-2 expression in RA FLS by blocking IL-6 signaling, likely via Janus kinase-STAT3 pathway downregulation. The IL-6-soluble IL-6Rα-gp130 complex is hyperactive in RA and OA. M10 may be the basis for a novel RA treatment drug.

ERp29 inhibits tumorigenicity by suppressing epithelial mesenchymal transition in gastric cancer.

ERp29 is a novel endoplasmic reticulum (ER) protein that plays an important role in protein unfolding and secretion. Recently, it has been reported to be widely implicated in control of tumorigenesis in some tumors. However, the potential function of ERp29 in gastric cancer remains poorly understood. In this study, we found that the positive rate of ERp29 in gastric cancer tissues was significantly lower than that in adjacent non-tumor tissues. And tumor with high ERp29 expression had inclinations towards smaller tumor size and earlier TNM stage. The in vitro experiments indicated that over-expression of ERp29 in gastric cancer cells significantly suppressed the proliferation and migration of tumor cells, which is consistent with the result of the in vivo animal experiments. Furthermore, our mechanistic investigations revealed that ERp29 reversed EMT process in gastric carcinoma, and its effect was related to the inactivation of ERK1/2 and AKT phosphorylation. Thus, we conclude that ERp29 acts as a tumor suppressor gene in gastric cancer, and is expected to become a novel target of the treatment of GC.

p21-activated protein kinase 1 induces the invasion of gastric cancer cells through c-Jun NH2-terminal kinase-mediated activation of matrix metalloproteinase-2.

Gastric cancer (GC) is one of the most common malignancies worldwide. The prognosis of GC is poor, mostly due to widespread metastasis. p21-activated kinase 1 (Pak1), the best characterized member of an evolutionarily conserved family of serine/threonine kinases, plays an important role in the regulation of cell morphogenesis, motility, mitosis and angiogenesis. By qRT-PCR and Gelatin zymograph assay, we demonstrated in the present study that stable overexpression of Pak1 induced matrix metalloproteinase (MMP)-2 mRNA expression and activity in the human MKN45 GC cell line. Conversely, knockdown of endogenous Pak1 expression by small interfering RNA (siRNA) decreased MMP-2 mRNA expression and activity in the MKN45 GC cells. Activation of c-Jun N-terminal kinase (JNK) was required for Pak1-induced upregulation of MMP-2 mRNA level and activity. Moreover, upregulation of MMP-2 by Pak1 via the JNK pathway notably promoted the invasion of MKN45 GC cells. Overexpression of MMP-2 mRNA was once again confirmed to be associated with GC metastasis. In conclusion, our results demonstrated for the first time that Pak1 stimulated MMP-2 mRNA expression and activity in MKN45 GC cells. The JNK signaling pathway was involved in Pak1 modulation of MMP-2, which was important for MKN45 GC cell invasiveness.

Aurora kinase A revives dormant laryngeal squamous cell carcinoma cells via FAK/PI3K/Akt pathway activation.

Revival of dormant tumor cells may be an important tumor metastasis mechanism. We hypothesized that aurora kinase A (AURKA), a cell cycle control kinase, promotes the transition of laryngeal squamous cell carcinoma (LSCC) cells from G0 phase to active division. We therefore investigated whether AURKA could revive dormant tumor cells to promote metastasis. Western blotting revealed that AURKA expression was persistently low in dormant laryngeal cancer Hep2 (D-Hep2) cells and high in non-dormant (T-Hep2) cells. Decreasing AURKA expression in T-Hep2 cells induced dormancy and reduced FAK/PI3K/Akt pathway activity. Increasing AURKA expression in D-Hep2 cells increased FAK/PI3K/Akt pathway activity and enhanced cellular proliferation, migration, invasion and metastasis. In addition, FAK/PI3K/Akt pathway inhibition caused dormancy-like behavior and reduced cellular mobility, migration and invasion. We conclude that AURKA may revive dormant tumor cells via FAK/PI3K/Akt pathway activation, thereby promoting migration and invasion in laryngeal cancer. AURKA/FAK/PI3K/Akt inhibitors may thus represent potential targets for clinical LSCC treatment.

ERp19 contributes to tumorigenicity in human gastric cancer by promoting cell growth, migration and invasion.

ERp19, a mammalian thioredoxin-like protein, plays a key role in defense against endoplasmic reticulum stress. It belongs to the protein disulfide isomerize (PDI) family, whose members have been implicated in development of breast, ovarian and gastrointestinal cancers. However, the role of ERp19 in gastric cancer (GC) remains undefined. Therefore, we sought to investigate the expression and prognostic value of ERp19 in GC patients, and to explore the role of ERp19 in tumorigenicity. Expression of ERp19 in gastric tissues was assessed by immunohistochemical staining and real-time PCR in clinical samples of GC patients. Statistical analysis of clinical cases revealed that the expression levels of ERp19 were higher in tumor tissues than non-tumor tissues. And the level of ERp19 expression was correlated with tumor size, lymph node involvement and poor clinical prognosis. Furthermore, ERp19 knockdown dramatically suppressed gastric cancer cell growth, inhibited cellular migration/invasion and down regulated the phosphorylation of FAK and paxillin, whereas ERp19 over-expression reversed these changes. We conclude that ERp19 contributes to tumorigenicity and metastasis of GC by activating the FAK signaling pathway, and may function as an oncogene in GC. ERp19 may represent a new diagnostic and prognostic marker and a novel target for the treatment of GC.

Epigenetic silencing of microRNA-149 in cancer-associated fibroblasts mediates prostaglandin E2/interleukin-6 signaling in the tumor microenvironment.

Tumor initiation and growth depend on its microenvironment in which cancer-associated fibroblasts (CAFs) in tumor stroma play an important role. Prostaglandin E2 (PGE2) and interleukin (IL)-6 signal pathways are involved in the crosstalk between tumor and stromal cells. However, how PGE2-mediated signaling modulates this crosstalk remains unclear. Here, we show that microRNA (miR)-149 links PGE2 and IL-6 signaling in mediating the crosstalk between tumor cells and CAFs in gastric cancer (GC). miR-149 inhibited fibroblast activation by targeting IL-6 and miR-149 expression was substantially suppressed in the CAFs of GC. miR-149 negatively regulated CAFs and their effect on GC development both in vitro and in vivo. CAFs enhanced epithelial-to-mesenchymal transition (EMT) and the stem-like properties of GC cells in a miR-149-IL-6-dependent manner. In addition to IL-6, PGE2 receptor 2 (PTGER2/EP2) was revealed as another potential target of miR-149 in fibroblasts. Furthermore, H. pylori infection, a leading cause of human GC, was able to induce cyclooxygenase-2 (COX-2)/PGE2 signaling and to enhance PGE2 production, resulting in the hypermethylation of miR-149 in CAFs and increased IL-6 secretion. Our findings indicate that miR-149 mediates the crosstalk between tumor cells and CAFs in GC and highlight the potential of interfering miRNAs in stromal cells to improve cancer therapy.

Exogenous IFN-beta regulates the RANKL-c-Fos-IFN-beta signaling pathway in the collagen antibody-induced arthritis model.

BACKGROUND: Although a variety of drugs have been used to treat the symptoms of rheumatoid arthritis (RA), none of them are able to cure the disease. Interferon ß (IFN-ß) has pleiotropic effects on RA, but whether it can be used to treat RA remains globally controversial. Thus, in this study we tested the effects of IFN-ß on RA patients and on collagen antibody-induced arthritis (CAIA) model mice. METHODS: The cytokine and auto-antibody expression profiles in the serum and synovial fluid (SF) from RA patients were assessed using enzyme-linked immunosorbent assay (ELISA) and compared with the results from osteoarthritis (OA) patients. Exogenous IFN-ß was administered to RA patients and CAIA model mice, and the therapeutic effects were evaluated. Endogenous IFN-ß expression in the joint bones of CAIA model mice was evaluated by quantitative real-time PCR (qRT-PCR). The effects of exogenous IFN-ß on CAIA model mice were assessed using a clinical scoring system, hematoxylin eosin and safranin-O with fast green counterstain histology, molybdenum target X-ray, and tartrate-resistant acid phosphatase (TRAP) staining. The RANKL-RANK signaling pathway was analyzed using qRT-PCR. The RAW 264.7 cell line was differentiated into osteoclasts with RANKL stimulation and then treated with exogenous IFN-ß. RESULTS: The expression of inflammatory cytokines (IFN-γ, IL-17, MMP-3, and RANKL) and auto-antibodies (CII antibodies, RF-IgM, and anti-CCP/GPI) were significantly higher in RA compared with OA patients. After IFN-ß intervention, some clinical symptoms in RA patients were partially alleviated, and the expression of IFN-γ, IL-17, MMP-3, and OPG) returned to normal levels. In the CAIA model, the expression of endogenous IFN-ß in the joint bones was decreased. After IFN-ß administration, the arthritis scores were decreased; synovial inflammation, cartilage, and bone destruction were clearly attenuated; and the expression of c-Fos and NFATc1 were reduced, while RANKL and TRAF6 expression was unchanged. In addition, exogenous IFN-ß directly inhibited RANKL-induced osteoclastogenesis. CONCLUSIONS: Exogenous IFN-ß administration immunomodulates CAIA, may reduce joint inflammation and, perhaps more importantly, bone destruction by inhibiting the RANKL-c-Fos signaling pathway. Exogenous IFN-ß intervention should be selectively used on RA patients because it may only be useful for RA patients with low endogenous IFN-ß expression.

RhoGDI2 confers resistance to 5-fluorouracil in human gastric cancer cells.

Resistance to 5-fluorouracil (5-FU) in patients with gastric cancer is a serious therapeutic problem and major efforts are underway to understand the underlying mechanisms. We have previously identified RhoGDI2 as a contributor to 5-FU resistance in colon cancer cells using 2D electrophoresis and mass spectrometry and the current study aimed to further investigate this role. The expression of RhoGDI2 in seven gastric cancer cell lines was positively correlated with resistance to 5-FU. Lower 5-FU sensitivity of isolated tumor cells from patients with gastric cancer was also associated with higher RhoGDI2 expression. Ectopic expression of RhoGDI2 in gastric cancer cells increased the resistance to 5-FU and reverted low dose 5-FU-induced G2/M phase arrest without affecting the population of sub-G1 cells. Overall, these findings suggest that RhoGDI2 is associated with 5-FU resistance and is a potential therapeutic target for enhancing chemotherapy efficacy in gastric cancer.

Emerging roles of the ribonucleotide reductase M2 in colorectal cancer and ultraviolet-induced DNA damage repair.

AIM: To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair. METHODS: Immunohistochemical staining of tissue microarray was performed to detect the expression of RRM2. Seven CRC cell lines were cultured and three human colon cancer cell lines, i.e., HCT116, SW480 and SW620, were used. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of RRM2, respectively. Cell proliferation assay, cell cycle analysis were performed. Cell apoptosis was evaluated by double staining with fluorescein isothiocyanate-conjugated Annexin V and propidium iodide (PI) using Annexin V/PI apoptosis kit. The motility and invasion of CRC cells were assessed by the Transwell chamber assay. Cells were irradiated with a 254 nm UV-C lamp to detect the UV sensitivity after RRM2 depletion. RESULTS: Immunohistochemical staining revealed elevated RRM2 levels in CRC tissues. RRM2 overexpression was positively correlated with invasion depth (P < 0.05), poorly differentiated type (P = 0.0051), and tumor node metastasis stage (P = 0.0015). The expression of RRM2 in HCT116 cells was downregulated after transfection, and HCT116 cell proliferation was obviously suppressed compared to control groups (P < 0.05). In the invasion test, the number of cells that passed through the chambers in the RRM2-siRNA group was 81 ± 3, which was lower than that in the negative control (289 ± 7) and blank control groups (301 ± 7.2). These differences were statistically significant (P < 0.01). Our data suggest that RRM2 overexpression may be associated with CRC progression. RRM2 silencing by siRNA may inhibit the hyperplasia and invasiveness of CRC cells, suggesting that RRM2 may play an important role in the infiltration and metastasis of CRC, which is a potential therapeutic strategy in CRC. In addition, RRM2 depletion increased UV sensitivity. CONCLUSION: These findings suggest that RRM2 may be a facilitating factor in colorectal tumorigenesis and UV-induced DNA damage repair.

Etiology and antimicrobial resistance of community-acquired pneumonia in adult patients in China.

BACKGROUND: Appropriate antimicrobial therapy of community-acquired pneumonia (CAP) is mainly based on the distribution of etiology and antimicrobial resistance of major pathogens. We performed a prospective observational study of adult with CAP in 36 hospitals in China. METHODS: Etiological pathogens were isolated in each of the centers, and all of the isolated pathogens were sent to Zhongshan Hospital for antimicrobial susceptibility tests using agar dilution. RESULTS: A total of 593 patients were enrolled in this study, and 242 strains of bacteria were isolated from 225 patients. Streptococcus pneumoniae (79/242, 32.6%) was the most frequently isolated pathogen, followed by Haemophilus influenzae (55/242, 22.7%) and Klebsiella pneumoniae (25/242, 10.3%). Totally 527 patients underwent serological tests for atypical pathogens; Mycoplasma pneumoniae and Chlamydia pneumoniae infections were identified in 205 (38.9%) and 60 (11.4%) patients respectively. Legionella pneumophila infections were identified in 4.0% (13/324) of patients. The non-susceptibility rate of isolated Streptococcus pneumoniae to erythromycin and penicillin was 63.2% and 19.1% respectively. Six patients died from the disease, the 30-day mortality rate was 1.1% (6/533). CONCLUSIONS: The top three bacteria responsible for CAP in Chinese adults were Streptococcus pneumonia, Haemophilus influenza and Klebsiella pneumonia. There was also a high prevalence of atypical pathogens and mixed pathogens. The resistance rates of the major isolated pathogens were relatively low except for the high prevalence of macrolide resistance in Streptococcus pneumoniae.

microrna expression signature of gastric cancer cells relative to normal gastric mucosa.

micrornas (miRNAs) play an important role in a wide range of physiological and developmental processes by negatively regulating the expression of target genes at the post-transcriptional level. In this study, we investigated the differential miRNA expression signature between gastric cancer cells and normal gastric mucosa to determine changes in miRNA expression during gastric cancer development. We analyzed the global miRNA expression profiles of 9 gastric cancer cell lines and 6 normal gastric mucosa lines using miRNA microarrays. In addition, we performed quantitative real-time PCR (Q-PCR) to validate the results. Correlations between the miRNA expression profile and tumor clinicopathological parameters were analyzed. We found that 17 miRNAs were upregulated in gastric cancer cell lines and 146 miRNAs were downregulated compared to normal gastric mucosa. Using microarray data and Q-PCR validation, 15 miRNAs were finally selected. These candidate miRNAs were associated with gastric cancer clinicopathology to various degrees. High expression levels of hsa-miR-93 were found to predict poor survival (median, 16 vs. 40 months; log-rank test p<0.05). These findings suggest that miRNAs play vital roles in human gastric cancer. The findings may also provide clues toward understanding the molecular functions of miRNAs in various biological processes.

[Suppression of Aurora-A by RNA interference inhibits laryngeal cancer Hep-2 cell growth].

OBJECTIVE: To investigate the effects of knockdown of Aurora-A by RNA interference on laryngeal cancer Hep-2 cell growth in vitro and in vivo. METHODS: A plasmid containing siRNA against Aurora-A was constructed and transfected into human laryngeal cancer cell line Hep-2. Measurements included the CCK-8 assay for viability and proliferation, Transwell assay for invasion, colony formation assay for cell anchorage-independent growth. Western blot and immunohistochemistry assay for protein expression. Tumorigenicity was observed in vivo. RESULTS: In Hep-2 cells transfected by Aurora-A siRNA (designated as siRNA-3), protein expression of Aurora-A was suppressed by 52%. In CCK-8 assay, absorbance value of siRNA-3 cells (3.268 ± 0.106, (x(-) ± s)) was lower than that of Hep-2 cells (3.722 ± 0.152, F = 17.634, P < 0.001). In Transwell assay, the average invasive cells per field in siRNA-3 cells (110.0 ± 18.0) was less than that in Hep-2 cells (236.0 ± 26.0, F = 26.462, P < 0.01). In colony formation assay, the average colony number of siRNA-3 cells (31.0 ± 6.6) was lower than that of Hep-2 cells (104.0 ± 14.0). The average tumor size in siRNA-3 group was (127.77 ± 174.83) mm(3), which was less than Hep-2 cell group (837.26 ± 101.80) mm(3), (F = 28.187, P < 0.001). Silencing of Aurora-A decreased the expression of focal adhesion kinase (FAK) and matrix metalloproteinase-2 (MMP-2), key regulators in cell adhesion and invasion. CONCLUSIONS: The knockdown of Aurora-A inhibits the growth and invasiveness of Hep-2 cells in vitro and in vivo, which may be a promising therapeutic strategy for LSCC.

[Interaction of SerpinB5 and MAFbx in gastric cancer cell and its action site].

OBJECTIVE: To explore the interaction between SerpinB5 and MAFbx in gastric cancer cell and to identify the interaction sites. METHODS: The interaction between SerpinB5 and MAFbx was screened and validated by yeast two-hybrid screening and co-immunoprecipitation. The expression of MAFbx was analyzed after SerpinB5 expression being modified by RNA interference and pGBKT7-SerpinB5 transfection. The impact of SerpinB5 on the expression of MAFbx was studied in gastric cancer cell line SUN-16. A model of MAFbx was constructed by homology modeling. The related residues for interaction were analyzed by Autodock4.0. RESULTS: The interaction between SerpinB5 and MAFbx was validated. The expression of MAFbx changed along with SerpinB5 expression. Amino acids including PRO261, ASN361, and LYS362 were key residue in the interaction of SerpinB5 and MAFbx. CONCLUSION: SerpinB5 interacts with MAFbx in gastric cancer cell. Amino acids including PRO261, ASN361, and LYS362 are potential binding sites.

P21-activated protein kinase 1 is overexpressed in gastric cancer and induces cancer metastasis.

P21-activated protein kinase (Pak1), a main downstream effector of small Rho GTPases, plays an important role in the regulation of cell morphogenesis, motility, mitosis and angiogenesis. However, the role of Pak1 in gastric cancer metastasis remains unclear. Here, we showed that Pak1 is overexpressed in gastric cancer tissues from 74 patients by immunohistochemistry. Overexpression of Pak1 was associated with metastasis and prognosis of gastric cancer. In addition, overexpression of Pak1 increased gastric cancer cell motility and invasion, whereas downregulation of Pak1 expression reduced gastric cancer cell migration and invasion. In further study, data showed that activated Pak1 inhibited stress fiber and focal adhesion complex formation in gastric cancer cells and led to the formation of motile phenotypes. Importantly, activated Pak1 elicited phosphorylation of the ERK and JNK-dependent pathway in gastric cancer cell lines. In conclusion, our results suggest that Pak1 is overexpressed in gastric cancer and plays an important role in the metastasis of gastric cancer. The mechanism by which Pak1 induces cancer metastasis may involve activation of ERK and JNK.

Knockdown of metallopanstimulin-1 inhibits NF-κB signaling at different levels: the role of apoptosis induction of gastric cancer cells.

The ribosomal protein S27 (metallopanstimulin-1, MPS-1) has been reported to be a multifunctional protein, with increased expression in a number of cancers. We reported previously that MPS-1 was highly expressed in human gastric cancer. Knockdown of MPS-1 led to spontaneous apoptosis and repressed proliferation of human gastric cancer cells in vitro and in vivo. However, how does MPS-1 regulate these processes is unclear. Here we performed microarray and pathway analyses to investigate possible pathways involved in MPS-1 knockdown-induced apoptosis in gastric cancer cells. Our results showed that knockdown of MPS-1 inhibited NF-κB activity by reducing phosphorylation of p65 at Ser536 and IκBα at Ser32, inhibiting NF-κB nuclear translocation, and down-regulating its DNA binding activity. Furthermore, data-mining the Gene-Regulatory-Network revealed that growth arrest DNA damage inducible gene 45ß (Gadd45ß), a direct NF-κB target gene, played a critical role in MPS-1 knockdown-induced apoptosis. Over-expression of Gadd45ß inhibited MPS-1 knockdown-induced apoptosis via inhibition of JNK phosphorylation. Taken together, these data revealed a novel pathway, the MPS-1/NF-κB/Gadd45ß signal pathway, played an important role in MPS-1 knockdown-induced apoptosis of gastric cancer cells. This study sheds new light on the role of MPS-1/NF-κB in apoptosis and the possible use of MPS-1 targeting strategy in the treatment of gastric cancer.

[Establishment of experimental angiogenic models with applications of quantitative digital image analysis].

OBJECTIVE: To establish experimental models for tumor neovascularization and to apply quantitative digital imaging analysis in the study. METHODS: An endothelial tube formation model was established by human umbilical vein endothelial cells (HUVECs). A vasculogenic mimicry model was established by SGC-7901 gastric cancer cell line. Fertilized eggs were used to establish a chorioallantoic membrane angiogenesis model. Using gene transfection experiment, IRX1 tumor suppressor gene was chosen as a therapeutic target. Image Pro Plus (IPP) analysis software was used for digital vascular images analysis with parameters including points, lines, angles and integral absorbance (IA) for the tubular formation or vasculogenic mimicry. RESULTS: Digital image analysis by IPP showed that HUVEC tubular formation was significantly inhibited in IRX1 transfectant, compared with controls. The tubular numbers in three groups were 12.80 +/- 3.83, 29.00 +/- 5.34 and 28.20 +/- 4.32 (P<0.01). The connection points of tubules in three groups were 13.20 +/- 2.59, 25.00 +/- 2.24 and 24.60 +/- 3.21 (P<0.01). The tubular lengths of three groups were (821.5 +/- 12.5), (930.9 +/- 13.5) and (948.4 +/- 18.1) microm (P=0.022). The IA values of PAS stain in three groups were 3606 +/- 363, 14 200 +/- 1251 and 15 043 +/- 1220 (P<0.01). In chick chorioallantoic membrane model, the angular numbers of tubules in three groups were 6.41 +/- 2.60, 10.27 +/- 2.65 and 9.18 +/- 1.99 (P<0.01). CONCLUSIONS: The endothelial tube formation model, vasculogenic mimicry model and chorioallantoic membrane angiogenesis model are useful for gene therapy and drug screening with targeting neoplastic vascularization. Professional image analysis software may greatly facilitate the quantitative analysis of tumor neovascularization.

Salinomycin can effectively kill ALDH(high) stem-like cells on gastric cancer.

Salinomycin is a novel identified cancer stem cells (CSCs) killer. Higher ALDH activity represents CSCs characterization. Here, we screened ALDH activities on several gastric cancer cell lines and divided them into ALDH(high) and ALDH(low) gastric cancer groups. ALDH(high) cancer cells (NCI-N87 and SNU-1) disclosed more CSCs characteristics, such as higher levels of Sox2, Nanog and Nestin, more floating spheroid bodies, more colony formation and more resistance to conventional chemotherapeutic drugs 5-Fu and CDDP, compared to these parameters observed in ALDH(low) cancer cells (P<0.01). Importantly, ALDH(high) cancer cells are relatively sensitive to salinomycin when compared to ALDH(low) cancer cells (P<0.01). Our results confirmed ALDH as functional marker of CSCs population on gastric cancer. Salinomycin might be selective therapy for CSCs fraction, which is resistant to conventional anticancer drugs 5-Fu and CDDP.

SerpinB5 interacts with KHDRBS3 and FBXO32 in gastric cancer cells.

Mammary serine protease inhibitor B5 (SerpinB5) is a potential oncogene in gastric cancer (GC); however, the molecular mechanism by which SerpinB5 promotes oncogenesis remains elusive. In this study, SerpinB5-associated proteins were selected based on yeast two-hybrid screening and microarray analysis after RNA interference and were validated using co-immunoprecipitation (Co-IP) and RNA Co-IP. The expression profiles of the interacting proteins were analyzed by Western blotting and immunohistochemistry. The effects of SerpinB5 on KHDRBS3 and FBXO32 expression in GC cells were analyzed using real-time PCR and Western blotting after the expression of SerpinB5 was modified. By yeast two-hybrid screening and microarray analysis, FBXO32 and KHDRBS3 were found to be SerpinB5-interacting proteins. The interactions were confirmed by Co-IP. An RNA co-immunoprecipitation assay found that KHDRBS3 interacted with FBXO32 mRNA. The expression of SerpinB5 was much stronger in the nucleus of GC cells. FBXO32 was expressed at higher levels in the cytoplasm of GC cells. KHDRBS3 was primarily detected in the nucleus of normal mucosal cells. SerpinB5 expression was modified in GC cells, KHDRBS3 mRNA levels remained stable, however, FBXO32 mRNA levels changed 24 h after changes in KHDRBS3 protein levels were detected. In conclusion, SerpinB5 interacts with KHDRBS3 and FBXO32, and KHDRBS3 can interact with FBXO32 mRNA.

Cell cycle dependent genes from the gastric cancer cell MKN45 can affect tumorigenesis.

BACKGROUND/AIMS: To investigate the cell cycle dependent genes involved in gastric tumorigenesis, possibly determining the relationship between the cell cycle and tumorigenesis. METHODOLOGY: MKN45 cells were collected every hour from Oh to 12h after release from G2/M and G1/S blocks. Nine samples (a-i), chosen at key times of the cell cycle, were prepared for RNA isolation and cDNA microarray analysis. RESULTS: In 2001 viable clones, 959 genes showed periodic variations during the cell cycle. Among 2001 genes that were clustered, a series of up-regulated genes were assigned to different cell cycle phases. Many periodically dependent genes in the cell cycle were ubiquitously expressed and participated in various cell physiological functions, such as transcription, translation, ubiquitination and signal transduction. These cell cycle dependent genes could affect cancer cell proliferation, apoptosis, activation of oncogenes and inactivation of tumor suppressor genes. CONCLUSIONS: We provided a comprehensive understanding of the gene expression profile involved in gastric cancer cell cycles and laid a foundation for further research on mechanisms of gastric tumorigenesis.